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Abstracts of Presentations at the 1989 Annual Meeting of 

The American Phytopathological Society 

August 20-24, 1989 

Richmond Marriott Hotel and Richmond Convention Centre 

Richmond, Virginia 

The number above an abstract corresponds to its designation in the program of the 1989 APS Annual Meeting in Richmond, VA, August 20-24. 

If a presentation was not given at the meeting, the abstract is not printed among the following pages. 

The index to authors begins on page 1224. 

1 lated peanut leaf and disease proportions are generated for 

INFLUENCE OF TILLAGE SYSTEMS ON THE INCIDENCE AND SPATIAL PAT- 

TERN OF TAN SPOT OF WHEAT. Wolgang Schuh, Department of Plant 

Pathology, The Pennsylvania State University, University Park, 

PA 16802. 

early leafspot, late leaf spot, both leafspots mixed or peanut 

rust. Pre-testing, drill and practice, and post-testing sessions 

were conducted in a 1-hr time period for introductory plant 

pathology students. Twenty of 22 students significantly improved 

their ability to estimate disease proportions of late leaf spot 

The incidence and spatial pattern of Pyrenophora triticirepentis 

was assessed at four locations (two conventional & two 

conservation tillage) two times during 1987 and three times 

during 1988. Tan Spot was detected earlier in 1988 and had a 

higher final disease incidence in both years under conservation 

tillage. Spatial pattern analysis, using Morisita's index of 

dispersion, revealed a shift from clumped to random distribution 

over time, indicating the importance of residueborne 

inoculum under conservation tillage systems. Spatial patterns 

under conventional tillage were random, indicating airborne 

inoculum 

iment suggest 

as the 

that 

source 

straw 

of infection. 

residue on the 

Results 

field 

from 

surface 

this 

will 

experlead 

tincreased thatlstofaPyresidueora therfieldcito 

increased 

r 

levels 

faent 

of 

i t 

Pyrenophora 

ll ery 

tritici-repentis, thereby 

counterbalancing 

tillage systems. advantages associated with the use of reduced 

(random lesion sizes) as measured by (i) a higher coefficient of 

determination relating the actual proportion (X) to the esti- 

mated value (Y) and/or (ii) a regression coefficient (slope) 

which was closer to 1.0 in post-tests compared to pre-tests. 

4 

EFFECTS OF MAIZE INTERCROPS ON ANGULAR LEAF SPOT OF BEANS. M 

eFFECTS OF MAItE ULArsity of oF Kenya. 

Boudreau, Crop Science Dept., University of Nairobi, Kenya. 

Angular 

which hind 

leaf 

been 

spot 

planted 

(ALS) 

alone severity 

and 

was 

simultsneously 

evaluated on 

with 

common 

maize beans 

at 

Kabete, Kenya in 

1987. 

Novembet 

Intercropping 

1986, 

reduced 

and 

the 

at 

area 

Kabete 

under the 

and 

disease 

Thika 

progress 

in April 

cuv curve (AUDPC) ADC by y2%i 26% in 1986 96ada and 

did 

at Thika hk in n18 1987 by y2% 24%, 

not 

but 

significantly reduce AUDPC at Kabete 

u 

in 1987. Additional 

treatments evaluating bean density and row versus random 

2 planting in 1986 had no effect on AUDPC, but fertilizer 

amendment in 1987 increased AUDPC by 64% at Kabete and 22% at 

TOBACCO GROWTH AS AFFECTED BY MELOIDOGYNE INCOGNITA AND SOIL Thika. Modifications in temperature, relative humidity, and wind 

MOISTURE. T. A. Wheeler, K. R. Barker, and S. M. Schneider, velocity in the bean canopy when maize was present suggest 

Department o-Tit Pathology, North Carolina State University, conflicting influences on ALS development. The results 

Raleigh, NC 27695-7616. corroborate other data indicating a significant but variable 

effect of maize intercrops on reducing ALS. 

Flue-cured tobacco (var. NC27-NF) was grown in microplots in a 

RCB design with a factorial arrangement of treatments. The main 

effects werq initial nematode population levels (0 to 20000 5 

eggs/500 cm- soil) and irrigation rates (none, low, moderate and 

high). Plants were destructively sampled throughout the growing A GENERALIZED STOCHASTIC SIMULATION MODEL OF THE INCREASE AND 

season. All stages of M. incognita were removed from the roots SPREAD OF ARTHROPOD-ECTORED PLANT VIRUSES. 

and soil at each sampling date. Tobacco with a moderate R.S.F~riss and P. H. Berger. University of Kentucky, Lexington, KY 40546 and 

irrigation rate had the highest growth rate (r=0.00899), University oflIdaho, Moscow, ID 83843. 

followed by low and no irrigation (r=0.00709 and 0.00679, 

respectively). The r parameter is from the logistic equation: A theoretical model was developed for illustration of the effects of virus transmission 

dH/dt=r*Ht (I-Ht) , where H=host leaf weight and time is in characteristics and vector activity on virus disease dynamics. The model simulates spread from 

degree-days. Tobacco yield potential was greatly impacted by a source plant in a 425-plant field. Input parameters include probabilities of virus acquisition 

irr igation rate. Yield suppression by N. incognita was and inoculation, lengths of latent periods in the plant and vector, length of the vector 

adequately fitted by quadratic regression. infectious period, number of vectors per plant, and three options for the type of simulation of 

vector movement. Decisions about acquisition, inoculation and vector movement are based on 

3 pseudo-random values. Output includes numbers of viruliferous vectors and positions of 

infected plants at each iteration. Model predictions are generally consistent with expected 

DISEASE . PRO : A COMPUTED PROGRAM FOB EVALUATING AND IMPROVING A natural spread. The model illustates the great effect that vector movement can have on disease 

PERSONS ABILITY TO ASSESS DISEASE PROPORTION. F. W. Mutter, Jr. dynamics and spatial distribution, particularly for diseases transmitted ins non-persistent 

and 0. Worawitlikit, Department of Plant Pathology, University manner. Although themodel will be difficult to fully validate, it provides a logically rigorous 

of Georgia, Athens 30602. way of integrating knowledge about the many proceases that affect virus disease epidemics. 

A computer program written in BASIC was developed for the purpose 

of evaluating a persons ability to estimate disease propor- 6 

tions. The program is similar to DISTRAIN but allows greater 

flexibility in the selection of lesion size (small, medium, A CLIMIATOLODGYOFAIRPARCELTRAjECTORIES RjELATDTO'fI}{ 

large, random mixture) and allows for regression analysis of ATMOSPHERIC TRANSPORT OF PERONOSPORA TABACINA.LM 

rating performance. A color monitor is used to display a simu- Davis and J.F. Monahan, North Carolina State University, Raleigh, NC 

27695 

Temperature and wind date from 5-yr of atmospheric soundings were used in 

Camera-ready abstracts are published as submitted. The abstracts conjunction with an atmospheric transport model to construct a trajectory 

are not edited or retyped in the APS headquarters office. climatology for the transport of the sporangia of P.. tab~acina. Daily receptor- 

Vol." 79, No. 10, 1989 1135

oriented trajectories, with the receptor point located in the mountains of North Winter oilseed rape (Brassica napus) grown on 17'000 ha is 

Carolina, were calculated for April through August and for the annual period. the predominant edible-oil crop in Switzerland. The moat 

The 12-, 24-, and 48-hr air parcel positions prior to arrival at the receptor point important fungal diseases are caused by Phoma lingam, Alterwere 

analyzed by examining the plots of the probability density function (pdf). naria sp., Botrytis cinerea and Sclerotinia sclerotiorum. 

By integrating the pdf over a region of interest the probability that the given Cylindrosporium concentricum, Verticillium dahliae and Pseureceptor 

site will receive air parcels from this region can be obtained. Results docercosporella capsellae are rarely found but have the 

indicate that the probability of the receptor point receiving air parcels from the potential to become more prevalent. 

tobacco producing region of south Georgia/north Florida, which is a potential Integrated crop management is employed to try to control 

inoculum source region, increases during the April to August period. This these diseases. It involves measures such as use of resistant 

increase can be attributed to the strengthening of the Bermuda high pressure cultivars, crop rotation and removal of infected residue. 

system. Fungicides are considered a last resort. None are registered 

at present. The identification of resistant cultivars is a 

top priority in our research. Special nurseries have been 

established. The potential use of biotechnology for this 

7 purpose will be investigated. 

GENETIC DIVERSITY AND POPULATION STRUCTURE OF THE RICE 

BACTERIAL BLIGHT PATHOGEN. J. Leach, M. Rhoads, C. Vera Cruzi, 

F. White, T. Mews, and H. Leung . Dept. of Plant Pathology, 11 

Kansas State University, Manhattan, Kansas and lInternational INFECTION OF STRAWBERRY ROOTS BY COLLETOTRICHUM 

Rice Research Institute, Los Bafios, Philippines. FRAGARIAE AND GLOMERELLA CINGULATA. C. M. Howard, C. 

A repetitive DNA element pJEL101 isolated from Xanthomonas K. Chandler, and E. E.. Albregts. Univ. of Fla., AREC, 

campestris pv. oryzae (Xco) was used to determine the popula- Dover, FL 33527. 

tion structure of Xco in the Philippines. pJELl01, when used Large roots ca 14 cm long were excised from rhizomes 

to probe EcoRl-digested genomic DNA of Xco isolates, revealed of strawberry plants that had wilted in Florida 

race-specific restriction fragment length polymorphisms (RFLP). fruiting rields because of anhthacnose crown rot. 

Of 94 isolates examined, 26 RFLP types were identified. Most Isolations from the thizomes yielded primarily C. 

of the variability (20 RFLP types) was found in race 1, 2, and fragariae from seedlings that previously had bean 

3, which were isolated from lowland areas. Three RFLP types grown n a Florida nursery and G. cingulata ( = C. 

were found among isolates from traditional cultivars grown at gloeosoorioides) from Chandlec p-ans that previoaly 

high elevation. The genetic diversity of the total Xco popula- had been grown in a Tennesse.e nursery. The roots were 

tion was 0.93, of which 44% was due to genetic differentiation cut into 1 c.m sections and every other, section 

between races. The genetic diversities of isolates collected beginning with the basal section was plateC on PDA. 

in 1972-1976, 1977-1981, and 1982-1986, were 0.89, 0.89, and Colletotrichum spp. or G. cingulzta (primarily C. 

0.91, respectively, suggesting a consistently high level of fragariae fros seedlingY, and . cinngulata and C. 

variability in the pathogen population over the past 15 years. acutatum from Chandler) were isolated from 45% of the 

basal root sect ions and from 11% of 'he thi]cd 

sections. These fungi were isolated from 1.4% to 2.&% 

8 of sections 5, 7, 9, 11, and 13. 

CULTURAL CONTROL OF SCLEROTINIA CROWN AND STEM ROT IN 

ALFALFA SEED FIELDS. R, G, Gilbert, USDA-Agricultural 2 

Research Service, P. 0. Box 30, Prosser, WA 99350. 

Cultural control of Sclerotinia in broadcast-planted alfalfa A 72KD PROTEIN ASSOCIATED WITH CERCOSPORIN SYNTHESIS IN 

seed fields were: 1) "fall-burn" of harvest residue; 2) 

"winter-burn"; and 3) "spring-beat-back." Fall-burn reduced 

CERCOSPORA KIKUCHII TOXIGENIC ISOLATE PR. Jeffery A. Rollins 

and Robert C. Upchurch. Department of Plant Pathology, N.C. 

sclerotia in the field by >95%. Sclerotia were killed in State University, Raleigh, NC 27695-7616. 

the residue and surface soil. Only 14% of the surface soil 

sclerotia were viable after the fall-burn in November and Comparative SDS-PAGE analysis of the protein extracts of C. 

during the next 6 months the number of surface soil kikuchii parental isolate PR and cercosporin-minus mutants 

sclerotia declined from 246/m2 to 7/mi, and the viability of derived by UV mutagenesis from this isolate revealed that a 

sclerotia was reduced from 12 to 0%. Winter-burn reduced prominent 72kD protein present in the parental isolate was 

surface residue and sclerotia numbers, but did not reduce absent in the mutants. Two-dimensional gel electrophoresis 

viability and survival of surface soil sclerotia. Spring- indicates that the protein is basic. Gel scans indicate that 

beat-back dried the soil surface and reduced the canopy the protein composes as much as 12% of the total extractable 

relative humidity, which established conditions that were protein of PR, but that it is present in lower concentration in 

unfavorable for Sclerotinia activity. Alfalfa seed yields other Cercospora species. Temporal analysis of toxin and 

in the fall-burn and spring-beat-back treatments were 862 protein synthesis in PR shows that the 72kD protein is 

and 716 Kg/ha for an increase of 43 and 13%, respectively, positively correlated with the synthesis of cercosporin. We are 

over winter-burn, interested in identifying this protein by cloning and 

characterizing its encoding gene because of the possibility of 

its involvement in cercosporin synthesis and/or regulation. 

9 

CONTROL OF PHYTOPHTHORA ROOT ROT ON CITRUS WITH SODIUM 

TETRATHIOCARBONATE. N.E. Natheron and J.C. Natej ka, 

1 

Univrsiy ofArionaYum Agrc. ente, Yma, Z 8364CHARACTERIZATION OF HYDROXAMATE SIDEROPHORES AND SIDEROPHORE- 

Greenhouse studies were initiated to determine the efficacy of -MEDIATED IRON IN GAUMANNOMYCES GRAMINIS VAR TRITICI. S. DoniI, 

sodium tetrathiocarbonate (STTC) for control of root rot on S. Solel 3 , Y. Kashman 2 and I. BarashI, Dept. of 3 plant Pathology, 

citrus caused by Phytophthora citrophthora and _P. parasitica. 

When roots of rough lemon seedlings were inoculated with 

ARO, The Volcani Center, Israel. 

zoospores of these two fungi in the presence of STTC at 245 

pg/al or metalaxyl at 10 pg/al, resultant fresh weights of 

Under iron-deficient conditions Gaumannomyces graminis var 

tritici (Ggt) produces hydroxamate siderophores designated as 

shoots and roots did not differ from noninoculated plants, A, B and C according to their electrophoretic mobility. Siderowhereas 

inoculated plants without chemical treatment suffered phores B and C were identified as dimerum acid and coprogen B 

significant loss of shoot and root weights. Treatment with STTC 

at 4900 pg/al was necessary to inactivate propagules of 

respectively. Siderophore A exhibited and identical NMR spectra 

to dimerum acid but was chromatographically distinct. The system 

P. parasitica in naturally infested soil. Rough lemon seedlings of siderophores mediating uptake of iron was characterized. It 

planted 7 days after treatment of soil to avoid phytotoxic exhibited an active transport and Michaelis kineties. Ggt could 

levels of STTC had root and shoot weights equivalent to those utilize iron effectively from siderophores produced by other 

grown in soil treated with 10 pg/ml metalaxyl. Apparently, 

can reduce the severity of root rot on citrus caused by 

STTC fungi. Competition experiments between coprogen B., dimerum 

acid and hydroxamate siderophores from other structural classes 

P. citrophthora and _P. parasitica, suggest a common transport system. Wheat seedlings were capable 

of utilizing iron from coprogen B and dimerum acid. The role of 

siderophores in survival and virulence of Ggt will be discussed. 

10 

CONTROL OF FUNGAL DISEASES OF WINTE OILSEED RAPE IN SWITZER- 

LAND. M. W. Andrea and W. E. Winter, Swiss Federal Research Highly virulent isolates of Fusarium selani from soybeans produce the Nectria ascoma 

Station for Agronomy, Reckenholzstr. 191, 80146 Zuerich, stage. T. S. Abney, T. L. Richards, A. J. Ivanovich, & D. H. Scott. USDA-ARS and 

Switzerland. Purdue University, W. Lafayette, IN 47907. 

1136 PHYTOPATHOLOGY 

14

Twenty Fusarium solani (Mart.) Sacc. isolates from soybeans with symptoms of mers' perceptions of pests and the actual pest distribution and 

sudden death syndrome (SDS) were compared in culture and evaluated for intensity. Farmers who applied pesticides did not have signivirulence 

on Spencer& Ripley soybeans. Initial colonies growing from soybean ficantly higher yield compared with farmers who did not (5.3 

root and crown tissues onto agar had macro- and micro-conidia typical of F. t/ha vs 4.5 t/ha). Pest intensity and diversity were low. The 3 

solani but did not consistently express a blue color. With subculture, the blue most prevalent insects were cutworm, leaffolder, and rice whorl 

pigmentation within fungal colonies was characteristic of isolates in a predomi- maggot; diseases were sheath blight, narrow brown leaf spot, and 

nately macroconidial (versus microconidial) 'form' or state of growth. All stem rot; and weeds were Echinochloa glabrescens, Monochoria 

isolates expressed a bluish color with subculturing. However, the nature and vaginalis, and Paspalum distichum. A first record of the nemaextent 

of the bluish colorvaried among isolates and with subculturing. Perithe- tode, Tylenchorynchus sp. was made. Rice tungro virus was deteccia 

and ascospores developed in subculture with two of the F. solani isolates. ted in 24.4% of fields using an ELISA test, even though symptoms 

Although, ascospores were not required for hypocotyl infection, virulence of were not observed. Farmers did not perceive any pest to be a 

the isolates that produced Nectria in PDA culture was higher than that of other major yield constraint but considered pesticide application a 

isolates. Typical SDS foliage symptoms developed 3 weeks after hypocotyl necessary production practice. Farmers generally fol lowed a 

inoculation with conidial (macro and micro) and mycelial inoculum only with calendar spraying program without considering pest populations. 

isolates that produced the ascoma stage in culture. Hypocotyl inoculations 

with ascospore inoculum also produced typical SDS foliage symptoms. 19 

15 EC PCID IYDI=A[loN DI!Sa _~aJIi MWO STRAINS TRANSR[T BY 

FULFILLING IPM STRATEGIES FOR CACAO BLACK POD CONTROL IN THE 

MMMLES SPP., VEU-C OF ASUR YEaZ ICN. I.-M. lee, R.E. 

Davis, T.-A. Chen, L.N. (hiykowski, J. Fletcher, and C. Hiruki. 

TROPICS. R. H. Fulton, American Cocoa Research Institute, Microbiol. and P1. Pathol. lab., Agricultural Research Service, 

7900 Westpark Drive, McLean, Virginia, 22102 USDA, Beltsville, MD 20705; Dept. of P1. Pathol., Cook College, 

ltgers University, t Brunawick, NJ 08903; AKjriculture Canada, 

Cacao, Theobroma cacao, is cultivated mainly on farms of 2 to 

6 acres where cash - flow is critical. In the Americas, 

Phytoohthora palmivora, is considered the major pod destroyer. 

Ontario, Canada; Dept. of P1. Pathol., Cklahlxxr State University, 

Stillwater, OK 74078; and Dept. of Plant Science, Univ. of Alberta, 

Alberta, Canada. 

For six decades a host of fungicides and varied spray 

schedules have failed to give consistent Black Pod control. 

Formerly in - depth etiological studies confirmed that the 

soil - tree root systems served as the massive inoculum 

reservoir. Today, 1PM cultural practices for lovering this 

inoculum source entails soil modifications via drainaoe and 

liming; while studies on pod surface characteristics, 

influence of canopy climate on infection has "reduced" the 

number of sprays. Trials employing surfactants and oil 

adiuvants oroved that growers could spray "less" copper 

fungicide with positive control results. 

Strains of rqrolaasmalike organisms (MLOs) transmitted by aster 

yellows (AY) insect vectcrs (Macrosteles spp.) were analyzed by 

nucleic acid hybridizations with biotin-labeled cloned DNA fragments 

from AY and tcmato big bud (BB) MIOs. All strains examuied were 

gaetically related at the level of ch ,rscme, but differential 

hybridizations distirnuished armon the strains. Distincticns did 

not omrpletely coincide with cnoqets of "eastern" and "western" AM. 

Sane of the data indicated that certain of the strains exmined may 

be more closely related genetically to BB MMI than to AY MLM. The 

results underscore ambiguities involved in classifying ML3s as 

strains of AY MID on the basis of biological properties. 

16 20 

LEAF REMOVAL FOR MANAGENENT OF DISEASES AND PESTS OF WINE LOCALIZATION OF SPIROPLASMA CITRI SURFACE PROTEINS USING 

GRAPES IN THE SAN JOAQUIN VALLEY. J. J. Stapleton#+, W. W. INDIRECT IMMUNOFLUORESCENCE. M. G. Morley and J. Fletcher. 

Barnett#+, K. M. Kelley+, G. M. Leavitt+, M. VK. Norton+, and 

P. S. Verdegaal+, Statewide IPM Project#, and Cooperative 

Department 

Siwater, of 

oK 

Stillwater, OK 

Plant 

7407,-9947i Pathology, 

74078-9947. 

Oklahoma State university 

Extension+, University of California, Modesto, CA 95355. The relative distribution of spiroplasma citri surface proteins 

Leaves surrounding clusters of several wine grape (Vitis 

Leaessurondngclutes f evra wie rae Viisdetermined 

vinifera) varieties vinfer) were removed reove vrieieswer after afer late ateblom bloom in i 6 Sandermnd anproteins 

Joaquin Valley vineyards in 1988. Comparisons of treated vs. 

control, bilateral cordon-trained vines showed that incidenceS. 

(p29, p58, p77, and p89) (Phytopathology 77:1726) was 

using antisera made against specific surface 

uig atsamde gist pcfc ra 

in indirect immuno-fluorescence. Glutaraldehyde-fixed 

p.ctei cl were incubat e nterum andthentee 

citri cells were incubated with antiserum and then treated 

and/or severity of Botrytis bunch rot, sour rot, or "total" rot 

at harvest was reduced (P=0.05 or P=0.01) after leaf removal in 

5 

canopy 

of the 

populations 

6 experimental vineyards. A trend toward reduced 

of leafhoppers and mites, and reduced 

cluster damage due to insect pests also was observed following 

leaf removal. The treatment did not significantly affect grape 

with fluorescein-conjugated anti-rabbit serum. Cells treatnd 

pro ateinsep p7repa 9 a showed fluecencewhcapeared 

proteins p29, p77, or p89 showed fluorescence which appeare 

evenly distributed along the spiroplasma cells; however, cells 

treated with preimmune or anti-p58 sera did not fluoresce. 

Fluorescence of entire spiroplasma cells with antisera to p 29 yield or quality parameters. 

, 

p77, and p89 indicated that these proteins are not clustered in 

one area of the S. citri membrane but are distributed over the 

18 

cell length. 

21 

PRODUCTION OF MONOSPECIFIC POLYCLONAL ANTIBODIES AGAINST TNE 

ASTER YELLOWS MYCOPLASMA-LIKE ORGANISMS (AYMLO) OF OKLAHOMA. 

0. Errampalli, J. Fletcher and J. L. Sherwood, Dept. of Plant 

Pathology, Oklahoma State Univ., Stillwater, OK 7407e-9947. 

Polyclonal antibodies prepared against partially purified AYMLO 

from infected periwinkle tested positive with AYMLO infected 

plants but also showed cross reactivity with healthy controls. 

AYMLO antibodies were further purified using electrophoretically 

separated and electroblotted 23K, 55K, and 70K 

AYMLO-specific proteins as specific immunoabsorbents. The 

nitrocellulose strips, each containing a specific AYMLO 

protein, were used repeatedly to trap the specific antibodies, 

which were then eluted from the strips by changing the pN. 

Such antibodies reacted positively on the blots of periwinkle 

infected with Oklahoma AYMLO, but not with healthy controls. 

The monospecific antibodies were used to screen plants infected 

with several isolates of presumed A'! from Oklahoma and other 

geographical areas. 

DEFINING THE SOCIO-BIOLOGICAL ENVIRONMENT FOR RICE PEST 22 

MANAGEMvEN~T USING SAMPLE-SURVEYS. P. 5. Tengj, F. A. Elazegui, 

B. M. Shepard, K. M. Moody, International Rice ResearchOFHMLD 

BIOCHEMICAL AND BIOPHYSICAL FACTORS AFFECTING FEEDING 

CACGUTA AVEOR FDIASS ASD 

Insitte MniaPhiipins.BY P.. ox93, 

XYLELLA FASTIDIOSA. P. C. Andersen, R. F. Mizell, 

An "Integrated Pest Survey" was initiated in the wet season of 

1987 to determine existing farmers 'pest control practices, far- 

B. V. Brodbeck and A. B.--Goulid. University of Florida, 

AREC-Monticello, Rt. 4 Box 65, Monticello, FL 52344. 

Vol. 79, No. 10, 1989 1137

Homalodisca coagulata is a polyphagous leafhopper Onion seedlings which were grown in a greenhouse were inocuvector 

of many diseases induced by Xylella fastidiosa, lated with VAM fungi by placing inoculum on the entire root 

although little is known concerning factors that system after the seedlings had grown for different periods of 

influence feeding. H. coagulata, confined to stems of time. Seedling age had a great effect on mycorrhizal colonizahost 

plant species produced copious quantities (i-2 tion. VAM colonization took place rapidly in 3-day-old onion 

ml/h) of dilute exudate (8-25mM) consisting mainly of and reached 51% after one week. Older plants were far 

inorganic 

less 

ions, characteristic of a xylem fluid- responsive. VAM colonization of onion and pepper reached a 

feeder. Total amino acids, organic acids and sugars maximum two weeks after inoculation and of cotton at 3 weeks 

were metabolized with > 99% efficiency; major organic after inoculation. In the field, comparisons of VAM colonizacompounds 

(glutamine, asparagine, malic acid and tion efficiency with respect to placement of the inoculum were 

succinic acid) with > 99.9% efficiency. Biochemical studied. Inoculum was placed 3 cm below seeds at planting and 

and biophysical plant factors, and leafhopper also 5 cm deep by 3 cm from one side of the root system 

responses 

and 5 

were manipulated by altering plant nutrient cm deep by 3 cm from both sides of the root system 2 weeks 

status and moisture stress. Feeding was not reduced at after planting. Maximum VAM colonization was achieved when 

a specific threshold xylem fluid tension. inoculum was applied 3 cm below seeds at planting. 

23 27 

THE PRODUCTION OF A POLYCLONAL ANTISERA TO THE BEET LEAFHOPPER EFFECTS OF OZONE EXPOSURE AND SOIL WATER DEFICIT ON GROWTH, 

TRANSMITTED VIRESCENCE AGENT. D. A. Golino*, B. C. Kirkpatrick, ECIXJMYCORRHIZAE AND NON-STRUCTURAL CARBOHYDRATES OF LOBLOLLY 

and G. A. Fisher. USDA-ARS*, Department of Plant Pathology, PINE SEEDLINGS. S. Meier and L. F. Grand, Department of Plant 

University of California, Davis, CA 95616. Pathology, North Carolina State University, Raleigh 27695. 

Antisera specific for the beet leafhopper transmitted virescence 

ntiseagsecific agent (BLTVA-MLO) fo webtlfoere were prepared byvinjectingrabbir- by transpited injecting rabbits 

with MLO-enriched extracts prepared from infected leafhoppers, 

Circulifer tenellus (Baker). When used in a F(ab)2/Protein A 

ELISA system thisantisera reacted positively with roots and 

leaves of symptomatic BLTVA-infected periwinkle (Catharanthus 

roseus), but not with periwinkles infected with Spiroplasma 

citri, X-disease or three strains of western aster yellows, 

ELISA also detected the BLTVA-MLO in infected radish (Raphinus 

sativus) and plantain (Plantago major). Western blot analysis 

of soluble proteins from BLTVA-MLO-infected periwinkle and C. 

tenellus revealed a single dominant antigen of approximately 

40 kd. 

Loblolly pine (Pinus taeda) seedlings from three full-sib 

d/week families 

for were 

6 or 

exposed 

12 wk). 

to 

Soil 

0, 50, 

water 

100, 

potential 

or 150 ppb 

was 

ozone 

maintained 

(5 h/d, 5 

near pot capacity (-0.03 MPa) or soil was allowed to dry to 

approximately -1.0 MPa and resaturated. Exposure to ozone and 

soil water deficit each resulted in less seedling volume growth 

and total dry weight. Exposure to increasing ozone 

concentrations resulted in a linear reduction in foliar starch 

but did not affect hexose or sucrose production. Soil water 

deficit lessened starch and sugar content in above- and below- 

ground plant parts. Soil water deficit did not affect numbers 

of ectomycorrhizal tips or percentages of roots that formed 

ectomycorrhizae. A linear dose-relationship between ozone and 

ectomycorrhizae was observed. The number of ectomycorrhizal 

24 

tips/cm long root and the percentage of feeder roots that formed 

ectomycorrhizae were lower as ozone concentration increased. 

PHYLOGENETIC RELATIONSHIPS OF THE WESTERN X-DISEASE MYCOPLASMA- 

LIKE ORGANISM (X-MLO) AS ESTABLISHED BY 16S rRNA SEQUENCE. 28 

B. C. Kirkpatrick and J. D. Fraser. Department of Plant 

Pathology, University of California, Davis, CA 95616. CHARACTERIZATION OF PROTEIN/ISOZYME PATTERN$ IN A 

TRIPARTITE SYMBIOSIS. J. S. Neck, J. B. Szerszen, and R. A. Taber, 

Two cloned DNA fragments, which together contain 97% of the X- Department of Plant Pathology and Microbiology, Agricultural 

MLO 16S ribosomal RNA gene (rRNA), were sequenced. One cloned Experiment Station, Texas A&M University, College Station 77843. 

fragment contains two putative promoters that are very similar 

to the 16S rRNA promoters of Bacillus subtilis, and 673 bp of Cosymbionts, Arachis hypogaea L. cv. Tamnut, Bradyrhizobium spp. (3 

the 5' end of the 16S rRNA. The second cloned fragment con- strain mixture), and Glomus etunicatum Becker and Gerd. were grown in 

tains 796 bp of the 3' end of the 16S rRNA gene. The G+C all combinations to assess potential plant (peanut) metabolic responses to 

content of the 16S rRNA gene is 45.5%, while the 5' flanking colonization by the rhizobial and vesicular arbuscular mycorrhizal 

sequence is 23%. These G+C values are among the lowest re- (fungal) components. Extractable host leaf/root tissue (50 day old plants) 

ported for prokaryotes. The X-MLO 16S rRNA gene is 78% homo- samples and pure culture rhizobial and fungal spore samples were 

logous to the 16S rRNA genes of B. subtilus and Mycoplasma electrophoretically (IEF-PAGE) separated and stained for buffer soluble 

capricolum, and 73% homologous to E. coli. These results general protein, glycoprotein, and isozyme patterns. Preliminary results 

suggest that the X-MLO is phylogenetically related to, but indicate some symbiotic combinations showed differential band patterns 

distinct from, other gram-positive prokaryotes. and/or band intensities. 

25 29 

CLONING AND SEQUENCE ANALYSIS OF THE 16S RIBOSOMAL RNA GENE OF PRODUCTION OF VA MYCORRHIZAL SPORES IN A SAND-VERMICULITE 

WESTERN ASTER YELLOWS MLO (AY-MLO). C. R. Kuske and B. C. MEDIUM. H. D. Liyanage and N. C. Schenck, Department of Plant 

Kirkpatrick, Department of Plant Pathology, University of Pathology, University of Florida, Gainesville, FL 32611. 

California, Davis, CA 95616. 

A 0.1 strength Long Ashton (nitrate type) nutrient solution of 

The 16S ribosomal RNA gene (16S rDNA) of the AY-MLO was identi- varying P levels (0.05 to 20 mg P/l) was evaluated in a sandfied 

by Southern blot analyses, using cloned fragments of the vermiculite (3:1, v/v) mixture as a substitute for a soil pot 

Western X-NLO 16S rDNA as heterologous probes. A 1.5 kbp Eco- culture medium. Two species of VA mycorrhizal fungi, Glomus 

RI/HindIII fragment, unique to AY-MLO DNA, hybridized with a etunicatum (isolate LETC 329) and G. mosseae (isolate LNSS 336) 

probe containing the 3' region of the X-MLO 16S rDNA. Two AY- were evaluated on alfalfa, bahiagrass and onion; in addition 

MLO-specific fragments (2.5 and 1.0 kbp) hybridized with a LETC 329 was evaluated on wheat. Colonized root lengths were 

probe containing the 5, end and upstream flanking sequences of maximum at 2 ag P/l for G. etunicatum and C. mosseae at 12 and 

the X-MLO 16S rDNA. The 2.5 kbp and 1.5 kbp fragments were 15 weeks, respectively. Increasing levels of P caused a more 

cloned in Ml3mp vectors and sequenced. The 2.5 kbp fragment rapid decline in root colonization by 0. etunicatum than by G. 

contains about 670 bp of the 5' region of the 16S rDNA and a- mosseae. At 0.5 mg P/l,_0. etunicatum--produced maximum spore 

bout 1.9 kbp of A:T rich DNA upstream. The 1.5 kbp fragment numbers per plant (39,500-43,400) on all hosts except wheat. 

contains 863 bp of the 3' region of the 16S rDNA, followed by a Glomus mosseae produced maximum spores on alfalfa and bahiagrass 

spacer region containing a tRNA gene and the 5' end of the 23S (990 and 4800) at 0.5 mg P/l and on onion (1381) at 10 mg P/l. 

rDNA. Sequence comparisons with the 16S rDNA of other pro- Thus, sand -vermiculite was an effective pot culture medium for 

karyotes and plant organelles indicate the AY-MLO is most close- these two species of VA mycorrhizal fungi. 

ly related to ~ci subtilis and Myc~opla.sma. ca~pric~oluma. 

30 

26 ACRONYMS FOR SPECIES OF VA MYCORRHIZAL FUNGI. Y. P~rez and 

N.C. Schenok, Plant Pathology Dept., Univ. of Florida, Gainesville, FL 32611. 

THE EFFECT OF ROOT AGE AND POSITION OF MYCORRHIZAL INOCULUM ON 

COLONIZATION OF COTTON, ONION AND PEPPER. U. Afek, E. A unique four-letter acronym for each species is proposed which can be 

Rinaldelli, d. A. Menge and E. L. V. Johnson. De-pt. of Plant used in lieu of epithets in computer databases, publication graphics, and 

Pathology, Univ. of California, Riverside, CA 92521. experimental notations. Other abbreviations occur in the literature, but the 

1138 PHYTOPATHOLOGY

means used to derive them were not described. Species can be assigned 

a unique acronym by following guidelines. The first letter of the acronym 

represents the genus and should not be one used previously to represent 

a genus, e.g. Acaulospora=A, Entrophospora=E, Gigaspora=G, Glomus=L, 

Sclerocystis=S, Scutellospora=C. The last three letters are from the species 

epithet. The second letter of the acronym is the initial letter of the species. 

The third and fourth letters are consonants, whenever possible, occurring in 

Specific proteins, obtained by PAGE (polyacrylamide gel 

electrophoresis) of mycelial extracts, were used to prepare a 

polyclonal antiserum against Verticillium dahliae. An antiserum 

obtained 28 days after first immunization reacted well with V. 

dahliae 

V.hniaerenceno;7V.esateritium; 

and to a lesser extent with 

Fusariumalxo-aorum 

V. albo-atrum but 

Rhizoctonia 

not wit 

solani and Colletotrichum sp. Double antibody sandwich enzymelainked 

immunosorbent assays (DAS-ELISA) were developed for 

the species name. The acronyms are written in upper case letters and 

without punctuation, e.g.ANCS=Acaulosporanicolsonii, ECLB=Entrophospora 

colombiana, GMRG=Gigaspora margarita, LDST=Glomus deserticola, SSNS 

= Sclerocystis sinuosa, CHTG=Scutellospora heterogama. The complete set 

of guidelines and acronyms for presently recognized species will be 

published elsewhere. 

detection and quantification of Verticillium spp. in potato 

tubers and plant tissue samples. The results of this study 

suggest that a relatively specific fungal antiserum can be 

produced using a soluble protein extract. The double antibody 

sandwich technique will be evaluated as a potential screening 

technique for identifying resistance to Verticillium in potato 

breeding lines. 

31 35 

DISCOVERY OF A SPORE OF SCUTELLOSPORA HETEROGAMA WITH TWO 

SUSPENSORS. 

Dept., University Rogerio 

of Florida, 

T. 

Gainesville, 

Almeida and 

FL 

N. 

32611. 

C. Schenck. Plant Pathology DEVELOPMENT 

PHYTOPHTH~ORA OF 

MEGASPERMA 

A DIRECT IMMUNOASSAY 

F.SP. GLYCINEA TO DETECT 

IN SOIL. .S.M. 

Miller, F. P. Petersen, S. A. Miller, J. H. Rittenburg, S.C. Wood and G. D. 

Nicolson and Gerdemann in 1968 (Mycologia 60:313-325) reported the 

occasional occurrence of spores of S. heterogama with two suspensors. 

Koske and Walker in 1985. (Mycologia 77:702-720) suggested that this 

observation could have resulted from a spore formed within a dead spore 

giving the appearance of a single spore with two suspensors. According to 

Koske (1984. Mycologia 76:853-862), spores forming within other spore 

"shells" is a common occurrence. A single spore of S. heterogama was 

observed in a soil sample from Alachua, Florida with a suspensor on 

opposite poles. The appearance of the spore was that of a zygospore of 

a typical zygomycetous fungus. The spore and suspensor contents 

appeared normal. No mycorrhizal pot culture resulted from this single spore, 

but pot cultures with S. heterogama spores from the same sample were 

established. The observation of a S. heterogama spore with two suspensors 

verifies the report of Nicolson and Gerdemann and indicates that not all 2- 

suspensor spores result from a spore-within-a-spore phenomenon. 

32 

Grothaus. Agri-Diagnostics Associates, 2611 Branch Pike, Cinnaminson, 

NJ 08077 

A simple, rapid technique has been developed to detect and quantitate 

Phytophthora megasperma f.sp. glycinea (Ping) in soybean soil. Ping oospore 

and mycelial components are extracted from the soil by a rapid water- flotation 

technique and fungal antigen is solubilized by grinding prior to analysis by 

enzyme-linked immunosorbant assay (ELISA). The ELISA incorporates 

monoclonal antibodies specific for Pmg soluble internal oospore antigen and 

soluble mycelial antigens. Relative levels of Pmg in soil samples are 

estimated by comparing the reactivity of the solubilized extracts to that of 

laboratory-prepared oospore standards. The presence of Ping in samples which 

gave positive results in the direct soil immunoassay has been confirmed using a 

bioassay method. This method directly measures Pmg antigen in the soil and 

does not require the germination of oospores for their detection. It has been 

possible to detect other soil-borne plant pathogens, including other 

Phytophthora spp.,Pythium spp. and Rhizctonia spp. by use of this technique. 

STIMULATION OF VESICULAR-ARBUSCULAR MYCORRHIZAL SPORE GERMINATION 

BY STREPTOMYCES ORIENTALIS. G. L. Tylka, R. S. Hussey, and R.W. 36 

Roncadori, University of Georgia, Department of Plant Pathology, DETECTION AND QUANTITATION OF PHYTOPHTHORA 

Athens, GA 30602 MEGASPERMA F. SP. GLYCINEA IN FIELD SOIL BY IMMUNOASSAY. 

Surface sterilized spores of Gigaspora margarita (G4G) and Glomus 

mosseae Surface (GNS) stwerilied were splted plated on 1.5%spoble 

1.5 % Noble m wrater water aga agar r and (GA)) (WA), oLankow, WA 

S.A. Miller, V. Korjagin, S.M. Miller, F.P.Petersen, M. Klopmeyer, R.K. 

Cinnaminson, and G.D. NJ 08077. Grothaus. Agri-Diagnostics Associates, 2611 Branch Pike, 

covering a layer of Streptomyces orientalis (SO) colonies 

suspended in WA (SO/WA), or SO/WA prepared with autoclaved SO A rapid, direct technique for detection and quantitation ofPhytophthora 

colonies (autoclaved SO/WA). GMG and GMS spores plated on SO/WA 

exhibited 10 to 70% greater germination than spores on autoclaved 

SO/WA or WA beginning 5 days after plating. GMG and GMS spore 

germination was also more frequent when spores were plated on 

SO/WA or WA in separate compartments of divided Petri plates 

versus germination of spores in plates containing WA alone. GMS 

megasperma f. sp. glycinea (Pmg) in soil was evaluated and compared with a 

baiting/immunoassay method (Phytopathology 78:1576). Samples were 

collected post-harvest from 198 fields, air-dried, pulverized, mixed thoroughly 

and subsampled for analysis. Both methods were effective in detecting and 

quantifying Pmg in the samples. Ping was detected in soybean fields and fields 

in which a rotational crop such as corn or wheat had grown immediately prior to 

germination was greater when plated on WA separated from SO/WA 

than when placed directly onto SO/WA. Thus, the stimulatory 

effect of SO on GMG and GMS appears to be volatile. Conversely, 

preliminary experiments indicate that Scutellospora heterogama 

spore germination is inhibited by SO. 

sampling. The baiting/immunoassay method was time-consuming (10days) 

due to the requirement for oospore germination. Direct tests of Pmg in soil 

were completed in less than one hour, and provided a quantitative measure of 

the total amount of Pmg antigen in the sample. Studies are currently underway 

to determine the relative risk of disease occurrence in fields with various levels 

of Pmg as determined by these methods. 

33 

USE OF ELISA KITS FOR DIAGNOSIS OF PHYTOPHTHORA 37 

CROWN AND ROOT ROT OF AZALEA. J. M. Mullen & A. K. DOUBLE-STRANDED RNA ASSOCIATED WITH MULTIFLORA ROSE EXPRESSING 

Hagan, Department of Plant Pathology, Auburn SYMPTOMS OF ROSE ROSETTE DISEASE. R. Di, A. H. Epstein, J.H. 

University, AL 36849-5409. Hill, Dept. of Plant Pathology, Iowa State University, Ames, IA 

During 1988 and 1989, azalea container plants with 

crown and root rot symptoms submitted to the Plant 

Diagnostic Lab at Auburn University were checked 

for Phytophthora spp. infection using apple 

baiting, isolations on a selective pimaricinvancomycin-Terraclor 

75WP medium (P 1 oVP), and 

serological assays with ELISA multi-well, dipstick, 

and rapid assay kits (Agri-Diagnostics 

Associates). Very good agreement was obtained with 

results of the apple baiting, culture, and ELISA 

multi-well kits. The dip-stick and rapid assays 

did not appear to be as sensitive as the multi-well 

50011-1020. 

Rose "rosette", a disease of unknown etiology which affects multiflora 

rose (Rosa multiflora, Thunb.) has been found in several 

areas of the United States. It is vectored by an eriophyid mite 

(Phylocoptes fructiphilus). Speculation is that the disease is 

caused by a virus; however, no virus-like particles have been 

observed in infected tissue. We have demonstrated the presence 

of double-stranded RNA (dsRNA) in extracts of diseased multiflora 

rose leaves using polyacrylamide gel electrophoresis. 

After digestion with DNase and RNase in high salt solution, 

three distinct dsRNAs were identified in diseased samples. Simi- 

lar dsRNAs were not detected in extracts from healthy control 

aind momuli-wl 

assy. idictedtha soltios th aple 

assaytr consistntly. deastectea 

aitrose tissue. Rosette disease can be lethal to rose and is being 

considered for development as a biological control for multiflora 

cinmn -,-,circl and "~rsiia rose, a serious pest on non-cultivated lands. 

34 38 

DEVELOPMENT OF 

Verticillium spp. 

ENZYME-LINKED 

IN POTATO PLANTS 

IMMUNOSoRBENT ASSAY FOR 

AND TUBERS. S. Sundaram and USE OF DNA PROBES TO DETERMINE PATHOGENICITY IN AGROBAcTERIUM 

E. E. Banttari, Department of Plant Pathology, 

Minnesota, St. Paul, MN 55108. 

University of ISOLATES 

Graves. 

FROM MUSCADINE. C-W. J. Sun, 0. 5. Luthe, and C. H. 

Dept. of Biochemistry and Molecular Biology, and Dept. 

Vol. 79, No. 10, 1989 1139

of Plant Pathology and Weed Science, Mississippi State Univ., 

Miss. State, MS 39762. 

42 

Cloned fragments of T-DNA from a wide host range Ti plasmid, 

pTi-A6, and from a limited host range plasmid, pTi-Ag63, have 

been used as probes in DNA hybridization experiments to determine 

the pathogenicity of isolates of Agrobacterium species from 

stems, roots and galls of muscadine. Several hybridization 

techniques including colony hybridization, slot blot, and Southern 

hybridization have been used. These techniques consistently 

identify known grape isolates, however, they are less sensitive 

when used to probe unknown samples from muscadine isolates. 

This suggests that the muscadine isolates may differ from Ag63 

in DNA sequence homology. Attempts are being made to use the 

polymerase chain reaction as a more sensitive detection technique. 

39 

EVALUATION OF SEED BIOPSY METHODS FOR NONDESTRUCTIVE SEED HEALTH 

TESTING. P. M. Higley, D. C. McGee, J. S. Burris, Seed Science 

Center and Dept. Plant Pathology, Iowa State Univ., Ames, IA 

A previous report described methoda for extraction and detection 

of pathogens from seeds for the purpose of developing nonde- 

structive seed health tests. Of particular value was the hiopsy 

method of coring tissue from dry seeds. The present work 

focuses on optimizing coring techniques to minimize damage to 

seed germination. Partial imbibition of corn, soybean, and 

Phaseolus seeds facilitated removal of cores, but the combined 

effect of imbibing and coring reduced germination compared to 

that of seeds that were imbibed only. Treatment of wounded 

tissue with a paraffin sealant was an ineffective means of 

correcting deleterious effects of coring on germination. 

However, reduction of the rate of dry-down by adjusting temperature 

and humidity improved germination in cored and imbibed 

soybean and Phaseolus seeds. Because corn germination was 

unaffected by coring treatments in this experiment, effects of 

COMPARISON OF THE ISOLATION OF XANTHOMONAS CAMPESTRIS PV. 

CAMPESTRIS FROM CRUCIFER SEEDS IN FOUR SEMISELECTIVE MEDIA. 

C. J. Chang', R. Donaldsoni, M. Crowley 

these corrective actions were not detected. 

2 , and D. 

Pinnow 

43 

2 . lDepartment of Plant Pathology, University of 

Georgia and 2 Seed Laboratory, Georgia Department of 

Agriculture, Griffin, GA 30223. 

INCIDENCE AND SURVIVAL OF SCLEROTINIA MINOR IN PEANUT 

D. M. Porter, R. A. Taber, and D. H. Smith. USDA, 

SEED. 

ARS, 

Four semiselective media, CS20ABN, NSCA, NSCAA, and F-S, were 

compared for their efficacy in isolating Xanthomonas 

campestris pv. campestris (Xcc) from crucifer seeds. Samples 

of 50,000 seeds per lot were washed for 2 hr at room temp in 

saline. The cheesecloth-filtered washings were centrifuged, 

and the resuspended pellets were diluted and pipetted onto the 

media. Saprophytic bacteria overgrew Xcc on NSCA and NSCAA 

but not on F-S or CS20ABN. Colony-forming units (CFUs) of Xcc 

ranged from 66-97 per plate at 10-1 or 37-259 at 10-2 

on CS20ABN. Moreover, 59-100% of recovered colonies on 

CS20ABN were Xcc, whereas only 4-29% were Xcc on F-S. Colony 

size of Xcc on CS20ABN was 2-4 times that on NSCAA or NSCA, 

but was 4-7 times that on F-S. 

Tidewater Agric. Expt. Sta., Suffolk, VA 23437; Dept. Plant 

Path. and Microbiology, Texas A&M Univ., College Sta., TX 

77843. 

The potential for seed transmission of Sclerotinia minor was 

assessed in peanut seed harvested from fields with Sclerotinia 

blight. The incidence of S. minor from 'VA B1B' peanut seed 

harvested and dried in Virginia according to recommended 

procedures was 4.2%. Seed with brown testa were colonized 

more frequently (5.7%) than seed with tan or normal colored 

testa (2.7%). The incidence of S. minor from seed with brown 

testa from five field sites was 3.6, 4.6, 5.0, 7.6, and 7.8%. 

The incidence from seed with tan testa from the same field 

sites was 1.2, 2.8, 2.8, 3.0, and 3.6%. The incidence of S 

minor from seed testa averaged 3.4% while the incidence from 

cotyledonary tissues averaged 0.1%. A recommended seed 

treatment (Botec 4 oz./cwt) reduced the incidence of S. 

to 0.1%. 

minor 

40 

MODIFIED TWEEN MEDIUM FOR DETECTING XANTHOMONAS CAMPESTRIS 

PV. VESICATORIA IN TOMATO SEEDS. H. MKlim and N.W. Schaad. 

Chonbuk Nat. Univ.,Chonju, Korea and Harris Moran Seed Co., San Juan 

Bautista, Ca. 95045. 

44 

MOISTURE VARIABILITY AMONG INDIVIDUAL SEEDS OF SOYBEAN FROM THE 

SAME POD BEFORE HARVESTING. F. A. Lazzari and R. A. Meronuck, 

Department of Plant Pathology, University of Minnesota, St. 

Paul, MN 55108. 

X2712 omsc strspv vesc orl (X v ) i s s e d b orn e in t omat o. 

Tween medium A has been described for Isolating Xv from fruit and 

leaves (McGuireR.G. etal. Pl. Dis. 70: 887-891). The authors describe 

the same medium plus boric acid and Increased cephalexin(Tween B) 

for assaying seeds. Dilution platings on test media with xv and 

tomato seed washings were done in comparison to KMB. Three of 8 

strains failed to grow on Tween B. All strains grew on Tween B only 

after reducing cephalexin, tobramycin and boric acid by 46,50 and 67 

%, respectively. 

incmaio 

The 

oKBrne 

recovery in cfu 

rm5-2 

of 8 strains 

ma 

on modified 

f7) euto 

Tween 

in seed flora on Tween B and modified Tween was similar (98 &97%). 

Moisture is important in storability of soybeans. Variability 

in moisture content (MC) among 

may affect 

single seeds 

susceptibility 

in stored 

to 

soybeans 

invasion by storage fungi. 

The moisture variability among single seeds of soybean from 

different positions inside the same pod was 

oven-dried 

determined 

single 

using 

The average MC of 

seeds 

individual 

at 103 

seeds 

C for 

from 72 

the 

hours 

respective 

in copper 

position 

cups. 

were: 13.20% (seed near pedicel), 12.60% (middle seed), and 

11.10% (farthest seed from pedicel). 

The fresh weight of single seeds was inversely 

to 

proportional 

seed), 

the MC, 

and 

i.e., 

0.1451 

0.1363 

g (farthest 

g (seed near 

seed 

pedicel), 

from pedicel). 

0.1430 

While 

g (middle 

moisture content was highest in seeds nearest the 

the 

pedicel, the 

fresh weight was lowest. 

We conclude that variation in MC exists in mature seeds 

inside the pod before harvest. 


45 

SUPPRESSION OF STORAGE FUNGI IN GRAIN WITH SOYBEAN OIL. 

0. C. McGee, A. lies and M. K. Misra, Seed Science Center, 

Iowa State University, Ames, Iowa. 

Effects of soybean oil, applied to grain at rates used to 

reduce dust in elevators, on storage fungi, were examined. 

Corn and soybeans at 17% and 15% moisture content, 

respectively, were treated with soybean oil at 200 ppm, alone 

or in combination with thiabendazole at 20 ppm, then stored 

on-farm in aerated metal bins, with a capacity of 250 kg. 

After 12 months, kernel infection by Penicillium and 

Asoergillus app was 83.0% and 63.7%, respectively, in 

untreated corn, compared to 60.0% and 46.2%, in soybean oiltreated 

corn, and 24.7% and 17.0%, in soybean oil + 

thiabendazole-treated corn. After 12 months, soybean seed 

infection by Penicillium and Aspergillus spp. was 45.7% and 

39.2% , respectively in untreated seeds, 17.7% and 8.2% in 

soybean oil-treated seeds, and 1.7% and 2.0%, in soybean oil + 

thiabendazole-treated seeds. The mechanism for reduction in 

storage fungal growth by soybean oil was not fungicidal.

46 

A COMPARISON OF THE EPIPRE AND KENTUCKY DECISION GUIDE 

PREDICTIVE SYSTEMS FOR SCHEDULING FUNGICIDE APPLICATIONS TO 

CULTIVAR RESISTANCE TO RICE KERNEL SMUT. Fleet N. Lee and G.S. 

Jones. University of Arkansas, P.O. Box 351, Stuttgart, AR 72160 

CONTROL LEAF DISEASES OF WHEAT IN THE NETHERLANDS AND IN 

KENTUCKY. R.E. Stuckey, J.C. Zadoks, D.E. Hershman, and W.P. Rice cultivars Mars, Lemont, Lebonnet and Bond appeared resis- 

Clinton. University of Kentucky, Lexington, KY 40546-0091 tant and cultivars Tebonnet, Newbonnet and Labelle were suscepand 

Agricultural University, Wageningen, The Netherlands. tible to kernel smut (Tilletia barclayana (Bref.) Sacc.& 

in a 3 year survey. Rough rice samples of the cultivars 

Syd.) 

aver- 

Predictive decision guides for scheduling fungicide applica- aged 4, 5, 6, 7, 20, 58, and 77 smutted kernels per kg, respection 

to control leaf diseases of wheat were developed in tively. Selected cultivars were inoculated with spore suspen- 

Kentucky (KDG) and in The Netherlands (EPIPRE) in response to sions .either by injection into the flag leaf whorl at threegrower 

interest in intensive wheat management. In The inch panicle development (stage 1), early swollen boot (stage 

Netherlands, treatment yields in replicated plots were 2), 1-2 days prior to panicle exsertion (stage 3); or by misthighest 

in the traditional protective (PROT) fungicide appli- ing the exposed panicle prior to anthesis (stage 4) or during 

cations, followed by KDG, EPIPRE, and non-sprayed control, anthesis (stage 5). Inoculated panicles had 12, 27, 28, 0.6, 

respectively. Dollar return per hi above control plots were and 0.2 % smutted kernels for growth stages I through 5, 

151, 126, and 79 for KDG, EPIPRE, and PROT, respectively. At respectively. Smut incidence in the inoculated tests did not 

Lexington, Kentucky, increased yields and economic returns 

for all fungicide treatments occurred such that: KDG and 

correspond with cultivar resistance observed in the survey. 

EPIPRE > PROT > non-sprayed control. The effectiveness and 

limitations of deploying computerized and in-field predictive 

systems in foreign countries are discussed. 

51 

PRIMARY INOCULUM SOURCES OF RICE BLAST (PYRICULARIA ORYZAE, 

47 

CAV.) IN ARKANSAS. Fleet N. Lee and G.S. Jones, University of 

Arkansas, P.O. Box 351, Stuttgart, AR 72160 

EFFECT OF TILLAGE AND ROTATION ON YIELD AND STALK ROT OF CORN International races IB-49 and IC-17 of Pyricularia oryzae Cay. 

GROWN IN POORLY DRAINED SOIL IN NORTHWEST OHIO. P. E. Lipps became widespread during 1986 and 1987. Seedling blast occurand 

I. W. Deep, Dept. of Plant Pathology, Ohio State Univ., rence in 1987 suggested that primary inoculum originated within 

Wooster, OH 44691. 

field plot was established in 1985 on Hoytville silty clay 

Arkansas. Random samples of crop stubble from fields, research 

plots and nearby native grasses were collected during the win- 

A fil pl othwest, to in on the silty ilay 

loam soil in Northwest, OH, to determine the effect of tillage 

(fall plow vs. no tillage) and crop rotation (continuous corn 

v.crin-soybeand rotation onaysield ofvand stalkirote of cgrass t 

during 1987 and 1988. Analysis of variance indicated that 

rotation, but not tillage, was significant (£= 0.05) for yield 

and tillage, but not rotation, was significant for incidence of 

stalk rot. Stalk rot incidence was positively (r=0.73, y 

54 58 

YIELD CONSTRAINTS IN WHEAT DUE TO SOILBORNE PATHOGENS. PERSPECTIVES OF DISEASE THREAT IN LARGE-SCALE PINUS RADIATA 

E.A. Milus and C.S. Rothrock, Dept. of Plant Pathology, MONOCULTURE -THE NEW ZEALAND EXPERIENCE. C.K.S.Chou, Forest 

University of Arkansas, Fayetteville, AR 72701. Research Institute, Rotorua, New Zealand. 

About 95% of New Zealand wood production today comes from exotic 

The importance of soilborne pathogens as constraints to plantations (mainly Pinus radiata) .For over 6 decades this forestry 

wheat yield and quality in Arkansas was determined in a monoculture,now 1.1 million hahas withstood the threat of dissplit, 

strip plot experiment. Main plots were raised or ease. In the late 1940's 25-35% of 20-25-year-old trees (unthinned 

conventional seed beds. Subplots were fumigated (Bromogas stands) were killed in a Sirex-Amylostereum epidemic.However the 

67), metalaxyl-treated seed, and a nontreated check. Sub- economic loss was insignificant.Aerial spraying to control 

subplots were planted with wheat (cv. Keiser, Florida 302, Dothistroma needle blight since 1966 has cost around $14.5 

or Caldwell) or oats (cv. Bob). In January plants were dug million. Losses caused by other diseases, i.e.,Sphaeropsis sapinea 

from the ends of each plot, growth parameters were measured, Armillaria root-rot,and Cyclaneusma needle-cast, have been 

and the entire crown and root system plated onto water agar. locally severe but tolerable overall. The current practice of waste 

Fungal colonies were transferred to PDA, identified to genus, thinning 75- 8 5% of initial stockings before age 10-12 allows a high 

and representative isolates were tested for pathogenicity. level of acceptable loss for diseases which mainly attack young 

Bedded plots had twice the number of plants infected with stands, while clear-felling at age 25-30 obviates diseases that 

Helminthosporium sativum. Plants from fumigated plots had affect older stands.However, New Zealand still has only a few pine 

more tillers/plant, greater top weight, and fewer plants pathogens and with the trend towards clonal forestry, genetic uniinfected 

with Pythium, Helminthosporium sativum, and formity, and reduction of initial/final crop stocking ratios,the 

Rhizoctonia. Pythium was the most commo .genus isolated. history of disease in this country is no way near its end. 

55 

HYPOVIRULENT STRAINS OF CRYPHONECTRIA PARASITICA IN 

RECOVERY 

NEW JERSEY. 

OF WHITE AND GREEN ISOLATES OF ASPERGILLUS FLAVUS P.J. Bedker. Department of Horticulture 

FROM 

and 

INDIVIDUAL 

Forestry, 

KERNELS 

Rutgers 

FROM DOUBLE-INOCULATED MAIZE EARS. University, New Brunswick, NJ 08903. 

Natale Zummo, USDA-ARS, Dept. of Plant Pathology and Weed 

Science, Mississippi State, MS 39762. Approximately 30 isolates of C. parasitica were collected from 

cankers 

Maize on ears were 

American chestnut inoculated at 4 locations. in the field The with virulence a white of 

each was determined by inoculating 

isolate of 

"Granny 

Aspergillus 

Smith" 

flavus 

apple 

at 

fruits. 

2 days after midsilk in Mean radial growth (MRG) was determined 

the shank or 

by 

through 

measuring 

the 

the 

husk 

length 

in the base of the cob and and width of lesions at 17 days after inoculation for 8 

re-inoculated 4 days later with a green isolate of the replications / isolate. Each apple was also inoculated with 

fungus through the husk at the top 

silk 

of the 

channel. 

ear or in the 

Reciprocal 

strain 

inoculations 

EP155 from Connecticut. 

were made. 

The MRG 

Ears 

for isolates collected 

from sites in Howell and Millstone Twps. 

were 

(Monmouth 

harvested at 

Co.), 

60 days after midsilk, dried, and Plumsted Twp. (Ocean Co.), and Sandyston Twp. (Sussex 

shelled. When 

Co.) 

kernels 

were 

from these double-inoculated ears 18.2, 19.1, 17.3, and 18.4 mm, respectively. The 

were 

MRG for 

assayed, 

37.5 

the 

% 

white isolate was recovered singly from (45/120) of the isolates was significantly 

8% of kernels, 

(P

62 

THE INCIDENCE AND IMPACT OF SPHAEROPSIS SAPINEA ON PINES IN 

SOUTH AFRICA. W.J. Swart andf N.J. WingfieldD5epartments of 

Plant Pathology and Microbiology, University of the Orange 

Free State, Bloemfontein 9300, South Africa. 

Surveys were conducted over a period of four years to determine 

the occurrence of Sphaeropsis sapinea diseases in plantations 

of exotic Pinus spp. in South ATrica. The pathogen was associated 

with siFoo-tblight, canker, root disease and blue stain. Dieback, 

resulting from shoot blight was the most common cause of 

tree death, with more than 70% of cases associated with hail 

damage and moisture stress. Infection associated with moisture 

stress was more common than with hail damage, but the latter 

had a far greater impact. The most extensive outbreak of dieback 

occurred after hail damage in a severely drought stressed 

plantation of P. radiata. Timber losses due to premature clearfelling 

in this instance were 28% of the volume and 54.5% of 

the value of potential production. Inoculation trials with S. 

sapinea indicated that P. radiata was the most susceptible -specdes, 

followed by P. piilaster and P. patula. The most resistant 

species was P. tae-?a followed by P-- elliottii. Our observations 

indicate that S. sapinea is the most important pathogen of 

Pinus spp. in Touth Africa. 

OAK DECLINE: GROWTH RING INDICATORS FROM A SOUTHERN 

BOTTOMLAND HARDWOOD SITE. F. I. McCracken and Ray Wolf*, 

USDA/Forest Service, Stoneville, MS 38776; *NOAA/National 

Weather Service, Stoneville, MS 38776 

Radial growth of Quercus phellos L. and Q. nuttallii Palm. in 

decline and control plots near Stoneville, MS were evaluated 

and related to weather data. Trees were 54 to 89 years old in 

a mixed hardwood stand on a Sharkey clay site. Between 1915 

and 1986, 18 years showed decreased radial growth and 16 of 

these years had below average annual rainfall. Radial growth 

was low in 1973-74 following extensive top breakage by ice, 

although rainfall was above average in both years. Radial 

growth was average or above in 5 years that had below average 

rainfall; however, precipitation was near normal during April- 

September. The mean basal area increment in decline plots was 

lower than controls from 1930 to 1986. Data shows short term 

63 

weather effects on radial growth and effects of unidentified 

long term factor(s). 

PREPARATION OF CELL-WALL-FREE PROTOPLASTS FROM THE CHESTNUT 

BLIGHT FUNGUS. F. H. Tainter, J. C. Jang, Clemson University, 67 

Clemson, SC 29634-1003, and W. L. MacDonald, West Virginia FUNGI ISOLATED FROM NEEDLES OF BLIGHTED EASTERN WHITE PINE IN 

University, Morgantown, WV 26506-6057. MAINE. T. Dreisbach, W. Merrill, Dept. of Plant Pathology, Penn State 

Certain isolates of Cryphonectria parasitica (Murr.) Barr con- Univ. University Park, PA 16802. 

tain cytoplasmically transmitted dsRNA which is believed to 

render mycelium hypovirulent. Practical utilization of hypovirulence 

in fungi is limited because of the general presence A blight of current-year needles on Pius strobus L. was documented in 

of mating incompatibility factors. A method of forcing vegeta- Acadia National Park, Maine, in 1983. From June 4 to August 4, 1988 

tive fusion, and hence possibly transfection of the dsRNA, in- three predominant symptom types were noted: chlorotic spots, 

volves use of electromanipulation. This research reports the necrotic tips, and chlorotic spots with necrotic centers. From June 13 

protocol necessary to produce cell-wall-free protoplasts. Two to August 4, fungi were isolated from all symptom types as well as 

strains were used. E7 is white in culture and is a hypovirulent from green, asymptomatic needles. A black yeast was recovered 

strain which produces large titers of dsRNA. Strain 591 is throughout the period. A previously undescribed hysteriaceous fungus 

virulent, is dsRNA free, and produces a yellowish-brown culture. and a LeQtostroma sp. were recovered from July 8-July 25 and July 8- 

The cell walls of the E7 isolate were easy to remove enzymati- July 29, respectively. A white, nonsporulating hyphomycete was 

cally. E7 yielded approximately 10 times as many protoplasts recovered beginning July 1, Hendersonia pinicola beginning July 25, 

as did 591 under identical growth conditions. Protoplasts from Truncatella truncata and a Seatoria sp. beginning July 29, all in 

591, however, were larger in overall size and had larger vacu- increasing amounts up to August 4. Thus, a succession of fungal species 

oles than did E7. The cytoplasm of E7 appeared much denser. appeared to colonize needles during the isolation period. 

64 

EFFECT OF STAND CONDITIONS ON ADVANCE OF PHELLINUS WEIRII IN 68 

DOUGLAS-FIR PLANTATIONS. W.J. Bloomberg, Pacific Forestry THE RELATIONSHIP OF DOUBLE-STRANDED RNA TO VIRULENCE AND 

Centre, Forestry Canada, 506 W. Burnside Rd., Victoria, B.C. MORPHOLOGY IN TYPE A AND TYPE B SPHAEROPSIS SAPINEA. Nyan-Tsz 

Canada, V8Z 1M5. Wu], M. Palmer 2 , and G. Adams 1 . IMichigan State University, 

Department of Botany and Plant Pathology, E. Lansing, MI 

Advance of Phellinus weirii in 62 infection centers during five 48824 and 2 pacific NW Research Station, Corvallis, OR 97331. 

consecutive 5-6 yr periods was studied in relation to stand 

conditions in three Douglas-fir plantations. Stocking level Sphaeropsis_ sapinea causes Diplodia shoot blight and canker 

(number of trees/ha) and average tree diameter were higher in of many conifers. Two populations, type A and type B have 

areas in which the fungus advanced than in those in which it did been reported that differ in morphology and virulence. Our 

not. Advances were more strongly related to tree diameter, objective was to determine whether the slower growth rate, 

whereas failures to advance were more strongly related to appressed colony morphology and low virulence of type B was 

stocking level. Presence of other tree species or trees dead correlated to the presence of dsRNA. We examined 40 isolates 

from other causes was also associated with failure to advance, from pines for differences in morphology and virulence on 

The fungus advanced unevenly among 450 sectors of centers. Only four species of pine. Of these, ten of 20 isolates of type 

1-3% of advances in a sector followed an advance during the A and six of 20 isolates of type B contained dsRNA. In both 

previous period, and 76-96% of non-advances followed groups, a portion of dsRNA-containing isolates were low in 

non-advances in the previous period. There were signif~icant virulence whereas others were not. Single-spore cultures 

differences in advances among plantations, centers and stand yielded a small portion of dsRNA-free conidia. The cured 

ages. Incidence of P. weirii in plantations was related to both subcultures of dsRNA-containing isolates maintained groupstocking 

level and degree of tree aggregation. 

specific colony morphology. 

66 

69 

Relationship of seedling height and dolcinitic lime application 

to black cherry leaf spot severity in northern Pennsylvania. 

G. Stanosz, PA Bur. For., 34 Ai~rport Dr., Middietw, PA 17057, 

and L. Aucr~d, USDA For. Serv., Box 928, Warren, PA 16365. 

I~afspot, caused by Blu~meriella iaapii (Rehm.) v. Arx, causes 

defoliation and death of black cherry (Prunus serotin Ehrh. ) 

seedl~ings. Temia (

deposition or unfavorable conditions for spore germination and photosynthetic rates and lower stomatal conductances at ambient 

infection. Low severity in limed plots may result fran reduc- C0 2 (340 ppm), and significantly reduced levels of active 

tion of primary inoculumn from leaf litter or nutrient-related RUBISCO. Protein extrayion and subsequent total activation of 

differences in susceptibility. Results suggest little disease extracted RUBISCO with `C indicated a significant reduction 

impact on regeneration after seedlings achieve heights >30 cm. in the total amount of RUBISCO in infected leaves. 

70 74 

ALLOZYME DIFFERENTIATION AMONG INTERSTERILITY GROUPS OF 

HETEROBASIDION ANNOSUM ISOLATES FROM EUROPE. W.J. Otrosina, 

T.E. Chase, F.W. Cobb, Jr. and K. Korhonen. USDA Forest 

Service, Pacific Southwest Forest and Range Experiment Station, 

P.O. Box 245, Berkeley, CA 94701; Department of Plant 

Pathology, University of California, 

Institute, 

Berkeley; Finnish 

Helsinki, 

Forestry 

Finland. 

EFFECTS OF PLANT GROWTH REGULATORS ON ACREMONIUM COENOPHIAWUM 

GROWTH IN CULTURE. G. E. Huff, K. D. Gwinn, and C. E. Sams. 

Departments of Entomology and Plant Pathology and Plant and Soil 

Science, University of Tennessee, P.O. Box 1071, Knoxville TN 

37901. 

Growth of the fungal endophyte (Acremonium coenophialum Morgan- 

Starch gel electrophoresis was conducted for isolates from 

Finland, Germany, and Italy representing "P', "S", and "F" 

intersterility groups. No clear differences in mobilities were 

found between the "S" and "F" groups in any of the 12 loci 

analyzed. In general, mobilities for the MDH-2 locus for all 

European isolates were indentical to the North American "S" 

group. The ADH, IDH, and GDH locus showed consistent 

differences between the "P" and "S" or "F" groups. Other loci 

exhibit shared alleles among the intersterility groups, which 

is in contrast to the nearly total fixation present in most 

loci betweeen the "S" and "P" groups in western North America. 

Within the "S" and "P" groups, allele frequency differences are 

apparent between isolates from both continents. 

Jones and Gams) is stimulated 

fescue 

during 

(Festuca 

the transition 

arundinacea 

of 

Schreb.) 

tall 

from the vegetative to 

reproductive state. This study was initiated to determine if 

plant growth regulators elicit a growth response by the 

endophyte. Plant growth regulators, dicamba and gibberellic 

acid, had no significant effect on the growth rate of the 

endophyte in culture. Kinetin completely inhibited growth of 

the endophyte at a concentration of 100 4M, and reduced 

endophyte growth by 75-80% at 80 pTM. Other plant growth 

regulators are currently being examined to determine in vitro 

effects on endophyte growth. 

75 

A cultivar-specific elicitor of the hypersensitive response in soybean has been identified 

which is associated with expression of the avirulence gene, avrD. Mark 71 M. Stayton*, 

Stanley J. Tamaki**, and Noel Keen§ *Department of Molecular Biology, University 

SEEDLING Wyoming, 

of 

RESPONSE OF TWO FOREST TREE SPECIES TO Field 

Laramie, 

Station, 

WY 

Richmond, 

82071; "*Cleargene, 

CA 94804-4698; 

Inc., 

§Department 

University of 

of 

California-Richmond 

VARYING DOSES OF OZONE 

Plant 

USING 

Pathology, 

OPEN-TOP 

University of 

CHAMBERS IN California at Riverside, Riverside, CA 

NORTHCENTRAL 

92521-0122. 

Davis, Dept. of Plant PENNSYLVANIA. Pathology, The M. Pennsylvania Simini, J.M. State Skelly, University, and D. D. A bacterially produced, low molecular weight compoand, has been idenitified which is a 

University Park, PA 16802 

cultivar SE compound specific 

is elicitor 

associated (SE) of 

with 

the hypersensitive 

the 

response 

expression 

in soybean. The 

of 

presence 

the 

of the 

avirulence 

Open-top chambers were 

gene, 

established 

avrD, 

at three sites in northern 

originally 

PA. Four 2- cloned 

year-old 

from Pseudomoassyringe 

seedlings each of Prunus 

pv. tomato 

serotina 

(Kobayashi 

Ehrh. and 

et. 

Liriodendron 

al. PNAS 86:157, 

tulipifera 

1989). 

L. were 

Expression 

planted 

of the 

in 1987 

avD 

in 

gene 

each 

in Ps. 

of 16 

pv. glycinea 

plots in a 

R4 

randomized 

alters the interaction 

complete 

of 

block 

the bacteria 

with 

design 

the cultivars 

at each 

IHarosoy' 

site. In 

and Norchief 

1988, the 

from 

seedlings 

compatible 

were 

to 

exposed 

incompatible. 

to ambient 

The presence 

air (no 

of 

chambers) 

the avirulence 

or to air 

gene, 

in filtered 

however, 

chambers 

does not 

which 

alter the 

contained 

compatible 

94%, 

interaction 

59% or 42% 

with the cultivar 

'Acme'. Furthermore, 

of 

E. 

the 

coli 

ozone 

cells overexpressing 

(03) in ambient 

the 

air. 

ayrD 

The 

gene 

number 

elicit the 

of hours 

hypersensitive 

which 03 exceeded 60 response 

ppb at the 

on exactly 

three 

the 

sites 

same 

was 

set of cultivars 

600, 646, 

as does 

and 

Ps. 

451 

pv. glycinea 

hrs, respectively. 

R4-avrD. The SE 

Ozone- compound 

induced 

can be 

stippling 

isolated from 

occurred 

cell-free 

on 

culture 

the adaxial 

supernatants 

leaf surface 

of E. coli, 

of 

P.s. 

both 

pv. tomaa 

species 

and 

at all !!.s. pv. glycinea 

sites. Differential 

R4 which express 

sensitivity 

the avrD 

was 

gene. 

found 

In 

among 

partially 

treatments 

purified extracts, 

and sites. 

the 

Injury 

SE 

was correlated compound causes a systemic 

growth 

positively necrosis 

were 

with only 

correlated 

ozone dose. in cultivars 

negatively 

Total height and basal diameter strains expressing the avrD gene. We have 

which 

purified, 

are 

by 

incompatible 

with 03 dose. reverse-phase 

with 

HPLC, 

bacterial 

active SE and have initiated an analysis of its chemical 

a biologically 

structure. 

72 

DISTRIBUTION AND INSECTICIDAL ACTIVITY OF ALKALOIDS IN FUNGAL 

ENDOPHYTE-INFECTED GRASSES. , L.P Bush, N. Fannin, G.C.M. Latch, 

D.D. Rowan, and B.A. Tapper. Plant Pathology, University of Kentucky, Lexington, Ky. 

40546; DSIR Palmerston North, New Zealand. 

A model for the gene-for-gene interaction between P.syringae and soybean. 

Tamaki*, 

Staley.,L 

Mark M. Stayton** 

California-Richmond 

and Donald Kobayashi§ 

Field 

*Cleargene, 

Station, 

Inc., 

Richmond, 

University 

CA 

of 

94804-4698; "*Department of 

Molecular Biology, University of Wyoming, Laramie, WY 82071; §Department of Plant 

Pathology, University of California at Riverside, Riverside, CA 92521-0122. 

The distribution of N-formyl and N-acetyl lolines, peramine, lolitrem and ergovaline was 

determined in Acremonium spp.-and Epichloe typ/i'na-infected cultivars and species 

Lolium 

of 

and 

Festuca, 

other grass genera. thirty of 34 host-fungus combinations produced alkaloids, 

peramine and ergovaline were the most common found alkaloids, while lolines and lolitrem 

were the least common. Three alkaloids (lolines, peramine, and ergovaline) 

A. coenophialum-infected 

were recovered from 

tall fescue and perennial ryegrass, while peramine, lolitrem, and 

ergovaline infected F. were longifolia. present 

Other 

in A. 

host-fungus 

lolii infected perennial 

combinations 

ryegrass, tall fescue, and in 

produces 

E. typhina 

only one or two of these 

alkaloid types. Aphid bioassays, using infected grsss stems, indicated that loline alkaloids were 

toxic to Rhiopalosiphusm padi (Rp) and Schu'zaphiis graminum (Sg); peramine was toxic only to 

Sg and ergovaline was not toxic to either aphid. Lolitrem was not toxic to Rp, but it could not 

be determined if the alkaloid was toxic to Sg as peraimine was always present in the same 

infected grasses. The relationship of alkaloid synthesis in infected grasses and resistance to 

In gene-for-gene systems the interaction between a dominant 

corresponding 

resistance 

avirulence 

gene in 

gene 

the host 

in the 

and 

pathogen results in an incompatible interaction 

characterized by the hypersensitive response (HR). We have isolated a low molecular 

cultivar-specific 

weight 

elici'or (SE) from a phytopathogenic bacterium; suggesting that the 

primary gene product of the avirulence gene is not directly involved in the recognitional 

event. Rather, the avirulence protein is predicted to possess a catalytic 

the synthesis 

activity involved 

of the 

in 

specific elicitor. The 

receptor, 

current 

the resistance 

models 

gene product, 

hypothesize 

which interacts directly with 

the 

the 

presence 

specific elicitor 

of a host 

triggers the 

and 

cascade of events manifested as an HR. Alternatively, the target within the plant 

for the specific elicitor may not be a dedicated receptor but may play other roles in plant 

metabolism. The biological effect of the SE shows parallels those of host-specific toxins 

produced by various IIelm~ialisnthsoriuim spp. and Alsnr spp. We will discuss the 

similarities in biological activity between the specific elicitor and various host-specific 

toxins and propose a working hypothesis which may account for both types of specificity. 

herbivory will be diacussed. 

73 OXALIC ACID, K 2 HP0 4 AND K 3 PO 4 AS INDUCERS OF SYSTEMIC 

RESISTANCE AGAINST DISEASES CAUSED BY FUNGI, BACTERIA, AND 

F-OIDSYNTmESIS AND STOM4ATAL RESPONSE IN A SUSCEPTIBLE ALFALFA VIRUSES IN CUCUMBER. Mucharromah and J. Kuc, Dept. of Plant 

CLONE INFETE WITH VERFTICILI.JtI4 ALBO-ATRUM. B.W. Pennvpacker, Pathology, University of Kentucky, Lexington, Kentucky 40546 

D.P. Knievel, K.T. Leath and E.J. Pell, Penn State Univ. and 

USDA~-ARS, U.S. Regional Pasture Jab, Univ. Park, Pa. 16B02. Oxalic acid, K 2 HPO 4 and K 3 PO4, induce systemic resistance to 

Phiotosynthesis was measured on a susceptible cucumber 

alfalfa 

anthracnose 

clone caused by Colletotrichum 

effectively as 

lagenarium 

induction with 

as 

infected with V_. albo-atrums 

_C. 

and 

lagen_, 

compared 

arium. 

to 

Spraying 

that of control 

with 

either oxalic acid, K2 HPO4 or 

plants. 

K3 PO4 on 

Infected 

the abaxial 

plants surface 

had symptoms of Verticillium wilt, but of leafI also induced 

photosynthesis 

systemic resistance 

was determined 

against 

only on asymptcomatic leaves. The Cladosporium cucumerinum, 

leaf being measured 

tobacco necrosis 

was placed 

virus, 

in a sampling ch1amber and Sphaerotheca fuliginea, 

exposed Pseudomonas to ca. 750 lachrvmans, 

ppm CO2. Sequential measurements were Mycosphaerella 

taken 

melonis, 

at 15 sec. 

cucumber 

intervals mosaic 

for 

virus 

20 min. and Erwinia 

as 032 was depleted by tracheiphila. 

photosynthesis. This evidence 

A/Ci supports 

response the 

curve hypothesis 

analysis that 

was used, which plants have the potential 

allowed for 

in vivo 

resistance 

assessment to many 

of the 

pathogens, 

amount of active RUBISCO in and that defense mechanisms can be induced by stress as well 

the leaf. Leaves on infected plants had significantly lower as by infection. 


76

79 83 

82 

SELECTION AND USE OF OXALATE MINUS MUTANTS TO STUDY 

PATHOGENICITY OF SCLEROTINIA SCLEROTIORUM, CAUSE OF BEAN WHITE 

MOLD DISEASE. G. Godoy, J.R. Steadman, R. Dam, and M. Dickman, 

Univ. of Nebraska, Dept. of Plant Pathology, Lincoln, NE 

68583-0722. 

Mutants of a Nebraska isolate of S. sclerotiorum were derived 

from UV irradiation of ascospores. Deficiency in oxalic acid 

production (OA-) was screeened by color change on potato 

dextrose (PD) agar containing 50 mg/l bromophenol blue. 

Determinations by enzymatic method, GC and HPLC indicate that 

the five selected mutants do not produce oxalic acid when grown 

on PD broth, nutrient agar, bean agar, or bean blossoms. In 

growth chamber experiments using leaves and stems on Phaseolus 

vulgaris plants, and in tests using detached leaves and pods, 

these OA- mutants were non-pathogenic. Lack of pathogenicity 

remained after over 30 generations of laboratory subculturing 

while the wild type remained pathogenic. Although the mutants 

and wild type were phenotypically alike, none of the mutants 

produced sclerotia. The role of oxalic acid in white mold 

disease will be discussed. 

DYNAMICS OF THE INTERACTION OF THE TERPENOID PHYTOALEXIN STUDIES ON HOST-PARASITE RELATIONS IN OPHIODOTHELLA VACCINII. 

DESOXYHEMIGOSSYPOL AND VERTICILLIUM DAHLIAE. M. E. Mace, R.T. Hanlin and C.W. Mims, Department of Plant Pathology, 

R. D. Stipanovic, M. H. Elissalde, and A. A. Bell, USDA, ARS, University of Georgia, Athens, GA 30602. 

Southern Crops, and Veterinary Toxicology and Entomology 

Research Labs, Rt. 5, Box 805, College Station, TX 77840. Ophiodothella vaccinii causes a leafspot disease of Vaccinium 

arboreum, an understory shrub in the southeastern United 

Desoxyhemigossypol (dHG) is the principal phytoalexin in States. Infection occurs in early summer, and although not 

Verticillium dahliae-infected Gossypium barbadense cotton, observed, penetration appears to occur directly through 

About 75% of dHG is absorbed from nutrient solution by V. epidermal cells. Thin-walled fungal hyphae spread throughout 

dabliae con~dia in 1 min. Metallic trace elements such as the leaf, forming a loose network of inter- and intra-cellular 

Fej+ and Mn + increase the rate of oxidation of mycelium. When a hypha reaches a cell wall, it passes through 

dHG to its aldehyde product hemigossypol. The toxicity of by means of a slender penetration peg, which expands to 

dHG, assayed by a tetrazolium technique, to a defoliating normal size on the other side of the wall. Invaded epidermal 

isolate of V. dahliae was not significantly changed by the cells overlying conidiomata become somewhat flattened and 

addition of trace elements. Evidence for the presence of the hyphae in them secrete a dark pigment, forming a shiny 

the toxic hydroxyl free radical, a putative decomposition black clypeus. As the season progresses, hyphae proliferatg 

product of dHG, is equivocal, to completely fill the leaf tissues. These hyphae develop 

thick walls and many lipid globules, and serve to carry 

the fungus through overwintering and development of ascomata 

the following spring. 

80 

EFFECTS OF VOLATILE LEAF SURFACE COMPOUNDS ON GERMINATION OF 84 

PERCNOSPORA TABACINA SPORANGIA. Mary L. Menetrez, Dept. of 

Plant Pathology, David A. Danehower, Dept. of Crop Science, INDENTIFICATION OF THE GENE FOR INDOLE ACETIC ACID LYSINE 

N.C. State University, Raleigh, NC 27695 and Harvey W. Spurr, CONJUGATION FROM PSEUDOMONAS SYRINGAE PV. SAVASTANOI. F. F. 

Jr., USDA-ARS, Crops Research Lab., Oxford, NC 27565. White, H. J. Klee, R. Nordeen, and F. Roberto. Department of 

Plant Pathology, Kansas State University, Manhattan, KS 66506 

In vitro microbial bioassays were done to determine the U.S.A. 

influence of plant volatile oils on germination of P. tabacina 

sporangia. Aqueous spore suspensions were exposed to vapors The open reading frame (orf) for the gene directing the 

from authentic compounds of major components of the volatile conjugation of indole acetic acid to the epsilon nitrogen of 

mixture emitted by tobacco. Germination was completely lysine was identified. The region conferring activity was 

inhibited when freshly harvested spores were exposed to identified in pLG87 and sequenced. A 1185 bp orf was 

volatile ketones and aldehydes for 4 hours. Short exposures functional for conjugation activity and directed the synthesis 

(30 sec-15 mins) to 3-octanone, nicotine, 1-methyl naphthalene of a 40 kd protein when introduced into the T7 promoter 

or 2-ethyl-l-hexanol resulted in slight stimulation of expression plasmid pT7-7. The gene is in the process of being 

germination. Significant stimulation of germination occurred introduced into tobacco plants under the control of the 35S 

when spores which were not freshly harvested were exposed to CaMV promoter. The presence of the iaaL gene as well as auxin 

beta ionone, methyl salicylate or nicotine. Leaf surface biosynthetic genes in various Pseudomonas pathovars will be 

interactions appear more complex since observing that leaf discussed. 

surface components may alter germination by pathogens. 

81 85 

EFFECT OF CULTURE FILTRATE AND NYCELIAL HONOCENATES OF PHYTO- 

EVIDENCE AGAINST POTATO AND TOMATO HOST SPECIFICITY IN PHTMORA INFESTANS ON VIABILITY OF POTATO PROTOPLAST. U. 

PHYTOPHTHORA INFESTANS. L. J. Spielman, B. J. McMaster, and W. Roongruangsree, R. J. Young, and K. L. Deahi. Dept. of Plant 

E. Fry, Cornell University, Ithaca, NY 14853. Pathology, West Virginia University, Norgantown, WV 26506- 

6057, USDA-ARS, Bldg. 04, BARC-West, Reltsville, ND 20705. 

We have investigated host specialization in P. infestans by 

comparing pathogenicity on susceptible potato and tomato Reaction of leaf protoplasts (PP) to culture filtrates and 

cultivars, using detached-leaflet tests. Of 54 isolates from mycelial homogenates (MN) was studied using PP from Solanuis 

potato cultivars and wild Solanum species (tested less than 1 tuberosum and fungal components of Phytophthora infestans 

year after collection), 47 were pathogenic on both hosts, 3 (pin). comparisons were made between 2 r-gene cultivars 

were pathogenic on potato but showed low pathogenicity on (cvs.), cvs. with R-genes, and a non-host, S. carolinense. 

tomato, and 4 showed low pathogenicity on both hosts. Three culture filtrates and NM Pin were prepared from lime bean 

isolates from tomato were highly pathogenic on both potato and broth and agar plates. Viable PP were assessed by fluorescetomato. 

Progeny of sexual crosses between parents pathogenic in diacetate at 0, 10, and 30 minutes. Culture filtrates had 

on both potato and tomato were also tested. The coincidence no effect on reducing PP viability. NH reduced viability of 

of pathogenicity on potato with pathogenicity on tomato ranged PP from all cvs. The mean number of viable PP after 10 and 

from 92-96% for three separate crosses. This evidence does 30 minutes reaction time for R-gene cvs. was 68% and 42%, 

not support the hypothesis of host specialization in P. respectively, whereas viability in r-gene cvs. was 58% and 

infestans, and indicates that potato and tomato patho- 34% for the same reaction period. R-gene cvs. appeared to be 

genicities are largely controlled by the same genes, less sensitive to MN treatment than r-gene cvs. 

Vol. 79, No. 10, 1989 1145

86 when root numbers were lowest. This indicates that citrus 

roots may be able to compensate for root damage or reduced 

THE `FECT OF CXMPOSTED MUNICIPAL SEWAGE SLUDGE (CIVISS) ON numbers with increased efficiency of root uptake. 

PHYTOPHTHORA ROOT ROT AND SCLEROTINIA CROWN AND STEM ROT OF 

ALFALFA. R. P. Woodward and R. B. Carroll, Department of Plant 

Science, University of Delaware, Newark, DE 19717-1303. 90 

Saranac alfalfa was planted into 15.2 cm pots containing autoclaved 

silt loam soil amended with C(SS obtained from three 

municipalities. Experimental design was a randomized complete 

block with five replications. Treatments were loading rates of 

11.2, 22.4, 44.8, 112.0, and 224.0 Mg/ha of each CMSS. 

Phytophthora megasperma f. sp. medicaginis (Pmm) and 

Sclerotinia trifoliorum via ixig (St) ito inocula oil achamed rio were toplatin. applied separately Aterresistance 

via mixing into each amended soil prior to planting. After 

nine weeks, disease development was rated. No significant 

differences (P=0.05) were noted with respect to 41455 origin, 

Significant Pmm disease suppression occurred at 44.8 and 

112.0 Mg/ha. Suppression of disease caused by St occurred at 

all loading rates. Isolations from infected root and crown 

tissues indicated less Pmm was recovered as the CMSS rates 

increased but St remained constant. 

87 

SOILS SUPPRESSIVE TO BLACK ROOT ROT OF BURLEY TOBACCO IN 

WESTERN NORTH Carolina. Julie R. Meyer and H. D. Shew. Dept. 

of Plant Pathology, North Carolina State University, Raleigh, 

NC 27695-7616. 

SoiLs suppressive to black root rot were identified by th 

abs supprese in blac rotrter identified bythe 

absence of disease to black in root fields rot planted and containing in cultivars 2-500 with cfu/g low soil of 

Thielaviopsis basicola. Suppressiveness was confirmed under 

controlled environmental conditions with several pathogen 

isolates and host cultivars. 

ies 

Suppressiveness 

and sultivaro. Suppressive e 

was 

s 

not 

of T. 

the 

result of reduced survival of the chlamydospores of T. 

basicola. The suppressive factor appears to be abiotic and 

associated with soil acidity. Soil amendments were used to 

separate the effects of different components of acidity (soil 

pH, base saturation, exchangeable Al) on disease. The results 

suggest that soil Al may be the mechanism of suppressiveness in 

these soils. 

EFFECT OF INSECT DEFOLIATION ON SEVERITY OF FUSARIUM CROWN- 

ROT OF ALFALFA. P. D. Colyer,I J. W. Lee, 2 and S. S. 

Quisenberry. 91 

2 Louisiana State University Agricultural Center, 

Louisiana Agricultural Experiment Station, Red River Research 

Station,I Bossier City, LA 71113 and Department of Entomology, 

COLONIZATION OF SOLANUM TUBEROSUM L. BY COLLETOTRICHUM COC 

2 

Baton Rouge, LA 70803. 

(WALLR.) HUGHES A.W. Barkdoll and J.R. Davis, Univ. of Idaho 

R&E Center, Aberdeen, ID 83210 

Alfalfa, Medicago sativa L. variety 'Florida 77', was inoculated 

with three different isolates of Fusarium and defoliated 

to varying levels with yellowstriped armyworms, Spodoptera 

ornithogalli (Guenee), to determine the effect of insect 

defoliation on the development of crown-rot under greenhouse 

conditions. There were no significant interactions between 

short-term insect defoliation and Fusarium crown-rot on forage 

quality, yield, or root carbohydrate reserves. Although 

insect defoliation alone did reduce plant height, yield, 

and maturity (18,33, and 30%, respectively) at the first 

harvest, no significant effects were observed at two subsequent 

harvests. Fusarium oxysporum Schlecht was the most virulent 

of the three isolates tested. 

Colletotrichum coccodes can be found in roots, tubers, stem 

bases and apices of potato in the field. Tubers from foliar 

inoculated potatoes contained significant differences (P=O.Ol) 

in colony forming units (cfu's) of C. coccodes of 2000 and 300 

in stem and bud ends respectively. Surface disinfestation of 

tubers with 10% clorox did not reduce cfu's. In view of the 

high cfu's of C. coccodes in tubers and stems an experiment was 

conducted to evaluate fungal colonization of potato originating 

from infested seed tubers. Russet Burbank mini-tubers wgre 

inoculated with a conidial suspension (50 ul of 9.2 x 10 

conidia/ml) of C. coccodes and were planted in the greenhouse. 

The resulting pl-ants were destructively sampled three times 

during the season. Stem apices and bases, roots and soil were 

sampled for C. coccodes at each period. At no sampling period 

88 

was C. coccodes detected in stem bases or apices. In contrast, 

C. coccodes in soil increased from zero to above 0.2 cfu/g soil. 

Root infection at the last sampling increased from 0 to 15%. 

CORRELATION BETWEEN SAMPLES FOR ESTIMATION OF INOCULUM DENSITY 

OF CYLINDROCLADIUM CROTALARIAE IN SOIL. A. K. Culbreath, r1. 

K. Beute, and B. B. Shew, Depts. of Plant Pathology, Coastal 92 

Plain Univ., Expt. Raleigh, Stn., NC Tifton, 27695. GA 31793 and North Carolina St. 

COLONIZATION AND PATHOGENICITY OF FUSARIUM OXYSPORUM AND F. 

Four quadrants, each consisting of 63 (13.7 mi 

SOLANI ON ESSEX SOYBEAN. G. M. Farias and G. J. Griffin. 

2 ) contiguous 

plots were established in a field naturally infested with 

Cylindrocladium crotalariae in Martin County, NC, in 1988. 

Half adjacent of the area field was was planted planted to peanuts. to corn the Two previous quadrants year and an 

(designated peanut or corn) were situated in each area. Two 

independen samplest e corn)weresisuting 

of 12 acm diam. To 

independent samples, each consisting of 12 (2.5 cm diam. x 

16.5 cm) soil 

density 

cores, were taken from all plots. Inoculum 

elutriation-selective 

(ID) of C. crotalariae 

medium technique. 

was estimated 

Both 

using 

samples 

an 

gave 

similar estimates of mean ID in each quadrant; ID was 

in 

greatest 

peanut quadrants. Estimates of ID from the two samples 

were highly correlated (p < 0.01) in peanut quadrants but were 

not significantly correlated in corn quadrants, 

Department of Plant Pathology, Physiology and Weed Science. 

Virginia Polytechnic Institute and State University. BlaCksburg, 

VA 24061. 

Colonization Clnzto of fEsxsyenctldn Essex soybean cotyledons by yFsru Fusarium oxysp xsou 

occurred 1 day after planting in naturally infested soil and the 

fungus was present at high frequency (25%) after 4 days. F. 

oxysporum and F. solani colonized the lower hypocotyl and - 

emerging 4 days both roots fungi 2 and were 3 days found after colonizing planting, t elongatin respectively. Afer 

portion of the hypocotyl. The hypocotyl-root transition 9 , upper zone had 

the highest frequency (20-28% of 2-mm tissue segments) 

nization 

of colo- 

by each species at 4 days. F. solani had higher 

colonization rates per unit of inoculum than F. oxysporum (0.042 

and 0.023 colonizations/m root/propagule/g soil after 4 days, 

89 

respectively). In soil-temperature tank tests at 20 C and -0.01 

MPa water potential, all of the four 

solani 

F. oxysporum 

isolates 

and four 

tested 

F. 

delayed seedlilig emergence and caused 

significant reductions in stem length and plant fresh weight. 

ROOT UPTAKE OF RUBIDIUM-86 BY CITRUS ROOTS AS AFFECTED BY ROOT 

PATHOGENS, SEASON AND IRRIGATION. Q. A. Menge, E. L. V. 

Johnson, E. Pond and H. Liu. Dept. of Plant Pathology, 93 

University of California, Riverside, CA 92521. 

THE EFFECT OF PLANTING DATE ON FUSARIUM WILT OF M4USKMELON IN 

Uptake of Rb" by 1-cm pieces of excised citrus root tips was CALIFORNIA. 0. J. Jacobson and I. R. Gordon, Dept. of Plant Pathmeasured. 

In a greenhouse experiment, roots from Troyer ology, University of California, Berkeley 94720. 

citrange inoculated with Phytophthora parasitica absorbed 36% 

less Rb" than roots not inoculated with P. parasitica. In the A randomized complete block design was employed with planting 

field, trees treated with nematicides and fungicides (oxamyl dates as blocks and susceptible and resistant cultivars as 

and metalaxyl) consistently exhibited a 27-57% greater uptake treatments; the experiment was conducted for two years. Cortical 

of Rb" than did non-treated trees. Most of this increase in root colonization by Fusarium oxysporum f. sp. melonis was 

uptake was observed July-October when P. parasitica and nematode measured on seedlings and at full fruit load. Rate of disease 

populations were high. Uptake of Rb" was greater in roots of progress and incidence at harvest were both higher during hotter 

citrus receiving irrigation which was 80% and 120% of the portions of the growing season. Root colonization by the 

evapotranspiration demand (ETD) than by those receiving 100% of pathogen was not affected by planting date; cultivar differences 

the ETD. Root uptake of Rb" fluctuated considerably on a were apparent only at full fruit load and may represent xylem 

seasonal basis and was greatest during the summer months and colonization of the susceptible host. Inoculumn varied sig- 

1146 PHYTOPATHOLOGY

nificantly among plots but there was no correlation (r 2 = 0.20) genomic DNA from 50 field isolates for restriction fragment 

between inoculum level and root colonization. In addition, root polymorphisms. Most of the plasmid probes hybridized preferentially 

colonization by the pathogen was not correlated to disease, 

Thus, the increase in disease with temperature cannot be/ 

with a subset of the total isolates, with little or no cross-hybridization to 

the remaining isolates. Sub-group specificity of random-cloned DNA 

attributed to initial infection; however, temperature may probes is concordant with previous observations of low genomic 

influence subsequent systemic spread or symptom development. DNA/DNA reassociation (30-40%) between the two AG 4 subgroups. 

Restriction polymorphisms within each subgroup were also evident. 

94 

Further studies using the plasmid library should permit us to determine if 

additional population substructure is evident in AG 4. 

COMPARISON OF MEDIA FOR ISOLATING RHIZOCTONIA SOLANI 

FROM SOIL. P. C. Vincelli and C. M-S. Beaupre, Dept. of Plant, Soil & 

Insect Sciences, University of Wyoming, Laramie, WY 82071. 98 

Radial growth of R. solani AG- 1 through AG-5 at 27 C was fastest on water COMPARATIVE GERMINATION OF CULTURE-PRODUCED AND PLANTagar 

(WA; 14.9 mm/day), equal or slightly slower on Ko & Hora's PRODUCED SPORANGIA OF PYTHIUM ULTIMUM IN RESPONSE TO 

medium containing 5 ul/1 prochloraz 40EC (KHp; 1 1 . 3 mm/day), and greatly SOLUBLE SEED EXUDATES AND EXUDATE COMPONENTS. Eric B. 

reduced on ethanol-potassium nitrate medium containing 2% ethanol (EPN2; Nelson and Cheryl M. Craft, Department of Plant Pathology, Cornell University, 

3.6 mm/day). KHp and EPN2 were both highly effective in inhibiting 

Ithaca, 14853. 

indigenous fungi and bacteria from three Wyoming agricultural soils. At least Sporangia of Pythium uldmum were produced on conventional culture media and 

97.9% and 98.8% of soilborne fungi in three soils were inhibited on KHp on media amended with germinating seeds and excised radicles of several plant 

and EPN2 respectively, after 7 days at 19-22 C; and 99.9% of soilborne species. When produced on culture media commonly used for the cultivation of 

bacteria were inhibited on both media. WA inhibited at least 70.0% of bacteria Pythium species, sporangia germinated in response to certain sugars and amino 

after the same period but did not significantly inhibit fungi. No difference was acids as well as cotton seed exudate. When produced in association with plant 

observed between media in estimates of populations of Rhizoctonia-like fungi tissue or on media amended with ct-phosphatidyl choline, however, sporangia failed 

(RLF; characterization of isolates in progress) from three soils. Although to germinate in response to any sugar or amino acid tested, despite their ability to 

efficiency of recovery of RLF from soil was equal among media, it was easier germinate in response to cotton seed exudate. It is believed that sporangia 

to locate and identify colonies of RLF on the two selective media than on WA. produced on plant-tissue amended media more closely reflect the behavior of those 

It was concluded that the high cost of materials for EPN2 (approx. $22/1) as produced on diseased plant tissue in soil than do sporangia produced on 

compared to KHp (approx. $1/1) is not offset by any marked advantage, conventional culture media. Therefore, molecules other than sugars and amino 

acids are probably responsible for activating sporangia of P. ultimum and 

establishing host-pathogen interactions under natural conditions. 

95 

EFFECTS OF SOIL BULK DENSITY ON WILT/ROOT ROT OF CHICKPEAS. 99 

M A. Bhatti and J. M. Kraft, Washington State University and 

USDA-Agricultural Research Service, Irrigated Agriculture EFFECT OF IRRIGATION OF SEEDLINGS ON SEVERITY OF PHYTOPHTHORA 

Research and Extension Center, P.O. Box 30, Prosser, WA 99350. ROOT ROT OF SOYBEANS. A. F. Schmitthenner, Dept. of Plant 

Bulk densities of 1.2 g/cm 3 (loose) and 1.5 g/cm 3 (compacted) Pathology, Ohio State Univ., Wooster, OH 44691. 

were produced to determine the effects of compacted soil on Over a 3-yr period in soil infested with P. megasperma f.sp. 

root disease severity and root growth of chickpeas (Cicer qlycinea, irrigation with 100 mm water 3 days after planting 

arietinum). The susceptible cultivar JG-62 was grown in soil was compared with no irrigation on three cultivars, with and 

infested with Fusarium oxysporum f. sp. ciceri, F. solani f. without metalaxyl applied in the seed furrow at planting time 

sp. pisi, Pythium ultimum, and/or Thielaviopsis basicola at rates of 560 g/ha. In nonirrigated plots, stand and seed 

separately and in various combinations under controlled growth yield was significantly increased by metalaxyl in low-tolerant 

chamber conditions. There was more severe root disease and cv. Sloan but not in high-tolerant cv. Asgrow 3127 or resistant 

less root growth in the compacted soil than in loose soil when (Rps 1-k) Century 84. Stands and seed yields of Asgrow 3127 

JG-62 was grown in soil infested with these pathogens were significantly lower in the irrigated treatments without 

individually or in combination. The effects of these root metalaxyl. Sloan was severely damaged in the irrigated 

pathogens on severity of wilt and root rot of chickpea were treatments and metalaxyl provided only partial control. 

additive when roots were exposed to various combinations. Irrigation had no effect on stand or yield of Century 84. It 

Compaction had no effect on wilt severity caused by F. was concluded that severe Phytophthora rot can be consistently 

oxysporum f. sp. ciceri. Root growth was inversely related to induced by early irrigation and controlled by resistant 

soil compaction. cultivars or by metalaxyl soil treatment of high-tolerance 

cultivars. 

96 

INFLUENCE OF SOIL MOISTURE ON WILT/ROOT ROT OF CHICKPEA. 

M. A. Bhatti and J. M. Kraft, Washington State University and SUSCEPTIBILITY TO HELMINTHOSPORIUM CARBONUM RACE 1 in IOWA STIFF 

USDA-Agricultural Research Service, Irrigated Agriculture 

Research and Extension Center, P. 0. Box 30, Prosser, WA 

STALK SYNTHETIC MAIZE. K. M. Tubajika*, C. A. Martinson*, A.R. 

Hallauer 0 99350. 

, and K. R. LamkeyO, *Dept. of PlantPathology, "USDA/ 

ARS Dept. of Agronomy, Iowa State University, Ames, Iowa 50011. 

The susceptible chickpea (ricer arietinum) cultivar JG-62 was 

grown in soil infested with Fusarium oxysporum f. sp. ciceri, 

Iowa stiff stalk synthetic (BSSS) populations are important 

F. solani f. sp. pisi, Pythium ultimum, and/or Thielaviopsis 

basicola. These tests were conducted in greenhouse pot 

sucso omrilmiegrpam 

(=Bipolaris zeicola), which is 

ae1o .crou 

specific for the hm allele, seculture 

at 12, 18, and 25% soil moisture. Root rot or wilt verely attacked progeny in 59% of S2 lines of BSI3(S)C5 in 1987. 

increased with soil moisture as did rhizosphere populations of Differential inbreds Pr and Prl were susceptible and resistant, 

each pathogen. Soil infested with equal proportions of each respectively. Three of 16 inbred progenitors of BSSS were suspathogen 

together had as much or more disease when planted ceptible. BSSS populations have been improved through recurrent 

with JG-62 as in soil infested with an individual pathogen. selection (RS) since 1939. The intermated populations from CD 

The exception was a combination F. oxysporum f. ap. ciceri and through C7 cycles of BSSS (MT) (half-sib RS), CO through CS cycles 

P. ultimum where wilt severity decreased. Rhizosphere of BSI3(S) (Sl RS) and C4, C8, and Cll cycles of BSSS(R) (recippopulations 

of F. oxysporum f. ap. ciceri were much higher rocal RS) were assayed for frequency of susceptible plants; 4 to 

than populations of other pathogens at the duration of each 54% of plants in each cycle were susceptible. Modifying alleles 

testand genes were common. Maize breeders need to assess their BSSS 

test.germplasm for susceptibility to this race of N. carbonum. 

100 

97 101 

DNA RESTRICTION POLYMORPHISMS BETWEEN INTRA- 

SPECIFIC GROUPS OF AG 4 FROM RHIZOCTONIA SOLANI. EFFECT OF RELATIVE HUMIDITY ON GERM TUBE ELONGATION AND 

R. Vilg~alvs, 1 D. Gonzalez, 1 T. B. Brenneman, 2 D. R. Sumner, 2 and A. S. APRSOILFPAINO•ECSOAZA-ADS AndREC.ORA. NarYTInONet of CROPlRan PatholoYDIoaStat P.R• hro U.Tosni 

Csinos . -Department of Botany, Duke University, Durham, NC 27706, and 2 Coastal varsity,an .A Ames,MatnsnIa 5001etl1. ofPatahlgyIwaSteUi 

Plain Exp. Station, Dept. of Plant Pathology, University of Georgia, Tifton, GA 31793. 

Plasmid probes containing cloned DNA fragments from an isolate of R. 

Cercospora zeae-maydis, the cause of gray leaf spot of maize, 

has been reported to be favored by prolonged high R.H and leaf 

solani anastomosis group (AG) 4 were used to screen Southern blots of wetness. Germ tube elongation and appressorial formation of C. 

VOl. 79, NO. 10, 1989 1147

zeae-maydis were examined after exposure to different RH with growth chamber and field studies. Steamed and nonsteamed 

regimes. Conidia were atomized onto polysulfone membrane filter soil was used in growth chamber studies. No significant interdiscs 

and allowed to germinate for 6 hr. Discs were then sus- actions were found between herbicides and plant growth parspended 

above glycerol solutions maintained at 25.0 C to obtain meters in steamed soil. Significant interactions between 

39-100% RH. Germ tube elongation and appressorial formation herbicides and inoculum treatments affecting root and hypocotyl 

were observed microscopically after staining the discs with acid weight occurred in nonsteamed soil. Root and hypocotyl weights 

fuchsin in lactophenol. Appressoria formed in 2 to 3 days when were significantly lower in inoculated soil than in noninocugerminated 

conidia were maintained at 95% RH. Appressoria did lated soil in the absence of herbicides. No similar significant 

not form at 90% RH even after 15 days. However, when these differences were observed in treatments where herbicides were 

conidia were then transferred to 95% RH, appressoria formed in added to the soil. In field studies R. solani infested oats 

2 to 3 days. Conidia held at 39-80% RH for 15 days did not con- placed in furrow at planting significantly reduced plant growth 

tinue development when transferred to 95% RH. parameters. No significant interactions were observed between 

herbicides and inoculum treatments. Effects of R. solani 

inoculum were significantly reduced with carboxin-pentachloro- 

102 nitrobenezene seed treatments. 

THE EFFECTS OF NITROGEN ON CHARCOAL ROT DEVELOPMENT IN 

SORGHUM BICOLOR. G.L. Cloud and J.C. Rupe, Dept. of Plant 

Pathology, University of Arkansas, Fayetteville, AR 72701. 106 Withdrawn 

Colonization of sorghum by Macrophomina phaseolina was 

determined at nitrogen fertilization rates of 0, 50, 100, 

and 150 lbs/acre in a randomized complete block design with 

five replications. Plant tissue and roots were sampled 

monthly to determine total nitrogen and percent root 

segments colonized by natural infection. Sorghum stalks 

also were inoculated with toothpicks infested with M. pLhaseolina, 

and four plants were rated monthly for lesion 

length. There were no significant differences between the 

level of soil nitrogen and incidence of root colonization. 

There was a positive correlation between the length of stalk 

lesions from the point of inoculation and plant tissue 

nitrogen levels at 119 and 139, but not 79 days after 

planting. 

103 

AMBIGUITY OF THE DESIGNATION RACE 4 FOR ISOLATES OF CERCOSPORA 

SOJINA. D. V. Phillips and H. R. Boerma, Departments of Plant 

Pathology and Agronomy, Georgia Station, University of Georgia, 107 

Griffin, GA 30223. CHARACTERIZATION OF COLLETOTRICHUM GRAMINICOLA ISOLATES 

Race 3 and Race 4 of Cercospora sojina were described in 1968 WITH 

TROPHORESIS 

AMINOPEPTIDASE 

OF SOLUBLE 

PROFILES AND 

PROTEINS. 

POLYACRYLAMIDE 

Ali, 

GEL 

M.E.K., 

ELECto 

designate isolates from North Carolina. In 

H.L. 

recent 

Warren, 

years, 

D.M. Huter 

_C. E.tROPHORES sOFSOBLPR S A atholEgyH 

sojina 

anrenDMurI 

isolates from the southern U.S. include Race 5 and F.E. Lytle, K. Hughes. USDA-ARS, Botany & Plant Pathology, 

several 

and 

of 

Che0istry 

Race 4. 

De- 

Since Race 4 was originally tested on only partments; Purdue University, W. 

16 

Lafayette, 

cultivars, 

IN 47907. 

additional cultivars were tested for reaction to Filter aminopeptidase and gel electrophoresis methods 

isolates 

were compared 

of this 

with 

race. 

a laser 

Several cultivars reacted differently enhanced aminopeptidase technique to characterize 

to 

isolates 

isolates 

of Colletotrichum 

designated 

gram 

as Race 4, indicating that the 16 inicola from three hosts. Filter and laser aminopeptidase profiles 

cultivars 

and gel 

used 

electro- 

to describe Race 4 are not adequate to phoretic patters of soluble protein indicate that 

distinguish 

isolates of C. graminicola 

Race 4 

from 

from other apparently new races. The sorghum, corn and barnyard grass can be differentiated 

reaction 

based 

of 

on 

additional 

their host speci- 

cultivars to Race 4 cannot be determined ficity. The laser aminopeptidase profiles further separated 

because 

sorghum 

an 

isolates 

authentic 

to 

culture is not available. Since at least races, similar to their reactions on sorghum 

3 isolates 

plants. Corn 

designated 

isolates showed 

as Race 

less 

4 can be separated from each variability, which is consistent with results from pathogenicity 

other and 

tests. 

from 

The 

Races 

percent 

1, 2, 3, and 5 by cultivar reaction, the hydrolysis of f?-napthylamides was highest in sorghum 

designation 

followed 

Race 

by 

4 

corn 

is 

and 

ambiguous and should not be used. barnyard grass. The aminopeptidase assay is a powerful method which is sensitive 

104 

enough to provide a rapid method of differentiating between isolates of C. graminicola 

from sorghum, corn and barnyard grass. Laser enhancement of the aminopeptidase 

system permits differentiation of races of C. graminicola pathogenic to sorghum. 

STABILITY OF PLASMA MEMBRANE OF WHITE BEAN ASSOCIATED WITH 

WHITE MOULD RESISTANCE. J. C. Tu, Agriculture Canada, Research 

Station, Harrow, Ontario NOR IGO. 

Stability of plasma membrane of white bean to oxalic acid 

secreted by Sclerotinia eclerotiorum was associated with 

disease resistance. Leaf tissues of a susceptible (Fleetwood) 

and a resistant (ExRico 23) cultivar were treated with 

different concentrations of oxalic acid for thin sectioning and 

freeze fracturing. In thin sections, at a given oxalic acid 

concentration, the plasma membrane and chloroplasts of 

Fleetwood were affected more and ruptured quicker than those of 

ExRico 23. In replicas of freeze-fractured plasma membrane, 

protrusions, wrinkles and breakages increased with increasing 

oxalic acid concentration. The degree of damage was distinctly 

more severe in the plasma membrane of Fleetwood than ExRico 

23. Conductivity measurements of the bathing water of leaf 

discs showed that Fleetwood had a higher conductivity reading 

than that of ExRico 23, indicating that the plasma membrane of 

Fleetw~ood was less stable than that of ExRico 23. 

105 

108 

OCCURRENCE AND IMPACT OF VIRAL DISEASES OF 

WINTER WHEAT IN NEW YORK STATE. N. R, Miller, G. C. 

Bergstrom, and S. M. Gray. Dept. of Plant Pathology, Cornell 

Uiest taaY183 

Tedsrbtoadicdneocmovrsdsae fetnsf 

whie winteri whenatd iniNew York Staten (NYus) wierses dfeterined in 

whtwierhatnNwYokSae( S)eedtrmedn 

1988 and 1989. Over 100 randomly selected fields were assessed for 

wheat spindle streak mosaic(WVSSM), barley yellow dwarf(ByD),soil- 

borne wheat mosaic, and wheat streak mosaic using ELISA 

techniques and symptom expression. At early stem extension, 

symptoms of WSSM were severe in fields planted to susceptible 

cultivars. BYDVwas detected byELISAin many fields at this growth 

stage, although no symptoms of the disease were observed. At spike 

emergence, BYDVwas the only widespreadwheatvirus. In surveyed 

fields, as well as in field plot experiments, the cultivar Geneva 

consistently incurred a low incidence of WSSM. These findings, 

together with experiments examining the effect of WSSM on wheat 

yields, contribute to our understanding of the impact of virus diseases 

on wheat production in NYS. 

INTERACTION OF DINITROANILNE HERBICIDES AND RHIZOCTONIA DISEASE 

ON SOYBEAN. E. N. Bauske and H. W. Kirby, 1102 S. Goodwin Ave. 109 

University of Illinois, Urbana, Illinois 61801 

MANGANESE TOXICITY IN THE MARBLE CULTIVARS OF CHRYSANTHEMUM 

The effect of trifluralin, pendimethalin, and ethalfluralin and MORIFOLIUM. I. CYTOcHEMISTRY. R. H. Lawson anid7TM. M.Dienet. 

disease caused by Rhizoctonia solani on soybeans was determined UWfSDAARFlorist & Nursery Crops Lab., geltsville, MO. 

1148 PHYTOPATHOLOGY

Chrysanthemum morifolium (florists' chrysanthemum) cultivars Symptoms of the progressive decline of Arizona ash (Fraxinus 

of the Marble group are affected by necrotic spotting in lower velutina L.) in Arizona include chlorosis, leaf scorching, 

leaves and some veinal necrosis while 'Vero' appears progressive dieback of twigs and branches, and proliferation 

unaffected. Mn accumulation in necrotic lesions was determined of shoots along the main trunk. The incidence and severity 

by x-ray microanalysis and is similar to Mn toxicity in other of decline were observed in ash trees located at a study site 

species. Supplemental toxic levels of MnS0 4 increased the in Tempe AZ during 1985 and 1988. In 1985, about 30% of the 

incidence of necrotic lesions in the Marbles and induced trees had dieback in more than one half of the crown. By 

similar necrotic lesions on lower leaves as well as a top 1988, this percentage had increased to 55% including 16% of 

systemic chlorosis in 'Vero'. Aniline blue fluorescence in the trees which had died in the interim. Mortality 

cell walls adjacent to necrosis indicated deposition of progressed at a rate of 4.6% per year. Investigations into 

callose. There was no increase in callose in phloem from the etiology of this problem are continuing. 

leaves or petioles of necrotic Marble plants over symptomless 

'Vero' that would suggest the presence of an MLO. The DAPI 

stain for DNA did not reveal the presence of a fastidious 114 

prokaryote in Marble phloem. Leaf necrosis in the Marble EPIDEMIOLOGY OF DOGWOOD ANTKRAGNOSE IN CHEROKEE NATIONAL FORE 

group is associated with Mn toxicity. OF SOUTHEASTERN TENNESSEE. M. T. Windham. Department of 

110 

Entomology and Plant Pathology, University of Tennessee, P.O. 

Box 1071, Knoxville, TN 37901-1071. 

MANGANESE TOXICITY IN THE MARBLE CULTIVARS OF CHRYSANTHEMUM 

MORIFOLIUM. II. ELECTRON MICROSCOPY. M. M. Dienelt and R. H. 

Lawson, USDA-ARS, Florist & Nursery Crops Lab., BeTtsville, MD. 

Dogwood anthracnose was first observed in the Applachian 

Mountains of southeastern Tennessee in 1988. Twenty-one plots 

in 1988 (elevation 274- 915m) and 35 plots in 1989 (elevation 

Cell wall abnormalities associated with manganese-induced 

necrotic lesions in Chrysanthemum morifolium 'Pink Marble' 

(PM), 'Florida Marble' (FM), and 'Vero' were similar to those 

associated with necrotic lesions in untreated FM and PM. 

Although no pathogenic agents were observed, vesicles and 

filaments resembling MLO's occurred in new growth of all treated 

and untreated plants. These structures occurred in vacuoles of 

companion cells, gum duct epithelial cells and salt glands but 

not sieve elements. Transfer-cell-like wall ingrowths and 

papillae composed of membranes, phenolic substances (identified 

by the nitroso reaction) and a faintly stained amorphous matrix 

occurred in necrotic and adjacent tissue. Papillae often 

developed at plasmodesmata openings. Large papillae, corresponding 

to aniline blue fluoresence noted earlier, bordered 

necrotic cells. Papillae, which often occur in response to 

pathogenic invasion, may also reflect nutritional stress. 

250 - 915m) were monitored for incidence of dogwood anthracnose 

and disease severity. When plots were located more than 100m 

from water, disease severity values for trees within plots 

increased as plot elevation increased. In plots located within 

50m of water, the disease was severe regardless of plot 

elevation. An average of 55% of the trees with trunk diameters 

> 5cm displayed symptoms of the disease and < 25% of the 

foliage was affected in infected trees. Disease incidence 

averaged 11% in trees with trunk diameters of less than 1.3cm; 

40% of the foliage was affected in infected trees. 

1 

ALTERNARIA LEAF SPOT OF GOMPHRENA GLOBOSA. Karen K. Rane and 

Robert L. Wick, University of Massachusetts, Suburban 

Experiment Station, 240 Beaver St., Waltham, MA 02154. 

11 

A severe leaf spot disease of Gomphrena globosa was found in a 

commercial cut-flower field in Massachusetts. Virtually 100% 

DISEASE INCIDENCE AND BOTRYTIS CINEREA CONIDIAL CONCENTRATION 

AMONG GERANIUM STOCK PLANTS. M. K. Hausbeck and S. P. Pennypacker, 

Department of Plant Pathology, The Pennsylvania State 

University, University Park, PA 16802 

of the crop was affected and became unsalable as fresh cut 

flowers. Lesions consisted of dry, necrotic areas surrounded 

by red or purple margins, and ranged from 0.3 cm to 1.0 cm in 

diameter. Alternaria gomphrenae Togashi was recovered from 

lesions, and Koch's postulates were completed by foliar spray 

Hourly Botrytis cinerea conidial concentrations among geranium inoculation. A. gomphrenae was also recovered from two of four 

(Pelargonium x hortorum) stock plants within a commercial commercial gomphrena seed samples examined. This is the first 

greenhouse were estimated for selected time periods of the 1986 

and 1987 growing seasons using a Burkard recording spore trap. 

Disease incidence occurring during 1987 was also documented. 

Maximum concentrations of conidia typically occurred at midday 

report of this pathogen in the United States. 

or in association with grower activity including watering, 

pesticide application, and harvesting of cuttings. The number 

of conidia trapped per day and the amount of disease increased 

during the season. The maximum number of conidia trapped in 

May was 2,000 conidia/m 

116 

SUSCEPTIBILITY OF FLORIST'S CHRYSANTHEMUM TO TOMATO SPOTTED 

WILT VIRUS AND WESTERN FLOWER THRIPS. J. A. Matteoni, W. R. 

Allen and A. B. Broadbent. Agriculture Canada, Vineland 

3 air/hour and an average of 24% of the 

stems and 5% of the necrotic leaves were infected. Conidial 

concentrations and disease incidence continued to increase 

through July to a maximum of 14,000 conidia/m 

Station, Ontario, Canada LOR 2E0 

A wide range in susceptibility to tomato spotted wilt virus 

3 air/hour and an 

average of 71% infected stems and 88% infected necrotic leaves. 

(TSWV) and host preference of western flower thrips 

(Frankliniella occidentalis) was found among over 30 cultivars 

of florist's chrysanthemum (Chrysanthemum X morifolium). 

Significantly more plants became infected (P

were mixed-infected with both CyMV and ORSV; 27.2% were not 

infected. The indirect ELISA method was used for virus detection. 

118 

nase was not associated with immunization in Xanthi-nc 

tobacco. Total soluble carbohydrate increased in Kyl4 and 

Xanthi-nc immunized with P. tabacina but not in regenerants of 

immunized Kyl4 or Kyi4 immunized by TMV. 

PHENOL OXIDASES OF FIVE ARMILLARIA BIOSPECIES. 

P.M. Wargo, U.S.D.A. Forest Service, Hamden, CT 

S.F. Marsh and 

06514 

122 

ASSOCIATION OF PATHOGENESIS-RELATED PROTEINS(PRs), PEROXIDASE 

Three isolates each 

and 

of Armillaria 

VII were 

biospecies 

assayed 

groups I,III,V,VI 

for constitutive phenol oxidases after 11, 

(PER), 0-1,3-GLUCANASE(GL) AND CHITINASE(CH) WITH INDUCED 

RESISTANCE OF TOBACCO TO PERONOSPORA TABACINA BUT NOT TO 

18,25,32 and 39 days growth in liquid culture. Laccase was 

measured in the mycelium, rhizomorphs and extracellular culture 

medium. Tyrosinase was only found intracellularly. Averaged 

across isolates, intracellular laccase activity was highest at 

11 days while tyrosinase and extracellular laccase were highest 

at 18 days. Activities of laccase and tyrosinase were not 

different among biospecies groups because there were large 

differences between isolates within a biospecies group. Among 

rhizomo~ph-producing isolates (12), isolates that produced more 

rhizomorphs had higher laccase activity. Laccase activity was 

also present in isolates that produced no rhizomorphs. Preliminary 

results indicate that peroxidase activity may be present 

in some isolates. No relation between the enzyme activities 

and reported pathogenicity, pH of culture medium, phenol production 

or mycelial pigmentation has been found, 

SYSTEMIC TOBACCO MOSAIC VIRUS. X. S. Ye S. Q. Pan and J. 

Kuc. Dept. of Plant Pathology, University of Kentucky, 

Lexington, KY 40546-0091. 

Tobacco Ky 14 inoculated with TMV and held at 23C for 3-12 

days had localized necrosis on the inoculated leaves and was 

systemically protected against blue mold and TMV. Above 28C, 

systemic mosaic was apparent. Accumulation of PRs and activi- 

ties of PER, GL and CH increased in induced plants. When TMV- 

inoculated leaves were removed 12 days after inoculation and 

plants were challenged with TMV or P. tabacina and transfered 

from 23C to 28C one day after challenge, induced resistance to 

P. tabacina but not TMV was apparent. Accumulation of PRs and 

activities of PER, GL and CH were further increased after 

challenge in induced plants at 23C and 28C. Transfer of plants 

from 23C to 28C for 7 days after induction and removal of 

inducer leaves did not affect induced resistance to P. 

119 

tabacina and TMV when challenged and held at 23C for 7 days. 

ASSOCIATION OF PEROXIDASE WITH SYSTEMIC RESISTANCE TO 

CLADOSPORIUM CUCUMERINUM INDUCED BY COLLETOTRICHUM LAGENARIUM 

IN CUCUMBER CULTIVARS RESISTANT OR SUSCEPTIBLE TO C. 

CUCUNERINUM. R. Reuveni and J. Ku6. Department of Plant 

Pathology, University of Kentucky, Lexington, KY 40546-0091. 

123 

ENHANCED PEROXIDASE AND ITS PERSISTENCE IN CUCUMBER PLANTS 

IMMUNIZED AGAINST ANTHRACNOSE BY FOLIAR INOCULATION WITH 

COLLETOTRICHUM LAGENARIUM. R. F. Dalisay and J. Kuc, Dept 

Cucumber cultivars susceptible or resistant to C. cucumerinum 

were systemically protected by infection of leaf 1 with C. 

lagenarium. The protection of leaf 2 was positively correlated 

with the concentration of C. lagenarium inoculum, negatively 

correlated with the concentration of challenge inoculum 

and was not overcome by 106 conidia/ml of C. cucumerinum. 

Peroxidase activity increased in protected-leaf 2. After 

challenge, peroxidase increased further in protected plants 

and was detected as early as 24 hr after challenge in resistant 

protected plants. The increase in peroxidase activity 

and tandte the apprteaned appearance pants.d and intensification int e icreastion operoxidase of peroxidase ativty isozymes 

occurred in the absence of visible damage. Peroxidase after 

of Plant Pathology, University of Kentucky, Lexington, KY 

40546-0091. 

Inoculation of the first true leaf of cucumber plants with 

conidia of C. lahenarium induces systemic resistance against 

theisa f fr-wkWen temiculate (inde 

the same fungus for 4-6 weeks. When the inoculated (inducer 

leaf was detached one week after inoculation, resistance was 

reduced but persisted for the same time period. Peroxidase 

activity was 3-10 x greater in induced plants than in plants 

treated with water on leaf 1 and was consistently higher in 

plants after leaf with 1 the was inducer induced leaf followed attached. the same Leaves pattern 4-12 for in the protec- bud 

to n eoiaeatvt sla .Rslssgetta 

challenge may be a useful marker for both induced and non- signal produc e dur ith fs 7aaf te iulation o 

induced resistance. 

120 

signal produced during the first 7 days after inoculation of 

leaf 1 affects protection and peroxidase activity of expanded 

leaves and leaves in the bud. Once triggered, the response 

persists in the absence of the signal. 

BIOSYNTHESIS OF SESQUITERPENOID PHYTOALEXINS IN 

BACTERIALLY INOCULATED COTTON LEAVES AND COTYLEDONS. 

M. Essenberg, H. Hamada, and G. D. Davis. Dept. of Biochemistry, 

Oklahoma State University, Oklahoma Agricultural Experiment Siation, 

Stillwater, OK 74078-0454. 

124 

PMG WALL GLUCAN IS AN EFFICIENT ELICITOR OF ISOFLAVONES BUT IS 

NOT AN EFFICIENT ELICITOR OF GLYCEOLLIN IN SOYBEANS. T. L. 

Graham and M. Y. Graham, Dept. of Plant Pathology, The Ohio 

Chemical degradation of 2,7-dihydroxycadalene (DHC) and 2-hydroxy-7methoxycadalene 

(HMC) produced in cotton cotyledons from [2-14C,5- 

3 

H]mevalonolactone following inoculation with an incompatible race of 

Xanthomonas campestris pv. malvacearum revealed that biosynthesis of DHC 

and 

probably 

HMC 

occurs 

involves 

as 

a 

hydrogen 

1,3-hydride 

transfer to the 

shift 

isopropyl 

during 

side chain, 

cyclization 

which 

of the farnesyl 

precursor. Gas chromatographic/mass spectrometric analysis of cotyledonary 

extracts showed the presence of inoculation-induced substances of molecular 

weights 204, 206, 216, 218, and 232, whose mass spectra suggest that they 

are intermediates on the pathway to DHC and HMC. High-resolution mass 

spectrometry and tH-nmr of the MW 218 compound indicate that it is 

C15H22O, 7-hydroxycalamenene. 

State University, Columbus, OH 43210. 

The cell wall glucans of Phytophthora megasperma f. sp. glycinea 

(PMG) elicit glyceollin at nearly hormonal (ng/ml) levels. 

However, they are non-race specific elicitors of glyceollin. 

Because we have demonstrated that preformed 

of 

pools 

the glyceollin 

of conjugates 

precursor, daidzein, are used in race 

specific accumulation of glyceollin in PMG infected tissues, we 

were interested to re-examine the specific role of the glucan 

elicitors in relation to these events. We discovered that PMG 

glucan is a highly efficient elicitor of daidze in, but is a 

very inefficient elicitor of glyceollin. Only 3-20% of the 

daidzein in PMG glucan treated tissues is converted to glyceollin, 

depending on factors such as light intensity, wounding and 

121 

tissue age. The results complement earlier studies in 

suggesting that effective glyceollin elicitation may require 

RELATIONSHIP OF f3-1,3-GLUCANASE AND TOTAL SOLUBLE 

either a second elicitor or a specific physiological state of 

the tissue perceiving the PMG wall glucan. 

CARBOHYDRATE TO THE IMMUNIZATION OF TOBACCO AGAINST BLUE MOLD 

CAUSED BY PERONOSPORA TABACINA. S. Q. Pan, x. s. Ye and J. 125 

Kuc, Department of Plant Pathology, 

Lexington, Ky 40546-0091. 

University of Kentucky, 

BACTERIAL AND PLANT GENE EXPRESSION IN POTATO SOFT ROT. Z. 

Stem injection of Kyl4 tobacco with sporangiospores of P. 

acina or leaf inoculation with tobacco mosaic virus (TMV) 

tab- 

Yfang, C.L. Cramer, and G.M. 

Biology of Plant Stress, VPI&SU, 

Lacy, Laboratory for Molecular 

Blacksburg, VA 24061-0330. 

systemically immunized plants against disease caused by both 

pathogens and systemically increased -1,3-glucanase activity. 

After challenge with P. tabacina, lI-1,3-glucanase also increased 

earlier and more rapidly in immunized plants and their 

tissue culture regenerants than in controls. One dominant 

intercellular isozyme of P-l,3-glucanase was associated with 

immunization before and after challenge and another with symptom 

expression after challenge only in controls. P-1,3-gluca- 

A membrane-separated system involving potato tuber slices and 

Erwinia carotovora subsp, carotovora (Ecc) was used to study 

simultaneous in planta regulation of bacterial pathogenicity- 

related and defense-related genes in soft-rot interaction. 

Northern hybridization showed increases in pathogenicity- 

related genes including endo- and exo-pectate lyases (PLs) and 

endo-polygalacturonase (PG) within 3 hr reaching maxima beteen 

6 and 12 hr. Most defense responses were monitored by phenylal- 

1150 PHYTOPATHOLOGY

anine ammonia lyase (PAL) and hydroxymethyl glutaryl CoA reduc- distribution of A across the leaf were tested for by 14C02 

tase (HMGR) activity and mRNA levels. Ecc induces rapid accum- autoradiography but not found. G and A of diseased leaves 

ulation of PAL mRNA and enzyme activity superimposed on wound were increased by increased relative humidity. In 

response. However, Ecc activates a specific HMGR isogene that Verticillium-infected plants and drought-stressed plants, low 

is not induced by wounding, which rapidly elevated expression G was correlated with low leaf water potential. However, 

of a second isogene. Therefore, HMGR represents a novel defen- leaves from Verticillium-infected plants had higher G at equal 

se-related gene useful in the study of molecular mechanisms of leaf water potentials. Pressure-volume curves showed that 

host defense responses. this was not due to osmotic adjustment. 

126 130 

ELECTROPHORETIC CHARACTERIZATION OF PEANUT ENZYMES AFTER SODIUN BISULFITE ENHANCES ELECTROLYTE LEAKAGE, PEROXIDASE 

INFECTION WITH ASPERGILLUS SPP. J.B. Szerszen, R. E. Pettit, J. S. Neck ACTIVITY AND SPORULATION OF BIPOLARIS MAYDIS RACE T (BMT) IN 

and R. A. Taber. Department of Plant Pathology and Microbiology, Agricultural MAIZE. M. Akhtar and M. 0. Garraway, Department of Plant Path., 

Experiment Station, Texas A&M University, College Station, Texas 77843-2132. OARDC and The Ohio State University, Columbus, OH 43210. 

Isozyme patterns of buffer-extractable cotyledonary proteins from 10 peanut Pretreatment (24 h at 28 C in the dark) of detached leaves of 

genotypes before and after infection with Aspergillus flavusor A. parasiticus were two isolines of the maize inbred W64A with an aqueous solution 

assayed electrophoretically using microprocessor-controlled native-PAGE (gradient (500 pg/ml) of sodium bisulf ite (NaHS0 3 ) increased electrolyte 

8-25%) and IEF-PAGE (pH 3-9). Aspergillus-inoculated and water control cotyledons leakage and peroxidase activity compared to controls. Also, the 

from viable kernels were incubated (dark, 32 C, 95% RH) and sampled every 6 hrs for Tms cytoplasm (susceptible) isoline of W64A treated with NaHSO 3 

3 days after inoculation. Non-inoculated cotyledons showed few inter-genotypic had significantly higher electrolyte leakage and peroxidase 

enzyme polymorphisms. Both fungi caused a rapid decrease of activity of alcohol activity than the comparably treated N cytoplasm (resistant) 

dehydrogenase and acid phosphatase, 12 and 24 hrs after inoculation, respectively. isoline. Moreover, after NaHSO 3 -treated leaves were inoculated 

After 24 hrs the fungi induced activity of alkaline phosphatase (not detected in water with BMT the NaHS0 3 -induced increase in sporulation was 

controls) and caused a rapid loss of activity of 6-P-gluconate dehydrogenase. with BMT the suceptible tn onlt i stas 

Variations in banding patterns and activities of malate dehydrogenase and isoline. Also trends in sporulation of BMT on amended agar 

glucose-6-P-dehydrogenase as well as increased activity of esterase were recorded isoli e sted siin rulation bHtwon aee agar 

30-72 hrs after inoculation with both fungi. Activities of catalase and P-glucosidase media suggested a positive relationship between the NaHSO 3 - 

were not changed. Minor inter-genotypic isozyme variations were detected in in maize leaves and the NaHS0 3 -enhanced sporulation. 

infected cotyledonary tissue. 

127 

DIVERSITY OF CUTINASES FROM FUNGAL PLANT PATHOGENS. EFFECTS OF XYLOSE ON PARTIALLY PURIFIED POLYPHENOLOXIDASE (PPO) 

Frances Trail and Wolfram K61ler, Department of Plant FROM BIPOLARIS MAYDIS. R. C. Evans and M. 0. Garraway. Biology 

Pathology, 

Experiment 

Cornell 

Station, 

University, 

Geneva, NY 

N. 

14456. 

Y. State Agricultural Dept., Rutgers Univ., Camden, NJ 08102 and Dept. of Plant Path.,. 

OARDC, The Ohio State University, Columbus, OH 43210 

Our study on the enzymatic diversity of cutinases from various 

pathogens has revealed a relationship between cutinase 

characteristics and organ specificity. Cutin hydrolysis by 

Cochliobolus heterostrophus , a leaf pathogen, is optimal at pH 

6.5, whereas cutin hydrolysis by Rhizoctonia solani, a stem- 

When Bipolaris maydis race T was incubated on a basal agar 

medium supplemented with xylose (CX medium), sporulation on 

to mycelia incubated on a control medium lacking xylose (cmedium). 

in0 was partially purified using fractional 

base pathogen, shows a pH-optimum of 9.5. pH-optima reported 

for cutinases isolated from leaf-infecting fungi Botrytis cinerea 

and Venturia inaequalis, and from stem-base pathogen Fusarium 

solani are in accordance with this relationship. Alternaria 

brassicicola and Colletotricum lindemuthianum, two pathogens 

that attack both stems and leaves, have cutinase pH optima at 

mecipitatio was ptially rified and fractionA 

precipitations with ethanol-chloroform and (NH4) 2d0 4 . A 

comparison of the (NH4)2So 4 fractions from C- and CX-grown 

mycelia indicated that the two sources of FF0 were similar in pH 

and buffer concentration optima, substrate specifity, response 

to inhibitors and electrophoretic mobility. Lineweaver-Burke 

both acidic and alkaline values. The cutinase activity of 

Alternaria an brassicicola ersnstoioyeoewith has been resolved each by pH chromatofocusing optimum. 

and represents two isozymes, one wbetween 

plots indicated that the FF0 from all fractions of GX mycelia 

was fractions non-competitively of C mycelia. inhibited Thus, the with difference respect to in PPO FPO from activity all 

G and GX mycelia may involve a xylose-mediated 

inactivation of PPO. 

128 

THE USE OF A STEROL INHIBITOR TO INVESTIGATE 

SUBSTRATE PARTITIONING IN THE ACETATE-MEVALONATE 

PATHWAY IN POTATO AFTER ELICITATION OF PHYTOALEXIN 

ACCUMULATION BY ARACHIDONIC ACID. M.N. Zook and 

J.A. Kuc, Department of Plant Pathology, 

University of Kentucky, KY 40546. 

132 

PLANT PARASITIC NEMATODES CONTAIN MULTIPLE ACETYL CHOLINESTERASE 

CLASSES. C.H. Opperman and S. Chang, Department of Plant 

Pathology, North Carolina State University, Raleigh, NC 27695. 

Multiple molecular forms of acetyl cholinesterase (AChE) are 

The 

in 

levels of 

potato tuber 

2, 3-oxidosqualene increaeed 

tissue 

ten-fold 

treated with ten micromolar 

UI66,an inhibitor of 2, 3-oxidosqualene cyclase, 

UT866sAecnetaino I66 niie 

stheri saleconeta tionl o acU166ationhi9%bited fd 

a 

steoideff yct oalaoi accumulation o byhii 90 authd 

nobieffelctinthedy accumlaioniof rihii n ) and 

lubiin rachdonc lictedby aid AA) Inclasses 

potato tuber tissue treated with UI8666A, 2, 3- 

common in many organisms. These forms often fall into discrete 

classes with similar biochemical parameters and responses to 

inhibitors. These classes, however, often differ markedly in 

these parameters. Experiments with Caenorhabditis elegans, 

Heterodera glcns Meloidogyne arenaria, and M. incognita have 

demonstrated that these nematodes all possess at least two 

classes of ACHE. There is a difference in distribution of 

between the Meloidogyne species and H. glycines which 

results in significant differences in in vitr-o enzyme 

oxiidoqaeed po 

nnelicitedptt 

accumulartisu scmaed toloe lvlsiAtisuber 

Tiesse aeuls comp ared to 

inhibition. Heterodera glycines AChE is less sensitive to 

carbamate nematicides than that of Meoioyne. The different 

non-lictedtisue.Thee rsule idicte hatkinetic 

AA alters the flow of substrate towards phytoalexin 

parameters of these classes may help explain the 

responses to inhibitors. 

synthesis and away from sterol synthesis. 

131 

129 133 

MECHANISM OF REDUCTION OF PHOTOSYNTHESIS BY VERTICILLIUM ACQUISITION OF GENETICALLY ENGINEERED PSEUDOMONAS STRAINS BY BEES 

DAHLIAE IN POTATO. R, L, Bowden 1 , 0. I. RouseJ ad T DURING FORAGING ON STRAWBERRY BLOSSOMS. T. V. Suslow. DNA Plant 

D. Sharkey , Dept. of Plant Pathology , Dept. of Botany , Technology, 6701 San Pablo Avenue, Oakland, CA 941608. 

University of Wisconsin, Madison, WI 53706. 

The potential for pollinator bees to acquire and vector applied 

The decrease in leaf net photosynthesis (A) of potato 'Russet biological control bacteria under field conditions was 

Burbank' infected with Verticillium dahlias was correlated quantitated for strawberry blossoms. Ice-minus deletion mutation 

with decreased stomatal conductance (C). The response of A to strains RGP36R2, Pseudomonas syringae, and GJP17BR2, _P. 

intercellular CO2 concentration (Ci) at saturating light fluorescens, were applied to strawberries and established 

showed that partial stomatal closure was responsible for populations on leaves and blossoms. Up to 160 individual bees 

reduced A. Errors in the Ci calculation caused by uneven were assayed on a given sampling date. On one date the relative 

Vol. 79, No 10, 1989Q 1151

abundance of pollen attached to a bee was recorded. Pollen 40-50 C compost was significantly reduced as compared to that 

concentration was not correlated to acquired Ice-minus population in media prepared with higher or lower temperature composts. 

densities. Bees acquired Ice-minus strains, non-nucleating Humicola isolates, unable to grow on PDA at 25 C, specifically 

bacteria, and Ice-plus bacteria from strawberry blossoms. The reduced efficacy of the biocontrol agent in paired biocontrol 

prevalence of strain GJP17BR2 on bees as compared to RGP36R2 was radish bioassays. Population development of T. hamatum 382 or 

inversely related to their relative population densities on of Rhizoctonia solani in media prepared with composts from 

blossoms from the same sampling period. Among the various temperatures did not differ. Results suggest that 

interpretations of this observation is the possibility that the compost process temperature impacts performance of Trichodermatwo 

strains may occupy spatially distinct habitats on strawberry fortified composts. 

blossoms that are reflected in the foraging habits of bees. 

134 

138 

INDUCED SYSTEMIC RESISTANCE TO PERONOSPORA TABACINA IN 

TENNESSEE 86 TOBACCO AND TISSUE CULTURE REGENERANTS OF INDUCED 

PLANTS. E. M. Nuckles and J. Kuc. Department of Plant 

Pathology, University of Kentucky, Lexington, KY 40546. 

INCREASE OF SCLEROTINIA SCLEROTIORUM AND VERTICILLIUM DAHLIAE 

FOLLOWING CERTAIN FOLIAGE FUNGICIDE SPRAYS ON POTATO. Gene 0. 

Easton, Washington State University, Irrigated Agriculture 

Research and Extension Center, Prosser, WA 99350. 

The tobacco cultivar Tennessee 86 is resistant to tobacco etch 

virus and tobacco vein mottling virus but is highly susceptible 

to tobacco mosaic virus and to blue mold caused by f. 

tabacina. Stem injection with sporangiospores of P. tabacina 

induced systemic resistance to blue mold in Tn 86. Resistance 

was expressed as a reduction in number, size, and sporulation 

of lesions. Tissue culture regenerants of Tn 86 plants steminjected 

with P. tabacina were protected against blue mold 

compared to regenerants from plants stem-injected with water. 

Induction of systemic resistance may provide a technology for 

rapidly introducing resistance to plants resistant to one or 

more pathogens. 

135 

Fungicides were sprayed on potato foliage in plots infested 

with Colletotricum coccodes (Cc), Sclerotinia sclerotiorum 

(Ss), and Verticillium dahliae (Vd). Visual ratings of 

symptoms and laboratory propagule counts after culture from 

stems showed: 1) Sclerotinia stem rot was not different from 

the control in plots sprayed with vinclozolin and thiophanate 

methyl in 1986-1988, 2) Sclerotinia stem rot was 5- to 10-fold 

greater in 1987 plots sprayed with chlorothalonil or Bravo C/N 

and their combination and in 1988 plots sprayed with 

chlorothalonil or fentin hydroxide and their combination than 

the control, 3) significantly more visible microsclerotia and 

stem propagules of Vd were present in 1988 plots treated with 

chlorothalonil or fentin hydroxide than in control plots, and 

4) Cc was not reduced or enhanced by any spray treatment. 

Application of fungicides did not alter tuber yield. 

FIELD PERFORMANCE AND GREENHOUSE ASSAY OF FUNGI FOR BIOCONTROL 

OF RESIDUE-BORNE PYRENOPHORA TRITICI-REPENTIS. W. F. Pfender, 

W. Zhang, and A. Nus. Dept. of Plant Pathology, Kansas State 

University, Manhattan, KS 66506. 

Candidate biocontrol fungi (grown in bran/millet seed culture) 

were applied to field plots containing Pyrenophora-infested 

winter wheat straw, with the goal of reducing ascocarp (primary 

inoculum) production by the pathogen in the residue. 

Limonomyces reduced ascocarp production by 86% and by 60-80%, 

respectively, in two years of field tests. Among fungi tested 

in one year only, an unidentified fungus significantly reduced 

ascocarp production, Laetisaria gave inconsistent results, and 

several fungi were ineffective. To screen candidate biocontrol 

fungi which under 

Pyrenophora-infested controlled conditions, 

straws a 

are method 

inoculated was developed 

with test 

in 

fungi and placed on a greenhouse bench with intermittent 

wetting cycles. Test conditions (straw and inoculum types, 

wetting periods) have been adjusted to give results consistent 

with those in field teats with selected fungi. 

136 

139 

PROTECTION OF POTATO FROM RHIZOCTNIA-CANKER WITH BINUCLEATE 

RHIZOCTONIA-LIKE FUNGI. A. Escande and E. Echandi, Department 

of Plant Pathology, North Carolina State University, Raleigh, 

27695-7616. 

Fourteen isolates of binucleate Rhizoctonia-like fungi (BN 

wer t e d as of bincl te agent s f ungiot BURo 

were studied as potential biocontrol agents for protection of 

potato from Rhizoctonia-canker in greenhouse and potato fields 

naturally infested with Rhizoctonia solani (AG-3). Eight of the 

BNR reduced incidence and severity ofi reoctonia-canker by an 

average of 78 and 85%, respectively, in greenhouse experiments. 

In 

severity 

the field, 

of Rhizoctonia-canker six of the eight BNR 

by an 

reduced 

average incidence 

of 43 and 

and 

41%, 

respectively. In a field heavily infested with R. solani, 

selected BNR and the fungicide Ttps 2.5D (thiophanate methyl) 

were equally protective of potato from Rhizoctonia-canker. 

Cultivars 

Burbank, 

Atlantic, Irish 

and 

Cobbler, 

Superior 

Kennebec, Norchip, 

were 

Russet 

equally protected from Rhizoctoniacanker 

by selected BNR under field conditions. Isolates of BNR 

have potential as biocontrol agents for protection of potato 

from Rhizoctonia-canker. 

COMPATIBILITY OF SOME COMMONLY USED SOIL DRENCH FUNGICIDES AND 

INSECTICIDES WITH THE BIOCONTROL AGENT GLIOCLADIUM VIRENS. 

J. C. Locke and R. D. Lumsden, Florist and Nursery Crops Lab. 

and Biocontrol of Plant Diseases Lab., Plant Sciences Institute, 

USDA-ARS, Beltsville, MD 20705. 

The interaction of the biocontrol agent Gliocladium virens with 

fungicides and insecticides, which can be used as soil drenches 

in bedding plant production systems, was investigated. The 

pesticides tested included: Aliette 8OW, Banrot 40WP, Benlate 

50W, Subdue 2E, Terraclor 75W, Truban 25EC, Diazinon AG4E, and 

Vydate L. The pesticides were evaluated for their effect on 

both proliferation of G. virens and the degree of damping-off 

control achieved against Pythium ultimum and Rhizoctonia solani 

on zinnia seedlings. None of the pesticides evaluated, except 

Benlate applied prior to introduction of the biocontrol agent, 

altered proliferation of the biocontrol agent in a soilless 

growing medium. Similarly, none of the pesticides decreased 

nor increased efficacy against either Pythium or Rhizoctonia. 

These results demonstrate the compatibility of this biocontrol 

agent with these pesticides at their labelled application rate. 

137 

INTERACTIONS BETWEEN TRICHoDERMA HAMATUM AND THERMOPHILIC FUNGI 

IN BARK COMPOST IN SUPPRESSION OF RHIZOCTONIA DAMPING-OFF. Y. 

R. Chung and H. A. J. Hoitink, Dept. of Plant Pathology, Ohio 

State Univ., Wooster, OH 44691. 

141 

EFFECTS OF DE-W TEMPERATURE, DEW PERIOD, AND REPEATED 

A white zone of microbial growth typically is present in 

compost piles where process temperatures range from 40-50 C. 

Humicola spp. were the predominant fungal taxa isolated from 

this zone. The ability of Trichoderma hamatum 382 to induce 

suppression to Rhizoctonia damping-off in media prepared with 

INOCULATIONS WITH PUCCINIA JACEAE ON YELLOW STARTHISTLE. A. 

R. BENNETT and W. L. BRUCKART, USDA-ARS, Ft. Detrick, Bldg. 

1301, Frederick, MD 21701. 

Puccinia jaceae was evaluated for biological control of yellow 

starthistle (YST, Centaurea solatitialis) in greenhouse 

1152 PHYTOPATHO LOGY

studies. YST plants were uniformly inoculated with uredinio- resulting from biotic activity, was assayed by comparing 

spores 4 wk after planting and placed in dew chambers for 4, survival of Pratylenchus penetrans (PP) in raw (R) and sterile 

8, 12, or 16 hr at temperatures ranging from 10 to 30 C. (S) soils. PP were added to 100-g samples of orchard soil 

Disease severity (number of pustules) was evaluated 2 wk after (cherty silt loam) and incubated at 25 C. Percent recovery of 

inoculation, and the most pustules (1.6/cm 2 leaf area) viable PP was lower (P=0.05) in R than in S soils after 11 

developed after incubation at 20 C for 12 or 16 hr. No days of incubation. This difference in percent recovery was 

infection occurred at 10 or 30 C. Inoculation of YST plants greater at 20 than at either 14 or 30% soil moisture, and 

up to four times on a weekly basis beginning 4 wk after increased as initial inoculum was increased beyond 200 PP. 

planting resulted in mean shoot biomass values of 0.88, 0.60, This assay was used to evaluate NBA stimuiation by the 

0.52, 0.46, and 0.41 g for 0, 1, 2, 3, and 4 inoculations, nematicide Clandosan. Mean percent recovery was significantly 

respectively. Under suitable conditions, significant lower in R amended with 22-g of Clandosan per L (0%) than in 

reduction of YST biomass can occur from infection by P. jaceae. unamended R (17%), and, in Clandosan-amended S (16%) than in 

unamended S (33%). The results of this assay are inconsistent 

with an enhanced NBA mode of action for Clandosan. 

146 

PREVENTION OF AFLATOXIN CONTAMINATION WITH STRAINS OF 

ASPERGILLUS FLAVUS. P. J. Cotty, USDA, ARS, Southern Regional 

Research Center, P.O. Box 19687, New Orleans, LA 70179. 

Cottonseed can be infected by strains of Aspergillus flavus 

Link that do not produce aflatoxins. Furthermore, virulence of 

the fungus is not correlated with aflatoxin production. These 

observations suggest that strains of A. flavus which do not 

produce aflatoxins may exclude other strains from crops and 

prevent aflatoxin contamination. Greenhouse studies confirmed 

that non-toxigenic strains of A. flavus can prevent aflatoxin 

contamination of cottonseed by toxigenic strains. When 

simulated exit holes of the pink bollworm were inoculated with 

a strain which did not contaminate cottonseed with aflatoxins, 

subsequent contamination of developing cottonseed by two 

toxigenic strains was prevented. Inoculation with toxigenic 

and non-toxigenic strains simultaneously resulted in at least 

100 fold reductions in toxin contamination. Non-toxigenic 

strains may be useful as protective agents in all susceptible 

crops. 

143 

TRICHODERMA HARZIANUM USED IN INTEGRATED CONTROL OF BOTRYTIS 147 

CINEREA ROT ON APPLE. Arne Tronsmo. Department of Microbiology. POSTHARVEST BIOCONTROL OF GRAY MOLD OF PEAR BY PSEUDOMONAS 

Agricultural University of Norway. N-1432 Aas-NLH. Norway. GLADIOLI. G. H. Mao and R. A. Cappellini, Department of Plant 

By the use of the fungal antagonist Trichoderma harzianum we Pathology, Rutgers University, New Brunswick, NJ 08903. 

have been able to reduce Dry Eye Rot (Botrytis cinerea) on Pseudomonas gladioli and its cell-free filtrate (CFF) inhibitapple. 

However, we are not able to perform biological control 

of all other diseases and pests in commercial fruit growing, 

ed growth of Botrytis cinerea in vitro. The bacterium was 

tested in vivo for biocontrol of gray mold of Anjou pears. 

Integrated control with fungicide resistant antagonists is 

therefore of interest. By testing several Trichoderma spp. 

isolates for fungicide resistance, we were able to select 

isolates with much higher tolerange to Carboximid and other 

fungicides than the parent strain. One isolate, Trichoderma 

harzianum 220 were able to control Dry Eye Rot in a field with 

commercial spray program against other pest and diseases, both 

alone and together with reduced dosage of Vinclozolin. Even 

ifinerae cnro it . azinm n Vnlooinws o 

if integrated control with T. harzianum and Vinclozolin was not 

significantly more effective than biological control on its 

own., the combination of two different control methods will 

probably give safer control, and less danger of resistance 

development in the pathogen. 

Bacterial suspensions were applied to wounded pe rs. ýhe 

pears were then inoculated with B. cinerea (5x10 -5x10 /ml) 1 

hr later and incubated for 7-9 days in a moist ghamber at 

23-25 C. P. gladioli at concentrations of 1xl0 ) colony-form- 

ing units/rl (CFU/ml) retarded disesse development on pesrs 

challenged with B. cinerea spor e suspensions. When the 

antagonist was appliedat Ixi0 CFU/ml, the pathogen did not 

produce lis onpears. Contr l of g ay hold was not 

produce lesions on pears. Control of gray mold was not 

obtained on wounded pears applied with CFF at concentrations 

of 20-80 units/ml at the same pathogen inoculum levels. 

144 

BIOLOGICAL AND CULTURAL MANAGEMENT OF ROOT INFECTING FUNGI. Brad 

Melvin, Joe Vargas, Jr., Lee Berndt and Ron Detweiler. Dept. of 

Botany and Plant Pathology, Michigan State University, East 

Lansing, Michigan. 45823. 

Management of necrotic ring spot, a disease caused by an 

ectotrophic root infecting fungus (Leotosohaeria korrae), was 

obtained on Poa pratens~is receiving daily irrigation and/or bioorganic 

fertilizer treatments. Highly significant reduction of 

necrotic ring spot was achieved with a 2.5mm per day irrigation 

treatment compared to an 80% evapo-pan irrigation treatment or no 

supplemental irrigation treatment. Significantly less disease 

incidence occurred with the bio-organic fertilizers, Turf Restore 

and Sustane in the 80% evapo-pan irrigation treatment compared to 

the untreated control. A correlation between disease reduction and 

an increase in total thatch bacterial populations in daily 

irrigated plots during May and June was observed. The early season 

period of turf growth is the optimum time of year for infection of 

. Drtni by L- kore Total bacterial populations were higher 

throughout spring and early summer in the Turf Restore and Sustane 

plots compared to the untreated control. Many of the bacteria in 

these studies are antagonistic to I- kora in vitro. 

148 Withdrawn 

149 

145 CLONING OF THE BETA-TUBULIN GENE FROM BENOMYL-SENSITIVE AND 

A QUANTITATIVE ASSAY OF NEMATODE BIOCONTROL AGENT ACTIVITY AND BENOMYL-RESIsTANT FIELD STRAINS OF VENTURIA INAEQUALIS. 

ITS USE IN A CLANDOSAN AMENDED SOIL. J. C. Doney, Jr. and J. Harrie Koenraadt, S.C. Somerville and A.L. Jones, Dept. of 

B. Kotcon. Div. of Plant and Soil Sciences, West Virginia Botany and Plant Pathology and the Pesticide Research Center, 

univ., p. 0. Box 6057, Morgantown, WV 26506-6057. Michigan State University, East Lansing, MI 48824. 

Nematode biocontrol agent (NBA) activity, defined as mortality Widely differing levels of benomyl-resistance in Venturia 

Vol. 79, No. 10, 1989 1153

inaegualis has been attributed to allelic mutations in the tions were made from buds of cv. Red Haven, Crest Haven, 

beta-tubulin gene. To study this phenomenon at the molecular Correll and Sentinel bi-weekly from May 27 through September 

level, genomic DNA was isolated from 6-wk-old broth cultures 26, 1988. Buds were surface sterilized in 0.05% sodium hypoof 

a benomyl-sensitive (WC-S) and a benomyl-resistant (KV3C) chlorite in 10% ethanol, and plated on acid potato dextrose 

field isolate of V. inaequalis and partially digested with agar. Elevated isolation frequencies from 4% to 32% were 

the restriction enzyme Sau3A. Sucrose gradient fractionated recorded immediately after harvest for each cultivar. Cankers 

DNA (16-20 kb) and BamHI/EcoRI digested lambda EMBL3 DNA develop on bearing wood under the buds from December to 

were ligated with T4 DNA ligase and packaged to prepare a February. Twigs die immediately after bloom. Benomyl sprays 

library. The library was screened for clones with a hetero- (1 lb/A a.i.) on August 1 and September 1 reduced the 

logous Erisyphe graminis beta-tubulin probe. DNA sequence incidence of twig cankers in 1989 from 48% to 8%. 

analysis of the clones showed extensive sequence similarities 

with the probe thereby confirming that the beta-tubulin gene 

had been cloned. 154 

ISOLATION, PURIFICATION AND CHARACTERIZATION OF A PHYTOTOXIN 

15 ~*A.M. FROM LIQUID CULTURES OF LEUCOSTOMA PERSOONII AND L. CINCTA. 

Svircev, °A.R. Biggs, *N. Miles and *C. Chong. 

OVERWINTER SURVIVAL IN THE FIELD OF COLLETOTRICHUM *Horticultural 

ACUTATUM ON 

Research Institute 0 Ontario, Canada, 

of Ontario, 

LOR 2E0; 

Vineland 

Agriculture 

Station, 

STRAWBERRY 

Canada Research 

FRUIT IN OHIO. 

Station, 

L. L. Wilson, M. A. Ellis, and L. V. 

Madden, Dept. of Plant Pathology, Ohio State Univ., Wooster, OH Vineland 

44691. 

Station, Ontario, Canada, LOR 2E0. 

Liquid cultures of L. persoonii and L. cincta were grown in 2% 

Survival of Colletotrichum acutatum on infected strawberry fruit malt extract medium on a rotary shaker. Fourteen-day-old 

was evaluated in the was he field. valate iel. Infected Ifectd in fruit fuitwer were seaed sealed in n nylon nlon cultures separated were by ultrafiltration filtered and the into cell-free specific culture molecular filtrate size 

mesh bags (5 

was 

fruit/bag) and placed on and 5-8 cm beneath the soil fractions. 

surface 

Excised 

in Nov 

ltato intspefrsica 

1988. Both treatments 

shoot 

were 

tips 

covered 

(ca.i 

stra much.At nteval 1mo statin inDec with 8 cm of fr actin 

straw 

988onebag 

mulch. in wexcsed peah, 

At P 1-mo 

length) were used to test the runu crude p intervals, starting 

rica, fractions s 

in Dec 

oo 

1988, 

for tip 

one 

toxin 

bag activity. Peach 

from 

canker-like 

each of 

symptoms 

three replications 

were induced 

was 

only 

removed 

by the 

from on and beneath < 1,000 dalton 

the soil 

fraction. 

surface 

Further 

and fruit 

purification 

were assayed 

of the 

for 

phyto- 

viable C. acutatum. toxin by isoelectric 

From Nov 

focusing 

to Mar 

and 

1989, 

granulated 

ambient 

bed 

air 

electrotemperatures 

(above straw phoresis 

mulch) 

has 

ranged 

identified 

from 

the 

-18 to 

toxin 

20 C, 

as 

and 

a small 

soil temperatures 

polypeptide. 

ranged from Further purification and identification of the 

-3 to 

phytotoxin 

13.2 C. 

is 

C. 

in 

acutatum was recovered 

fruit 

from 100% 

from 

and 

on 

>95% 

and within 

of 

soil, respectively, for the first 3 mo. progress. tissue culture The program toxin is to being obtain tested peach as plants selective with agent an increased 

After 4 mo the fungus was recovered 

in the 

from 80 and 67% of the fruit resistance to Leucostoma spp. 

from on and within soil, respectively. Sampling will continue 

through May 1989. Results will be discussed. 

151 CYTOCHEMICAL PROCEDURES FOR ILLUSTRATING THE RESPONSE OF PECAN 

TO INFECTION BY CLADOSPORIUM CARYIGENUM. S. V. Diehl, C. H. 

CONTROL OF CYTOSPORA CANKER AND BACTERIAL CANKER IN A YOUNG Graves, and P. A. Hedin. Dept. of Plant Path. & Weed Sci. and 

SWEET CHERRY ORCHARD IN OREGON. LA. Spotts, T.J. Facteau, Crop Sci. Res. Lab, USDA, Mississippi State, MS 39762, 

and L.A. Cervantes. Oreg. St. Univ., Mid-Columbia Agric 

Research and Extension Center. Hood River, OR 97031. Several fungitoxic phenolics affect resistance in pecan to infection 

by C. caryigenum. Transmission electron microscopy can 

field study to evaluate control of Cytospora canker was be used to Tocate phenolics within leaf vacuoles. Distribution 

initiated in 1981 by planting sweet cherry trees cv. Bing. of phenolics within vacuoles varied depending upon the fixation 

Treatments included white trunk paint. 3 levels of nitrogen, procedure used. Localization and quantification of juglone, 

application of benomyl (1.35g/1) after dormant pruning or at isoquercitrin and condensed tannins in fresh microscopic tissues 

popcorn, petal fall, and shuck split. Trees were evaluated can be accomplished with the use of the Hoepfner-Vorstatz stain 

annually from 1982 to 1986 for active trunk cankers, and iso- and butanol-HCl and a microspectrophotometer. This technique 

lations made from margins of cankers. Between 1 and 7% of the showed that juglone was found consistently in higher concentratrees 

were infected with C. cincta each year. and 26% were tions in all greenhouse seedling leaf tissue than the other two 

infected by 1986. Bacterial canker, caused by Pseudomonas sy- compounds. Both infected and noninfected leaf tissue can be 

ringae, occurred in 13% of the trees in 1982 and 25% by 1986. compared with scanning electron microscopy. Combined use of 

Death of trees infected with C. cincta and P. syringae was 14 these procedures should give a composite illustration of host 

and 26%, respectively. Nitrogen or benomyl did not reduce response to infection by C. caryigenum. 

incidence of cankers. White trunk paint reduced the incidence r 

of both Cytospora and bacterial trunk cankers. Disease incidence 

was highest in trees close to an old cherry orchard. 156 

DIFFERENTIATION OF COLLETOTRICHUM SPP. PATHOGENIC TO STRAW- 

152 BERRY. P. S. Gunnell and W. D. Gubler, 

Pathology, 

Department 

University 

of Plant 

of California, Davis, CA 95616. 

PATHOGENICITY OF PHIALOPHORA SP. AND RHIZOCTONIA-LIKE 

ON CRANBERRY (VACCINIUM MACROCARPON). 

FUNGI 

Varney, and J. L. Peterson. 

L. P. Cn E.H. 

Plant 

The morphology 

Pathology, 

of both conidia and setae 

Rutgers 

produced on strawberry 

leaf 

University, 

piece agar 

New 

were found to 

Brunswick, 

be reliable 

NJ 

criteria 

08903. to 

distinguish Colletotrichum spp. pathogenic to strawberry. von 

Cranberry cuttings 

Arx 

inoculated 

proposedthaft 

with a high 

Cfragariae 

level of 

Brooks 

Phialophora 

was synonomnous 

gloeosporioides 

with C. 

developed 

Penz., however, 

severe 

conidia 

symptoms, 

of C. fragariae 

including leaf 

isoyellowing, 

lates, 

defoliation, 

including specimens 

desiccation, 

collected 

and 

by 

root 

Brooks, 

necrosis. Symptoms 

were 

were 

pre- 

dominantly clavate whereas 

significantly conidia 

milder of C. gloeosporioides 

at low inoculum were 

levels. Controls were predominantly cylindrical 

symptomless. 

with rounded-ends. 

There was a significant 

Conidia of 

difference 

C. 

in fresh fragariae 

weight 

were also 

and root 

longer 

length 

and narrower 

between 

than 

Phialophora-inoculated 

those of C.- 

and gloeosporioides. 

control Setae 

cuttings. 

of C. fragariae 

Rhizoctonia-like were brown, 

fungi 

several 

isolated from septate, somewhat 

cranberry 

sinuous, 

roots 

n-ot 

and 

tapered, 

shoots 

and 

had 

usually 

binucleate 

produced 

hyphae. Three conidia when mature. 

selected 

Setae 

isolates 

of C. 

(Rh-l, 


Rh-2, and Rh-3) 

although 

had different modes also brown and septate, 

of infection. 

were strongly 

Rh-i penetrated 

tapered, did 

and 

not 

colonized 

produce 

the epidermal and conidia, and 

cortical 

were 

cells. 

finely warted 

Rh-2 penetrated 

toward the 

and 

top. 

produced 

C. acutatum 

typical hyphae Simmonds produced fusiform 

and 

spores 

monilioid and 

cells 

comparativel-y 

in the 

short, 

epidermis. dark 

Rh-3 did not penetrate brown, thick-walled setae which were usually aseptate. 

the epidermal cells, but colonized the root surface. 

153 157 

PHOMOPSIS BUD AND TWIG BLIGHT OF PEACH. F. F. Hendrix, Jr., RELATIONSHIP OF TEMIPERATURE TO THE FUNGI INVOLVED 

Department of Plant Pathology, University of Georgia, Athens, IN CRANBERRY FRUIT ROT. F.L. Caruso, Cranberry 

GA 30602. 

Experinent Station, University of Massachusetts, 

East Warehamn, 

Phomopsis 

MA 

twig 

02538. 

blight of 

been 

peach, 

a major 

caused 

problem 

by Phomopsis 

in North 

sp., 

Georgia 

has 

for the last 5 years. More than 

The 

ten 

fungus 

different 

infects 

fungi 

through 

are 

healthy 

capable 

buds 

of 

after 

causing 

harvest. Isola- field or storage rot in cranberry fruit. Cranberriem 


155

(cultivars 'Early Black' and 'Crowley') with diverse caused by Penicillium funiculosum is not economically feasible, possibly 

rot symptoms were sampled in July, August, and Sep- because FCR can also be caused by Fusarium moniliforme f. sp. subglutinans. 

tenber (field collection) ,and in October and January Application of acaricides only when triggered by suitable 'predictors' has been 

(while in storage) . Individual berries were cut into proposed to reduce chemical application. The accuracy of prediction for any 

thirds, surface-sterilized, plated on three ACMA area will be limited by the natural variation of disease and the 'predictor' 

plates. Plates were incubated at 15, 22, and 30 C within this area. A survey was made of 10 fields to examine the correlation of 

for three weeks. Seven additional cultivars were pre- and post-force mite and fungal populations (P. funiculosum and F. 

sampled in February. Proper diagnosis of the primary moniliforme f. sp subglutinans) to FCR incidence at harvest. The use of entire 

causal agent is dependent on incubation temperature. fields as the statistical unit showed a correlation (r) between pre-force mite 

Godronia, Sporonema, and Apostrasseria preferred 

populations and FCR of 0.82. Due to variation within these fields, however, 

cooler temperatures whereas Phyllosticta, Physalo- 

the correlation dropped to 0.42 when the 60 subplots (6 within each field) were 

spora and Phomopsis preferred warmer temperatures. 

analyzed. Fungal isolations pre- and post-force showed high levels of F. 

There were diverse differences in fungi isolated at moniliforme f. sp. subglutinans but this was not statistically significant as a 

different sampling times among different cultivars. 

disease predictor. 

158 

POWDERY MILDEW OF PECAN: VARIETAL SUSCEPTIBILITY AND ROOT ROT OF KIWIFRUIT VINES CAUSED BY PYTHIUM SPP. 

EFFECTS ON KERNEL DEVELOPMENT. T. B. Brenneman and P. F. A. J. Latham, W. A. Dozier, Jr., J. M. Mullen, and H. L. 

Bertrand, Coastal Plain Experiment Station and Rural Campbell. Ala. Agric. Exp. Stn., Auburn University, AL 36849. 

Development Center, respectively, Dept. of Plant Pathology, 

University of Georgia, Tifton, GA 31793. Plantings of kiwifruit (Actinidia chinensis) have been 

made in several southeastern states. A planting on land 

Studies were conducted on powdery mildew (Microsphaera previously in long-time peanut production sustained over 50% 

penicillata) of pecan to monitor disease progress and loss to root rot following an extended wet period during 

determine its effects on kernel development. A total of 716 June. Pythium spp. were consistently obtained from rotted 

nuts (cv. Woodard) were rated six times during the season roots. One isolate cultured on corn meal agar at 25 C 

between July 10 and September 17, 1987. Severe powdery developed oogonia 23.4 um diam typical of Pythium ultimum 

mildew developed, and disease levels at the third through var. ultimum. Eight isolates cultured in the dark on a blue 

last evaluations were significantly negatively correlated grass-water medium at 22 C produced oogonia ranging from 

with kernel weight. The percent kernel weight was not 21.7 to 24.0 um typical of Pythium ultimum var. 

correlated with disease severity. Within a cluster, the sporangiiferum. Pure cultures were increased on corn 

further a nut was from the terminus, the less powdery mildew meal-sand mixtures and used to inoculate potted kiwi plants. 

it had. An additional study monitored disease progress on Within six weeks, both species caused root decay similar to 

14 pecan cultivars. Woodard was extremely susceptible but that observed in the field. 

the other cultivars had moderate to good resistance. 

159 

163 

REGULATION OF PECTIN LYASE (PNL) PRODUCTION IN ERWINIA CAROTOVORA 

EFFECT OF FLOOD DURATION ON SEVERITY OF PHYTOPHTHORA ROOT AND SUBSP. CAROTOVORA (Ecc) STRAIN 71 BY DNA DAMAGING AGENTS: 

CROWN ROT OF KIWIFRUIT IN CALIFORNIA. K. E. Conn and W. D. ISOLATION OF A NONINDUCIBLE MUTANT USING A pnlA-Ia:Z 

Gubler. Department of Plant Pathology, University of California 

Davis, CA 95616. 

TRANSCRIPTIONAL FUSION. James L. McEvoy. Russell 0. Nordeen. and 

Arun K. Chatterjee. Department of Plant Pathology, University of 

Missouri-Columbia, Columbia, MO 65211, U. S. A. 

Six month old kiwifruit seedlings grown for 3 months in soil artificially 

infested with Phytophthora citrophthora, P. crypto- To analyze the regulation of PNL production we cloned the 

gea, P. megasperma, or one of two unidentified Phytophthora app., structural gene, pnlA, and constructed pnlA-lacZ fusions using 

were subjected to biweekly flooding periods of 0, 6, 12, 24, or the mini-Mu element, P011734. In six of eleven insertions, lacZ 

48 hours. Root rot (RR) and crown rot (CR) caused by P. crypto- expression was controlled by the pnlA promoter. The induction of 

gea and an unidentified Phytophthora sp. increased with the B-galactosidase (B-gal) by mitomycin C (MC) occurred in RecA but 

length of the flooding period. With 48 hours flooding, consis- not in RecA- Ecc strains. A typical fusion was placed by marker 

tently severe RR (83-96%) was observed. In contrast, P. citro- exchange into the chromosome of a Lac- derivative of Ecc7l. In 

phthora, P. megasperma, and another unidentified Phytophthora this construct (AC5022), B-gal was induced 60-fold with MC. By 

sp. caused variable RR (13-80%) with 0 to 48 hours flooding EMS mutagenesis of AC5022 followed by screening for B-gal 

while CR developed only sporadically on seedlings flooded for production, we obtained a mutant (AC5023) that was noninducible 

48 hours. These results indicate that soil-water management that by MC. AC5023 was not defective in RecA nor was it. complemented 

avoids prolonged and repeated saturation may minimize losses by a PnlA' plasmid. These data suggest that in AC5023 there is a 

due to Phytophthora, depending on the species present. defect in 

production. 

pnlR, a gene locus that positively regulates PNL 

162 

164 

161 165 

CLONING OF eep GENES THAT DETERMINE EXTRACELLULAR ENZYME 

PRODUCTION IN ERWINIA CHRYSANTHEMI (EC16) AND E. CAROTOVORA 

SUBSP. CAROTOVORA (ECC71). H. Murata. W. Chun, and A. K. 

Chatterjee. Dept. of Plant Pathology. Univ. of Missouri, 

Columbia, MO 65211, U.S.A. 

During the isolation of pectinase-deficient mutants, we obtained 

Eep- mutants of ECI6 and Ecc71 that produced very low levels of 

pectate lyase, polygalacturonase, cellulase and protease 

activities. The ECI6 mutant also was mucoid and produced lower 

levels of phospholipase C. The Eep- mutants did nlot macerate 

potato tuber tissue. By mobilizing EC16 and Ecc71 gene 

libraries, we isolated clones that restored production of these 

enzymes and tissue-macerating ability in the cognate mutants. The 

Eep÷ plasmids did not restore extracellular enzyme production in 

export deficient (Out-~) mutants of EC16 or Ecc7l. Our data 

reveal the presence of a gene(s), other than out, that 

pleiotropically affects the production of extracellular proteins 

in these bacteria. 

VARIATION IN PREDICTION OF FRUITLET CORE ROT WITHIN AND 

BETWEEN PINEAPPLE FIELDS. JE, Yuen and G.Y. Taniguchi, Department 

of Plant Pathology, 3190 Maile Way, Honolulu, HI 96822. 

EVIDENCE FOR CATECHOL SIDEROPHORE PRODUCTION BY ERWINIA 

CAROTOVORA SUBSP. CAROTOVORA. C .T. Bull', C .A. Ishimaru', and 

J .E. Loper 2 . 'Department of Botany and Plant Pathology, Oregon 

2 

State University, USDA-ARS, HCRL, Corvallis, OR 97330. 

Routine application of acaricides for control of fruitlet core rot (FCR) and 

other fruit diseases (interfruitlet corking, eye inhibition, and leathery pocket) Erwinia carotovora subsp. carotovora strain W3C105, which 

Vol. 79, No. 10, 1989 1155

causes soft rot disease of potatoes, was investigated for 169 

production of a catechol siderophore. Catechol production was 

detected colorimetrically, by bioassay, and by homology to THE HYPERSENSITIVE RESPONSE 

genes 

IS 

determining 

ELICITED BY ESCHERICHIA 

biosynthesis 

COLI 

of enterochelin, the catechol CONTAINING A CLUSTER OF PATHOGENICITY 

siderophore 

GENES 

of 

FROM 

Escherichia 

ERWINIA 

coli. Results suggested that strain AMYLOVORA. S. V. 

W3C105 

BEER; 

produces 

C. H. ZUMOFF; 

a catzechol 

D. W. 

siderophore 

BAUER; B. J. 

with 

SNEATH; 

functional and R. J. LABY. Department of Plant Pathology, 

similarity 

Cornell 

to enterochelin. Genes involved in catechol University, Ithaca, NY 14853 U.S.A. 

production were identified from a genomic library of strain 

W3C105 in E. coli strain AN192, which is deficient in Hrp mutants of E. amylovora 

enterochelin 

are deficient 

production. 

in 

Three 

both pathogenicity 

clones producing a catechol to pear fruit and the ability to 

were 

elicit 

detected 

the hypersensitive 

on a universal siderophore-detection medium, response in tobacco. 

These 

A cosmid, 

clones 

pCPP430, 

provided 

containing 

iron to enterochelin-deficient 

wild-type 

indicator DNA of E. amylovora, was 

strains. 

identified 

The 

that 

role 

restores 

of catechol 

pathogenicity 

siderophore production in the and HR-eliciting ability to 18 transposon-induced 

pathogenicity and 

Hrpecology 

and two 

of E. carotovora will be evaluated, naturally occurring Hrp- mutants. pCPP430 contains a cluster of 

hrp genes, dispersed throughout a 45 kb region of chromosomal 

DNA. When Escherichia coli, strain DH5, containing pCPP430, was 

infiltrated into tobacco-leaf tissue, strong collapse occurred 

extremely rapidly. In addition, the cosmid conferred 

166 HR-eliciting ability (in tobacco), to several other species of 

CHARACTERIZATION OF AN rcsA-LIKE GENE OF 

Erwinia. 

ERWINIA 

Theseresults 

AMYLOVORA THAI 

clearly indicate 

all the 

that 

genes 

pCPP430 

needed 

contains 

for elicitation 

STIMULATES 

of 

EXTRACELLULAR 

the HR, and that 

POLYSACCHARIDE 

they 

(EPS) PRODUCTION IN are expressed in E. coli and in other Erwinia species. 

ERWINIA SPP. AND OTHER ENTEROBACTERIA. W. Chun, A. Chatterjee. 

R. N. Goodman and A. K. Chatterjee, Department of Plant 

Pathology, University of Missouri-Columbia, Columbia, MO 65211. 170 

EPS production by E. amylovora is required in the elicitation of CHARACTERIZATION OF PROMOTER-ACTIVE FRAGMENTS FROM 

the fire-blight disease in apples and pears. To examine the XANTHOMONAS USING A NEW BROAD HOST RANGE PROMOTER 

regulation of EPS biosynthesis, we obtained from an E. amylovora SELECTION VECTOR. S. Swarup, R. DeFeyter and D.W. Gabriel. 

cosmid library, several E. coli (HB1OI) clones that were mucoid Plant Pathology Dept., University of Florida, Gainesville, FL 32611. 

on agar plates. The complementation of an rcsA mutation in E. 

coli and the stimulation of EPS production in various A broad host-range (Inc Q) promoter selection vector, pUFC600, was 

enterobacteria (E. amylovora, E. stewartii, E. coli, and 

Salmonella typhimurium) associate the mucoid phenotype conferred constructed that enables a direct selection of promoter-active fragments. 

by the cloned DNA to an E. amylovora regulator gene. This pUFC600 is 9.4 kb in size, Knr, has a promoterless Smr gene, multiple 

rcsA-like gene along with its own promoter has been localized on cloning sites and tandem transcriptional terminators upstream of the cloning 

a-2.2 kb DNA segment. Nucleotide sequence homology determined by sites. In the absence of any promoter-active fragment, E. coil strain DH5x 

Southern hybridizations and functional complementation of the and Xanthomonas strains containing the plasmid were sensitive to 

mucoid phenotype by the cloned DNA indicate that the genes for streptomycin at 15 pg/ml in minimal and 25 pg/ml in complete media. A 

the regulation of EPS biosynthesis have been conserved in these genomic library (-4 kb insert size) of X. citri strain 3213 was constructed 

enterobacteria. in pUFC600. Clones were introduced into X.c. pv. citrumelo strain 3048 Spr 

at an average frequency of 10-4 per recipient. Promoter activity was 

selected or screened on both minimal and complete media with 

streptomycin and various classes of promoter active fragments were 

identified. The relationship of pathogenicity genes to promoter-active 

167 fragments which were induced on minimal media will be discussed. 

CLONING OF AN AVIRULENCE GENE FROM PSEUDOMONAS SOLANACEARUI 

STRAIN AWl AND ITS INVOLVEMENT IN HOST RANGE. B. F. Carney and 

T. P. Denny, Dept. of Plant Pathology, UGA, Athens, GA 30602. 171 

A locus responsible for the hypersensitive response (HR) in 

P. solanacearum strain AWl (avirulent on tobacco, but pathogenic 

on tomato) was cloned by complementation in P. solanacearum 

strain K601 (pathogenic on tobacco and tomato). Pseudomonas 

solanacearum K601 transconjugants [ K601(pBC73) and K601(pBC62) 

] were nonpathogenic on tobacco, suggesting that cloned locus had 

the potential to restrict the host range of K601. DNA analysis 

of the clones pBC73 and pBC62 indicated that a common 4.2 Kb 

EcoRI/BamHI fragment was responsible for the induction of HR in 

K601 transconjugants. Transposon mutagenesis of the wild type 

locus in strain AWl resulted in the loss of HR on tobacco but 

retention of pathogenicity on tomato, indicating that this locus 

contains an avirulence gene. Pathogenicity on tobacco was not 

acquired upon inactivation of this avirulence gene in AWl, 

suggesting that AWl lacks positive acting host range genes that 

are required for pathogenicity on tobacco, 

168 

1682 

CLONING OF TWO GENES FOR PRODUCTION OF 

POLYSACCHARIDE 

EXTRAC ELLULAR 

FROM PSEUDOMONAS SOLANACEARUM AND THEIR 

CONTRIBUTION TO VIRULENCE. S. -R. Baek and T. P. Denny. Dept. 

of Plant Pathology, University of Georgia, Athens, GA 30602. 

Tn5-induced mutations of Xanthomonas campestris pv. citrumelo 

affecting pathogenicity and host-species specificity. . 

Kingsley and D.W. Gabriel. University of Florida, Gainesville, 

Florida 32611 

Tn5-induced mutations affecting pathogenicity (PATH-) and host 

species specificity (HSS-) were recovered in X. campestris pv. 

citrumelo. All 3048::Tn5 exconjugants (including auxotrophs) 

were screened on both bean and citrus. Auxotrophic mutations 

had significant effects in planta. For example, uracil 

auxotrophy resulted in a path- phenotype, while isoleucine- 

valine auxotrophs were hss- in bean but were relatively 

unaffected in citrus. None of the Tn5-inserts affecting 

pathogenicity or host range appeared to be clustered by 

hybridization analyses, nor did the affected DNA regions 

hybridize with the P. solanacearum hrp cluster (ie. pVir2). A 

high level of polymorphism was observed between, but not 

within 12 species or pathovars of Xanthomonas probed with 

HSS-related DNA fragments, which may indicate a lack of 

conservation of the gene(s) present at the affected loci. 

17 

MOLECULAR CHARACTERIZATION OF A LOCUS REGULATING PRODUCTION OF 

EXTRACELLULAR POLYSACCHARIDE SLIME AND VIRULENCE IN PSEUDOMONAS 

SOLANACEARUM. S. M. Brumbley and T. P. Denny, The University of 

Previous research had marked two loci of P. solanacearum 

AWl that 

strain 

are involved in the production of extracellular polysaccharide 

(EPS) with Tn5 insertions. These Tn5 insertions and 

flanking DNA of P. solanacearum were cloned and used to locate 

eight cosmid clones that contained homologous sequences in a 

genomic library of P. solanacearum AWl. The cosmids were 

restriction mapped and found to contain unique and overlapping 

regions that spanned a total of 55 kilobases; the Tn5 insertions 

in the two loci were centrally located and 12.5 kb apart. The 

Tn5 inactivated genes in strains AWl-l and AWI-41 were 

designated epsA and epsB, respectively. Seven of the eight 

cosmids completely restored EPS production to strain AWI-41. 

Three cosmids partially restored EPS production to strain AWl-l 

and these transconjugants were more virulent on tomato than was 

strain AWl-l. These results support the idea that EPS has a 

major role in P. solanacearum causing wilt symptoms on tomato. 

Georgia, Athens, GA 30602. 

A well known phenomenon of P. solanacearum is the 

spontaneous mutation from a mucoid to a nonmucoid form. A 

variety of other traits, including virulence, are affected. A 

previously isolated Tn5 mutant (AWI-80) is identical to the 

naturally occurring spontaneous mutant AWl-A in every way tested. 

An EcoRI fragment containing the Tn5 plus flanking DNA from AWl- 

80 was cloned and used as a hybridization probe to identify two 

cosmids from a genomic library of the wild type P. solanacearum 

strain (AWl). These cosmids restored wild type traits when 

conjugated into AWI-80, AWl-A and several other spontaneous 

avirulent mutants strains of P. solanacearum. These results 

suggest that this locus (designated rpc) contains a regulatory 

element(s) with global functions. This system does not appear 

to be the same as that regulating the production of alginate in 

Pseudomonas aeruginosa. 

1156 PHYTOPATHOLOGY

173 177 

THE EFFECT OF EXPRESSION OF AGROBACTERIUM RHIZOGENES ROLB AND IDENTIFICATION AND NUCLEOTIDE SEQUENCE OF THE COAT PROTEIN 

C GENES ON ROOT INDUCTION AND GROWTH. F. Shaheen and F. F. GENE OF POTATO LEAF ROLL VIRUS. 0. P. Smith, USDA-ARS, 

White, Dept. of Plant Pathology, Kansas State University, Frederick, MD 21701 and K. F. Harris, Dept. of Entomology, 

Manhattan 66506. Texas A&M University, College Station, TX 77843 

The Ri plasmid of A. rhizogenes contains a set of unique T- The open reading frame (OaF) for the coat protein of potato 

DNA genes (rolA, B, and C) which, upon expression in plant leaf roll virus (PLRV) has been sequenced and identified using 

cells, induce hairy root syndrome. Each gene induces cloned PLRV cDNA. Verification was obtained by the expression 

independent effects in transgenic plants, while their combined of a portion of this ORF as an Escherichia coli B-galactosidase 

effects direct the hairy root syndrome. RoB gene causes root fusion protein followed by dot-blot ELISA analysis employing 

induction and flower hyperstyly. RolC gene expression results polyclonal antisera to purified PLRV. The predicted molecular 

in fast root growth, reduced plant height, small flowers, weight of the protein is 23 K (207 amino acids). Analysis of 

hyperstyly, poor fertility, and wrinkled leaves. Deletion of the protein revealed an amino acid sequence homology of 38% 

the DNA sequences that separate the rolB and C promoters with the 22 K coat protein of barley yellow dwarf virus (BYDV). 

decreases the rooting response on kalanchoe leaves. A 1.2 kb As reported for BYDV (Virology 165:306) the PLRV coat protein 

DNA fragment containing wild-type regulatory sequences was gene contains the complete overlap (+1 frame) of a second OR. 

fused to the reporter gene, 5-glucuronidase gene (GUS), in both This ORF encodes a potential protein of 17 K (155 amino acids) 

orientations to study the effect of rolB and C genes and shareg ca. 27% amino acid sequence homology with the 

expression. corresponding 17 K ORF of BYDV. 

174 178 

THE COAT PROTEIN OF TOBACCO MOSAIC VIRUS: AN ELICITOR MOLECULE THE ROLE OF PHENYLPROPANOID PATHWAY ENZYMES IN 

OF 

THE 

THE 

RESISTANCE 

HYPERSENSITIVE REACTION. J. N. Culver and W. 0. Dawson. RESPONSE OF SOYBEAN TO SOYBEAN MOSAIC 

Dept. 

VIRUS. C.W. 

of Plant 

Choi, 

Pathology, 

C. L. 

Univ. of California, Riverside, CA Cramer, D.C. Bays and S.A. Tolin, Department of 

92521. 

Plant Pathology, 

Physiology and Weed Science, Virginia Polytechnic Institute and 

State University, Blacksburg, VA 24061. 

Recently, specific nucleotide changes in the coat protein gene 

of tobacco mosaic virus (TMV) have been shown to be responsible In the soybean cultivar York, 

for 

which has 

the 

single 

induction 

gene resistance 

of hypersensitivity (HR) on Nicotiana to the type or GI strain of soybean mosaic virus (SMV), the G4 

sylvestris. These nucleotide changes resulted in amino acid strain induces local and systemic necrosis and the G5 strain 

substitutions in the coat protein. To determine if the altered induces systemic mosaic. Total mRNA isolated from 

RNA 

leaves 

or altered 

thro 

protein was responsible for the induction of HR, 72 hr after inoculation with SMV-G4 or -G5, separated on agarose 

the coat protein translational start was removed from a full- gels, and probed with cONA specific for each of two enzymes in 

length cDNA clone of mutant TMV 25, which normally induces HR the phenylpropanoid pathway, phenylalanine 

on N. sylvestris. 

ammonia-lyase 

Infectious 

(PAL) 

transcripts of the altered genome and chalcone synthase (CHS), was positive only for 

failed 

CHS by 48 

to 

hr 

induce HR on inoculated leaves of N. sylvestris. with SMV-G4. Enzymatic activity of PAL, 

However, 

assayed in 

infectious 

mock or 

viral RNA was recovered 10 days post- virus-inoculated leaves or hypocotyls, increased 

inoculation 

with both 

and Western blot analyses revealed the presence of SMV-G4 and -G5, but the time course 

the TMV 

differed. 

encoded 126 

The 

KD 

induction 

protein 

of 

and the absence of coat protein, defense responses in this host-virus system 

This 

will 

study 

be 

demonstrates 

compared to 

the coat protein of TMV to be the other pathogen strain-specific, induced host defense responses. 

elicitor molecule responsible for the induction HR on N. 

sylvestris. 

175 CHARACTERIZATION OF MAIZE CHLOROTIC DWARF VIRUS (MCDV) RNA. 

PHYSIOCHEMICAL ANALYSIS OF A SEROLOGICALLY DISTINCT TOMATO X. Ge, D. T. Gordon and R. E. Gingery (USDA), Dept. of Plant 

SPOTTED WILT VIRUS STRAIN. M. D. Law and J. W. Moyer, North Pathology, and M. D. McMullen (USDA), Dept of Agronomy, Ohio 

Carolina State University, Box 7616, Raleigh, NC 27695-7616. State Univ., Wooster, OH 44691. 

Tomato spotted wilt virus (TSWV) is the type member of a MCDV RNA isolated from virions was fractionated on oligo(dT) 

monotypic group of plant viruses (tomato spotted wilt group). cellulose into polyA+- and polyA-RNAs. The polyA+RNA containe 

We have isolated a TSWV variant from impatiens (TSWV-I). The predominantly 10-kb RNA, considered to be the full-length 

symptoms produced by TSWV-I were typical for TSWV but the host genomic RNA, while the polyA-RNA contained a range of small to 

range was limited to some ornamentals and Nicotiana sp. large (10-kb) RNAs with no discrete bands. The polyA-RNA 

Electron micrographs of TSWV-I infected tissue revealed hybridized to cDNA synthesized from the polyA+RNA. The two RNA 

cytopathic effects distinct from TSWV, consisting primarily of fractions directed synthesis of similar sets of proteins in 

filaments in a Lattice arrangement. TSWV-I is composed of cell-free translation systems. Northern hybridization analysis 

three distinct RNA species of approximately 8.3 Kb, 5.2 Kb and using cDNA synthesized from the 10-kb MCDV-virion RNA detected 

3.4 virions Kb,, which are composed comigrate of three with TSWV proteins, RNA. Purified approximately TSWV-I 78 KD only released full-length from polyribosomes MCDV RNA in by total either RNA, EDTA polyA+RNA, or puromycin and RNA 

viro, are composeand of three 

from 

proteins, aproxmi ately 7h KV MCDV-infected tissue. No subgenomic RNAs were detected. These 

(Gi),, proteins. 52 KD The (G2)., TSWV and and 28 TSWV-I KD (NC) nucleocapsid which comigrate proteins with were TSWVLA.-.!-.L-. not results support the hypothesis that MCDV-virion klMa RNA is a 

serologically related by Western blot analysis with polyclonal monopartite, positive sense, polyadenylated RNA which is 

antibodies. In contrast, TSWV and TSWV-I Gi and G2 proteins expressed as a polyprotein. 

were found to be related in Western blot analysis. 

179 

176 180 

SEROOGIAL F RLATONSHPS TE CASIDPROTINSOF TE TPEMAPPING OF POTYVIRUS-SPECIFIC AND GROUP-COMMON ANTIGENIC 

SEROOGIAL F RLATONSIPS TE CP~lDPROEIN OF 

ISOLATE 

HE YPEDETERMINANTS 

OF MAIZE CHLOROTIC 

WITH 

DWARF 

MONOCLONAL 

VIRUS 

ANTIBODIES 

(MCDV-T). 

BY 

C. 

WESTERN-BLOT 

M. Maroon, 

ANALYSIS 

AND COAT PROTEIN AMINO ACID SEQUENCE COMPARISONS. Ramon Jordan. 

0. T. Gordon and R. E. Gingery (USDA/ARS). Dept. of Plant USDA-ARS, Florist and Nursery Crops Laboratory, Beltsville, MD 

Pathology, Ohio State Univ., Wooster 44691.',, 

The reactivities of bean yellow 

MCDV-T 

mosaic 

consistently 

virus (BYMV)-specific, 

yielded three capsid proteins, designated BYMV subgroup-specific, and potyvirus cross-reactive monoclonal 

CP1 (MW=33.8 kD), CP2 (MW=25.8 kD) and CP3 (MW=23.6 kD), and antibodies (McAbs) were tested in immunoblot analysis with 

occasionally a fourth, CP4 (MW=19.6 kD), in SDS-PAGE. CPs 1, 2 SDS-PAGE separated coat protein subunit and enzymatically or 

and 3 were separated by SDS-PAGE, eluted, concentrated and used chemically generated peptide fragments. BYMV-specific antigenic 

to raise polyclonal antisera (PcAs) in rabbits. The dilution determinants were located in the 40-amino acid (aa) residue 

end points of these antisera, tested against 150 ng of the trypsin-cleaved N-terminal peptide. BYMV subgroup-specific 

homologous CP in Western blots (WB), were: CPl.- sites were located in the 18-aa C-terminal peptide and in less- 

PcAs=l"1,200,O00; CP2-PcAs=l'38,400; and CP3-PcSa=1:9600. In conserved regions of the 218-aa trypsin-resistant-core (TRC) 

WB, CPl-PcAs did not react with CP2 or CP3, whereas CP2-PcAs protein. Cross-reactive McAbs reacted with conserved regions 

and CP3-PcAs cross-reacted with CP2 and CP3, but did not react located in the TRC peptide. A highly conserved determinant, 

with CP1. We conclude that CP1 is distinct from CP2 and CP3 and recognized by a broad-spectrum McAb (which has reacted with 

hypothesize 

more 

that CP1 is expressed by a distinct viral gene. than 95 potyvirus isolates so far tested), was mapped to a 14-aa 

While appearing serologically related, we refrain from a residue peptide in the TRC. Progress in the ELISA analysis of 

conclusion on the relationship of CP2 and CP3; pending further overlapping synthetic octa-peptides, to further identify and 

study of cross-contamination of CP2 and CP3 preparations. delineate McAb-binding domains, will also be presented. 

Vol. 79, No. 10, 1989 1157

181 

THE EFFECT OF IN PLANTA PRODUCED HUMAN INTERFERON ON TYMY 

PRODUCTION OF TRANSGENIC TOBACCO CONTAINING COWPEA MOSAIC 

INFECTIONT 

VIRUS (CPMV) COAT PROTEIN GENES. D. L. NIDA and S. A. G.IA. de Zoeten, J. R. Penwick and T. Hohn. Friederich 

Ghabrial, Dept. of Plant Pathology, University of Kentucky, Giescher-Institut, P. 0. Box 2543, CH-4002, Basel, 

Lexington, KY 40546-0091 

Switzerland 

Preliminary data in our laboratory indicate that tobacco may 

serve as a model to study the potential of cross-protection 

against CPMV. ELISA results show that tobacco supports virus 

multiplication and cell-to-cell movement. Because CPMV coat 

proteins VP37 and VP23 are derived from a 60k polyprotein 

Cauliflower mosaic virus (CaMV) DNA was engineered to carry 

the human IFN ciD gene to the infected plant. Inoculation 

of turnip (Brassica rapa cv "Just Right") with CaMV strains 

carrying the IFN cLD gene resulted in the production of 

biologically active IFN oD in infected plants 

precursor by proteolytic processing, it was of interest to 

determine whether constitutive expression of the 60k 

polypeptide in transgenic plants would interfere with CPMV 

infection. Therefore, plant expression vectors were 

constructed by inserting cDNA representing the CPMV 60k 

biol (ca. 2pg/g g Fwt.) activ In nplanta In plan produced infeced IFN plants ciD did not 

hamper superinfection with a single stranded (+) RNA plant 

virus, turnip yellow mosaic virus (TYMV). 

precursor into the binary Ti vector pMON530. Constructs 

containing CPMV coat protein genes in sense and antisense 186 

orientations were generated. Transgenic plants are being 

produced via Agrobacterium cocultivation and selection on 

kanamycin-containing medium. 

GENOMIC CHARACTERIZATION OF BEET CURLY TOP VIRUS ISOLATES. 

Stenger and J. E. Duffus, USDA-ARS, Salinas, CA, 93905. 

0. C. 

Full-length, infectious DNA clones have been constructed for 

three distinct isolates of beet curly top virus (BCTV). 

182 Progeny virus derived from cloned genomes of the Logan (sev 

CELL-FREE STUDIES OF TEV 49KDA PROTEINASE PROCESSING: on Beta vulgaris, wide host range), Worland (mild on B. 

DIFFERENTIAL CLEAVAGE RATES AT DIFFERENT POLYPROTEIN vulgaris, wide host range), and Horseradish (narrow host range) 

JUNCTIONS. W, G, Dougherty and T. D. Parks, Dept. of isolates were transmitted by Circulifer tenellus, and displayed 

Microbiology, Oregon State Univ., Corvallis, OR 97331-3804. the same phenotypes as the original isolates. A fourth BCTV 

genome (severe, wide host range) was inadvertently cloned as a 

Tobacco etch virus (TEV) 49kDa proteinase recognizes a contaminant of the Horseradish isolate. Southern hybridization 

specific heptapeptide sequence at five locations on the TEV assays indicated that each cloned genome shared sequence 

polyprotein. This consensus cleavage sequence contains both relatedness with a full-length, infectious BCTV DNA clone 

conserved and nonconserved positions. In cell free studies, previously characterized by Stanley et al (EMBO J 5:1761). 

cleavage at the 50kDa/7lkDa protein junction proceeded at a Endonuclease restriction maps developed for the cloned BCTV 

slow rate relative to processing at the 58kDa/3OkDa cleavage genomes were distinct from one another. Infectivity assays 

site. Site directed mutagenesis of TEV cDNA sequences was determined that plasmids containing tandem repeats of BCTV 

performed, such that the nonconserved positions of the genomes were generally more infectious than excised linear 

50kDa/71kDa site were converted into those amino acids found BCTV DNA inserts. 

at the 58kDa/3OkDa cleavage site. Reciprocal mutations of 

the 58kDa/3OkDa site were also tested. In each case, 

processing of the 58kDa/3OkDa sequence proceeded at a faster 187 

rate. These data suggest that post translational regulation 

of gene expression in TEV may be possible via differential MUTAGENESIS OF GENE VI of CAULIMOVIRUSES RESULTS IN CHANGES IN 

proteolytic processing at various gene product junctions. THE DISEASE PHENOTYPE. E. P. Broglio and R. J. Shepherd. 

University of Kentucky, Lexington, KY 40506. 

A CaMV hybrid genome was constructed which contained unique 

183 restriction sites bordering gene VI that allowed its sequences 

DETECTION OF PLANT RNA VIRUS-SPECIFIC RIBONUCLEOPROTEIN to be subcloned, mutagenized and then ligated into the remain- 

COMPLEXES IN INFECTED CELL EXTRACTS. R French. USDA, ARS, der of the genome. Changes in the primary structure of gene VI 

Department of Plant Pathology, University of Nebraska, Lincoln, NE were made by linker-insertion mutagenesis. Of the several 

68583. mutants constructed, three were found to be infectious and were 

examined for disease phenotype on Brassica campestris and several 

solanaceous hosts. The infectious mutants and the parent 

Within cells, both cellular and viral RNAs probably exist as complex ribo- virus displayed a similar phenotype on B. campestris and two 

nucleoproteins. In order to detect RNA virus-specific ribonucleoproteins in Nicotiana species, N_. edwardsonii and N. bigelovii. In Datura 

a general way, antiserum to double-stranded RNA (dsRNA) was used to stramonium one of the mutants induced a-distinctly differen 

immunoprecipitate complexes fror barley stripe mosaic virus (BSMV, phenotype. This result supports previous reports that gene VI 

brome mosaic virus, or tobacco mosaic virus-infected barley protoplasts. is largely responsible for disease induction. 

Radiolabeled protein components of the complexes were then detected by 

SDS-PAGE and fluorography. Immunoprecipitates from mock-inoculated 

protoplasts contained little protein while those from infected cells revealed 

several proteins unique for each virus, including coat protein. Extracts 

from BSMV-infected cells contained polypeptides of ca. 140, 60, 25 

(coat), 20 and 18 kD. UV irradiation of cells prior to extraction increased 

the yield of protein suggesting that the immunoprecipitated proteins are 

intimately associated with RNA in yiVQ. Several types of complexes may be 

precipitated because synthetic dsRNA and BSIMV virion RNA were both 

partially effective in competition assays. 

184 

COMPARISON OF SYMPTOMS ASSOCIATED WITH EXPRESSION OF CaNV GENE 

VI WITH TWO PROMOTERS IN TRANSGENIC PLANTS. K-B. Goldberg, 

J.M. Kiernan, and R.J. Shepherd. Dept. of Plant Pathology, 

Univ. of Kentucky, Lexington, KY 40546. 

Datura innoxia and Nicotiana edwardsonii were transformed with 

gene VI of cauliflower mosaic virus (CaMV) strains CM1841 and 

04 with their homologous 19S promoters or a chimeric 35S 

promoter-gene VI of CM1841 using the Ti-plasmid vectors pGA472 

or pKYLX-7, respectively. These plants are not hosts for 

CH1841. Expression of P62, the gene VI protein, in 0. innoxia 

was associated with stunting accompanied by chlorosis or 

necrosis. The maximum levels of P62 in these plants with either 

a 19S or 35S promoter were similar, as determined by western 

blot analysis using antiserum to P62. The transgenic N. 1B8a 

edwardsonii were mildly chlorotic when expressing gene VI of MOLECULAR CHARACTERIZATION OF NON-ENVELOPED BACILLIFORM VIRUSES. 

CM1841 hut overall the levels of P62 were much lower than N. E. Olszewski, S. L. Medberry and B. E. L. Lockhart, 

observed with 0. innoxia. The accumulation of P62 in N." ... 

edwardsonii was at least ten times greater using gene VI in Uiest fMneoa t al N518 

combination with a 35S promoter compared to the 19S promoters. Gienoses of several non-enveloped bacilliform viruses have been 


185

shown to be double-stranded circular DNA. We have constructed Penicilliwm funiculosum, which showed antagonism to several 

genomic clones of two non-enveloped bacilliform viruses, Phytophthora spp. in vitro, suppressed azalea root rot caused 

kalanchoe top-spot virus (KTSV) and Commelina yellow mottle by Ph. cinnamnomi or Ph. parasitica in some, but not all, 

virus (CoYMV), and are using these clones to characterize these greenhouse experiments. The antagonist was grown in a 

genomes and the transcripts they encode. The CoYMV genome is 7.5 bran/peat medium and mixed into a planting medium [peat/perlite 

kb in size and each strand contains a single-stranded region (3:1)]; this was followed by inoculation with Phytophthora at 

(discontinuity). The CoYMV genome encodes a 7.6-7.7 kb 5-7 days. Results obtained over a 2-month period showed that 

polyadenylated transcript. DNA sequencing has identified a suppression of Ph. parasitica was in general greater than Ph. 

region of the genome with homology to the '-end of the cinnamomi. Introduction of the biocontrol agent by dipping the 

initiator tRNAmeT of wheat and bean. The tRNAme, homology and roots in a spore suspension was not effective. P. funiculosum, 

one discontinuity map to the same region. These features of however, improved the growth of azalea plants even in experi- 

CoYMV suggest that this virus replicates by a mechanism similar ments where Phytophthora was not suppressed. Plants in the 

to that of caulimoviruses. The host for CoYMV, Commelina antagonist-containing treatment grew better (70-220%) and had 

diffusa, contains a 1 kb transcript which shares homology with greener foliage than those in the control without the antagon- 

CoYMV DNA. This transcript occurs in both healthy and infected ist, possibly due to suppression of minor root pathogens 

leaves but its significance is unknown. present in the planting medium. 

189 193 

PROXIMITY OF PSEUDOMONAS FLUORESCENS STRAIN PRA25rif TO PEA INCREASE IN RHIZOSPHERE COMPETENCE OF TRICHODERMA HARZIANUM 

TAPROOTS. J. L. Parke and C. M. Liddell, Dept. of Plant FOLLOWING PROTOPLAST FUSION. A. Sivan and G.E. Harman, Dept. of 

Pathology, University of Wisconsin, Madison 53706. Horticultural Sciences, Cornell Univ., Geneva, NY, 14456. 

Pea seeds coated with a spontaneous mutant strain of P. Rhizosphere competence of strains of Trichoderma harzianum was 

fluorescens, PRA25rif, resistant to rifampicin, were sown in tested by treating seeds with conidial suspensions and assesssoil 

held at -6 kPa matric potential. Seven days later, ing colonization of roots of plants grown from inoculated seeds. 

PRA25rif populations associated with the pea taproot segment A strain (1295-22) derived by protoplast fusion exhibited a 

0.5-1.5 cm below the seed were assessed from dilution plate greater degree of rhizosphere competence than the parental 

counts. Root segments with various amounts of adhering soil strains (T12 and T95). T12 colonized mainly the upper third of 

(0-1200 mg cm" 1 ) were sampled to determine if soil mass the roots, while T95 was found primarily on the upper and the 

affects quantification of root-colonizing bacteria. The lower parts of the roots. However, 1295-22 showed a greater and 

amount of soil adhering to roots did not significantly affect more uniform colonization of the whole rhizosphere than did T12 

the number of bacteria recovered, and the density of PRA25rif or T95. Colony forming units of T95 and T12 along the lower half 

was greatest in the inner rhizosphere. After sonication of of corn roots were primarily confined to the rhizosphere. Conthe 

samples, 99.8% of the bacteria recovered were isolated versely, 1295-22 colonized both the rhizosphere and rhizoplane 

from the soil suspension, and only 0.2% were recovered from equally. Although population densities of the tested strains 

macerated roots. were, in general, lower in cotton rhizosphere than in corn, 

1295-22 colonized more cotton root segments than the parental 

types. 

190 

EFFECTS OF SOIL PH ON SUPPRESSION OF TAKE-ALL BY PSEUDOMONAS 

FLUORESCENS 2-79. B.H. Ownley, D.M. Weller, and L.S. 194 

Thomashow. USDA-ARS, Pullman, WA 99164-6430 INHIBITION BY PSEUDOMONAS CEPACIA, A POTENTIAL BIOCONTROL 

Pseudomonas 

AGENT, OF 

fluorescens 

SELECTED SOILBORNE 

2-79 suppresses 

PATHOGENS. 

Gaeumannomyces 

K. E. Conway, 

graminis 

C. J. 

Foor, D. Malvick, and C. Bender, Department of Plant Pathology, 

var. tritici (Ggt) which causes take-all of wheat. The major Oklahoma State University, Stillwater, OK 74078-9947. 

mechanism of suppression is phenazine-1-carboxylate; fluorescent 

siderophore and a second iron regulated factor have only A bacterium isolated from soil at the Oklahoma Forest 

minor roles. Mycelial growth of Ggt was inhibited by 2-79 on Regeneration Center in Washington, OK, exhibited strong anti- 

Kanner's agar (used for phenazine production) adjusted to pH biotic activity in culture against macrophomina phaseolina. 

4.5, 5.5, 6.5, 7.2, 7.6, or 8.5. To determine the effect of The bacterium was identified as Pseudomonas cepacia 

soil 

(Pc) 

pH 

by 

on take-all suppression, seeds were treated with 2-79 fatty acid profile analysis. In-dual culture on PDA, 

or mutants-deficient in production of phenazine (Phz-), sidero- strongly inhibited M. phaseolina, Rhizoctonia solani AG 1, AG 

phore (Sid ), antifungal factor (Aff-), or a combination and type 1, AG 3 and AG 4, FusariumR oxyporum, PythiumA irregula 

sown in a steamed Ritzville silt loam (pH 7.6, 29.6% sand, and Laetisaria arvalis, but not Sclerotium rolfsiir 

64.0% silt, 6.4% clay) with bulk soil pH adjusted to 4.9, 5.7, Trichoderma harzianum. However, Pc strongly inhibited S. 

6.2, 7.3, 7.6 or 8.0. Strain 2-79 and a Phz Sid Aff mutant - rolfsii in pairings on KMB. This indicated possible 

significantly suppressed take-all at all soil pH values. Phz siderophore production in addition to antibiotic production 

mutants were less suppressive and generally failed to suppress Eg. Potential of the bacterium as a biocontrol agent is being 

take-all at low (7.6) soil pH. assessed in soil systems. 

191 195 

BIOLOGICAL CONTROLOFRHIZOCTONIASEEDLINGDISEASES IN INFLUENCE OF WHEAT ROOT PATHOGENS ON MAINTENANCE OF POPUl.ATiONS 

BEDDING PLANT NURSERIES WITH THOUGHTS TOWARDS OF PSEUDOMONAS FLUORESCENS Q72a-80 AND 2-79 IN THE WHEAT 

COMIMERCIAL DEVELOPMENT. D.A. Schisler 1 , M.H. Ryder', R.G. RHIZOSPHERE. N. Mazzola and R.J. Cook, Dept. of Plant Path- 

Rowden 2 , 2 1CSIRO-Division of Soils, Glen Osmond, South Austraia and 

1n citec L td ., G ib so n Islan d , B ri sb an e, Q u een slan d , A u stralia.ol 

olg.n. 

g a n U S A R , W sh 

SaeUiesty .AA.,Wsigo ula 

g t n t t e n v r s y , P l m n 

96 

)9 6 

A cooperative research project between CSLRO and IncitecLtd. was iniiated to 

investigate tbe feasibility of using antagonistic microorganisms to control 

Rhizoctoniadiseases of seedlings in bedding plant nurseries. Fifty soil samples 

from 30 nursenies in South Australia were assayed for suppressiveness to 

Rhizoctonia SOlni. From these assays, 13 highly suppressive soils were 

selected and fungi, bacteria and actinomycetes were isolated from bulk and 

rhizosphere soils. Ninety-fsve prokaryotic antagonists were assayed against R. 

solani (anastomosis group 8) on Capsicum annuum cv "Green Giant" and 

Celosia argentea using commercial potting, seeding and watering equipment. 

More than one-third of the organisms significantly decreased disease (p

A biopesticide seed treatment using a strain (KRL-AG2) of T. Twelve isolates of Talaromvces flavus were examined for their 

harzianum produced by protoplast fusion is being developed-for capacity to"' parasitize pre-dried sclerotia of Scletrotium rolfsii 

control of plant diseases. In 1988, field trials were conducted on water agar (2% Bacto agar in tap water) and in natural sandy 

in five states, i.e. Florida, Idaho, Mississippi, New York and 

Texas. Crops included cabbage, canola, cotton, cucumber, snap 

loam soil adjusted to 70% MHC. The dried sclerotia 

a conidial suspension of T. flavus containing 10 

were dipped in 

spores/ml and 

bean, soybean, sweet corn and tomato. In general, seed treatment 

with KRL-AG2 and solid matrix priming provided reliable 

and effective control of seedrot diseases. Plant stands from 

seeds with biological treatments were equal or superior to 

incubated at 30 C for 3-4 days. All the tested isolates 

parasitized the sclerotia on water agar, but only 9 were capable 

of parasitizing the sclerotia in soil. Dipping the sclerotia in a 

heat-activated T. flavus ascospore suspension ( 10 7 those from seeds treated with chemical fungicides. Biological 

seed treatment increased yield in sweet corn and slightly increased 

effective yield mixtures in cotton of fungicides. 

relative to 

Expanded 

seed treatment 

trials, 

with 

including 

highly 

spores/ml), 

resulted in up to 5% mycoparasitism of the sclerotia in either 

water agar or soil, as compared to 5-100%, depending on the 

isolate and on the medium, obtained with T. flavus conidia. The 

differences in mycoparasitic capacity observed among the tested 

additional crops and states, are planned for 1989. T. flavus isolates were found to be correlated with their growth 

rates as well as with their glucose oxidase, 0-1,3-glucanase and 

chitinase activities. 

197 

EFFICACY OF BINUCLEATE RHIZOCTONIA AGENTS (BN) IN BIOCONTROL OF 201 

RHIZOCTONIA ROOT ROT OF SUGAR BEET IN THE FIELD. L. J. Herr, DIRECTEDENHANCEMENTOFBIOCONTROLINPSUEDOMONAS BY 

Dept. of Pl ant Pathology, Ohio State Univ., Wooster, OH 44691. CONSTITUTIVE ANTIBIOTIC BIOSYNTHESIS. W. HowŽie, D.Matsubara, N. Gutterson, 

and T. Suslow. DNA Plant Technology Corportation, Oakland, CA 94608. 

Similar field tests of three BN (as biocontrol agents) were run Constitutive biosynthesis of oomycin A in Pseudomonas fluorescens strain Hv37aR2 

on on88. a silty silt clay aygloam loam (SC) (SC) and and a a) plusand sandy loam loamt (SL) ( tol, 

soil during 

durig was achieved by placing a strong promoter 

.............. ..... 

1988. BN agents (1, 3 and 4) plus no agent controls, wereofhaulounstomcnbsyheiw 

sequence upstream of an introduced 

mntreyiomnsce 

copy 

banded over rows at two rates (11 .2 and 67.4 kg dried barl ey of the afuE locus. In situ oomycin A biosynthesis was monitored by bioluminescen 

inoculum/ha) in early June. On 15 June, beets were side- from a reporter afuE-Iux fusion in either a constitutive (WH157) or control (WH161) 

dressed win R.eslar ne. On- 2 barley Jne, beets u were side-strain. Biosynthesis of oomycin A ifn situ was determined over a 7-day period in both 

dressed with R. sol ani AG-2 ,T2 barley i noculum (45 kg/ha), strains. Biosynthesis in strain WHi157 was consistently 50 to 100-fold higher than in 

Harvest (10 Oct) data taken were % plant loss (% PL); disease strain WH161 on seeds, roots, hypocotyls, and cotyledons of 

rating 

cotton, 

(DR), 

Acala 

on a scale 

SJ2. 

of O=healthy to 5=80-100% rotted; and There were no significant differences in populations of the two strains on 

yield. 

any of 

On SC, BN-1 

the 

and 3 agent treatments (not diff.) had plant parts assayed. Seed treatment with WH157 resulted in an average of 15 to 20% 

lower % PL, DR, and a >50% increase in yield over the BN-4 increase in emergence and decrease in root infection by Pythiumultimum as 

agent and 

was 

no agent 

signi fi cantly was beter better 

(not ignficntl diff.) 

than hanthe11. the 

treatments. 

11.2 kgha kg/ha 

The rtehowverthe rate, 

67.4 

however 

kg/ha rate 

the compared rhizobacterial to strain strain WH1 can mitigate 61. Clearly, deficiencies 

constitutive 

in efficacy. 

antibiotic biosynthesis by a 

rate x agent interaction was non-significant. Whereas on SL, 

the rate x agent interaction was significant and BN-3 at 67.4 

kg rate ranked best in all categories, including a 383% 

increase in yield over the no agent treatment. 202 

A NEW BIOCONTROL FORMULATION FOR TRICHODERMA AND GLIOCLADIUM. 

EFFECT OF SUBLETHAL HEATING ON WEAKENING, AND ON HEAT SHOCK 

PROTEIN SYNTHESIS, IN PROPAGULES OF FUSARIUM. S. Freeman, C. 

Ginzburg* and J. Katan, Dept. Plant Pathology & Microbiology, 

Faculty of Agric., Rehovot 76100; and Dept. of Ornamental 

J.A. Lewis, G.C. Papavizas, and R.D. Lumsden, USDA, ARS, Horticulture, The Volcani Center, Bet Dagan 50250, Israel. 

Beltsville, MO 20705. 

Biocontrol formulations were prepared with vermiculite, wet fermentor 

biomass of isolates of Trichoderma spp. and Gliocladium 

virens, and dilute HC1. The mixture was air-dried and activated 

The weakening of propagules of F. oxysporum niveum (FON) by 

sublethal heating may be expressed in a delay in germination, 

and in reduced survival and disease incidence. Germlings of 

FON respond to heating by synthesizing a set of heat shock 

with wheat bran and additional dilute acid to develop germlings 

(actively-growing hyphae) of the fungi. Aseptic conditions did 

not have to be maintained during formulation and activation.Dry 

proteins (HSPs) corresponding to 95, 83, 80, 74, 70, 35 and 18 

kD. Vital fluorescent staining in germlings decreases in 

intensity which corresponds with appearance of HSPs. HSPs are 

preparations survived in storage at 5 and 25 C for at least 12 observed 10 min after heating. Recovery from a shock of 40 C 

wk. 9ungi in activated preparations proliferated in soil up to 

2x10 cfu/g of soil within 1 wk of amendment. Several preparis 

more rapid at 25 C than at 36 C. Thermotolerance to a 

lethal temperature of 44 C is acquired by preheating germlings 

ations significantly reduced survival ( >60%) of Rhizoctonia at sublethal levels, as determined by vital staining and 

solani in pathogen-infested beet seed in soil and prevented 

saprophytic growth in soil. Preparations containing various 

isolates significantly prevented damping-off of cotton (>60%) 

in R. solani-infested soil, damping-off and blight of bean 

survival. Solarization at sublethal temperatures may cause 

either a weakening or an acquired thermotolerance, but the 

direction is probably dependent on the environment into which 

the propagules are introduced. 

(>90%) in Sclerotium rolfsii-infested soil, and damping-off of 

zinnia in soilless mix infested with R. solani. 

199 

SELECTING FOR ALKALINE TOLERANCE IN TRICIHODERIVL HARZIANUM. 

G. Ruppel, USDA-ARS Crops Research Lab, 1701 Center Avenue, 

Fort Collins, CO 80525. 

E. 

203 

IDENTIFICATION OF HORTICULTURAL TRAITS FOR PREDICTING BROCCOLI 

CULTIVAR 

Dept. of 

SUSCEPTIBILITY 

Entomology and 

TO 

Plant 

BACTERIAL 

Pathology, 

SOFT 

University 

ROT. C. H. Canaday, 

of Tennessee, 

West Tennessee Experiment Station, Jackson, TN 38301. 

Triohoderma spp. are favored by soist, acid soils but do persist 

in western calcareous soils at low population densities, 

which may indicate genetic diversity for alkaline tolerance, 

Selections from over 200 random isolates of TrichodeŽ'sno sPP. 

were made in vitro at increasing medium pH. One isolate of T. 

harzianum (TpH) grew and sporulated on pH 11 medium. At soil 

pH 6.7 with six consecutive plantings, TpH and a known biocontrol 

isolate of the fungus (THW) at 106 cfu/g significantly 

Regression analyses of disease incidence versus horticultural 

traits identified three traits useful for predicting the 

susceptibility of broccoli cultivars to bacterial soft rot 

caused by Pseudomonas marginalis pv. marginalis and Erwinia 

carotovora. Data on eight traits thought to influence cultivar 

susceptibility were collected in a field evaluation of 25 

cultivars and lines maturing simultaneously under disease- 

conducive conditions. The percentage of harvested heads with 

soft rot (12.2-85.7%o) was regressed on the horticultural traits 

suppressed sugar beet damping-off in the first two plantings, 

By the sixth planting, treatment effects were nonsignificant. 

In soil at pH 8.2 with three plantings, no significant dampingoff 

suppression occurred at planting 1, but both isolates suppressed 

disease at plantings 2 and 3. At pH 6.7, TpH was not 

of 13 cultivars. The best model was head 2 tightness, doming, 

and looseness versus disease incidence (R =0.9352; P

in the greenhouse for resistance to powdery mildew caused by 

Sphaerotheca fuligenea. Seedlings were inoculated by spraying 

the cotyledons and the first true leaf with conidia suspended 

in a solution of distilled water and Tween 20 (2 drops/l). A 

suspension containing 5 x 105 conidia/ml gave a very uniform 

infection. Twenty-nine Pl's were resistant and 46 additional 

PI's had intermediate resistance. Over 50% of the resistant 

PT's were from China. Eight resistant accessions were selected 

to study disease progression. Disease progression, measured by 

a weekly analysis of spore populations on leaves, was significantly 

slower on the resistant PI's than on the cultivars 

'Marketer' (susceptible) and 'Ashley' (intermediate). Distinct 

differences were also detected in disease progression among 

some of the resistant accessions. Powdery mildew developed 

most slowly on the PI's 197088, 288238, 321006, and 390258. 

Moderately resistant (MR) and susceptible (S) celery had higher 

Fusarium yellows disease ratings when grown in the greenhouse in 

muck soil from a field monocropped to celery than in soil from an 

area in the same field rotated into leeks for one summer. Rotating 

another celery field into onions for I yr reduced the pre-planting 

population (popn) of Fusarium oxysporum f. sp. apii race 2 from 

80 propagules/ gram of soil (ppg) to 26 ppg, but growing celery 

the following yr increased post-harvest popn to 160 ppg. Race 2 

popn in an infested celery field rotated into other vegetables for 

5 yr was too low to measure, and remained low after 1 yr back 

into celery. An apparently suppressive soil (SS) was identified 

in a field with a low Fusarium yellows incidence on a farm where 

all other fields were highly infested. MR and S celery had lower 

disease ratings when grown in the greenhouse in the SS than in 

soil from a conducive field on the same farm. Race 2 popn was 

120 ppg in the conducive soil, but too low to measure in the SS. 

205 

DYNAMICS OF A WHITE MOLD (WHETZELINIA SCLEROTIORUM) EPIDEMIC 209 

IN FLORIDA CABBAGE. D. P. Weingartner, AREC, Hastings,FL 32045. ETIOLOGY OF CARROT CAVITY SPOT IN CALIFORNIA. E. Vivoda, R. M. 

Temporal and spatial development of white mold was studied on Davis. Department of Plant Pathology, University of California, 

a 140 ha commercial cabbage farm during the 1987-88 and 1988- Davis, CA 95616. 

89 cabbage seasons. Apothecia were observed Dec to April, 

however, were most numerous 10-14 days following periods of Pythium violae and P. ultimum were isolated from cavity spot 

rain or irrigation. Successive "crops" of apothecia were lesions on carrots from the San Joaquin Valley. Healthy, mature 

produced in individual fields, many from sclerotia buried 5-8 carrots were inoculated with the fungi and incubated in moist 

cm in soil. Abundant apothecia were present during infection chambers. Symptoms developed after 7 days. Either P. violae 

periods. New infection, however, did not always occur when or P. ultimum were reisolated from inoculated carrots exapothecia 

were present. Two apothecia were observed on current hibiting cavity spot lesions. Fourteen cultivars of carrots 

season's sclerotia during 1988. During 1989 stipes were were susceptible to P. violae but only ten to P. ultimum. In 

produced within 30 and 43 d, respectively, at 12 and 8 C, by growth chamber studies 3 month old carrots were transplanted 

sclerotia formed on current season's infections. Degree of into soil infested with either Pythium spp. (200 cfu/g soil) 

aggregation was assessed by mapping distribution of infection at 15, 20 and 250C. Symptoms developed after 2 weeks. P. 

in nine different fields. Data suggest that most infection is violae caused the greatest number of lesions per carrot at 

due to within field inoculum and that frequent cultivation may 15'C while the optimum temperature for disease development for 

be an effective control measure. Significantly more 'Savoy' P. ultimum was 20'C. Overall, more carrots were infested by 

plants were infected than 'Bravo'. P. violae than P. ultimum. The role of inoculum density, soil 

moisture and carrot age was also investigated. 

206 210 

EFFECT OF OIL AND ENDOSULFAN ON SPREAD OF POTYVIRUSES IN FALL- COLONIZATION OF CROP RESIDUE BY FUSARIUM OXYSPORUM F. SF. 

PLANTED WATERMELON. S. E. Webb and P. Groves, University of COLONIZ AT HOGENIC BY FU F. OXYSPORUM. SP 

Florida-IFAS, CFREC, 5336 University Ave., Leesburg, FL 32748 GORON, De PArtmentIo P T P l UieSity of 

GORDON, Department of Plant Pathology, University of 

We evaluated oil and endosulfan (to control colonizing aphids) California, Berkeley, CA 94720. 

alone and in combination in large plots (0.13 ha) of fallplanted 

(18 August) watermelon in Central Florida to determine 

if virus spread could be delayed. Plants showing mosaic 

symptoms were mapped every three days and were periodically 

tested for watermelon mosaic virus 2 (WMV-2), papaya ringspot 

virus type-W (PRSV-W), and zucchini yellow mosaic virus (ZYMV) 

using ELISA. In mid-October, plots treated with oil or oil and 

endosulfan averaged 50% fewer virus-infected plants than those 

treated with endosulfan alone, but by the end of October after 

a large aphid flight all plots were almost 100% infected. In 

late September, 91% of plants tested (n=150) were infected with 

WMV-2 and 15% with PRSV-W. In mid-October percentages were 

equal for WMV-2 and PRSV-W. By early November, 84% (n=860) 

were infected with WMV-2 and 97% with PRSV-W. Fewer than 0.1% 

were infected with ZYMV. 

Colonization of crop residue, by Fusarium oxysporum f sp. O 

melonis (FON) and nonpathogenic strains of F. oxysporum (FON), 

was studied in an agricultural field soil. Inoculum densities 

of the two fungi in this soil were approximately equal. Shoot 

tissue incubated for five days in 'moist soil was heavily 

colonized by both pathogenic and nonpathogenic strains. Tomato 

shoots supported significantly higher levels of FOM than FON, 

while the reverse was true for wheat. Residue colonization 

averaged over seven crop species showed significantly higher 

levels of the pathogen relative to the nonpathogenic strains. 

Five days after shoot removal, FOM was present at significantly 

higher levels than FON on roots of both muskmelon and tomato. 

However, the proportion of total colonization (FOM + FON + F. 

equiseti -t- F. solani) represented by FOM and FON was the same 

on living roots as on roots five days after shoot removal. 

207 211 

ETIOLOGY OF TOMATO DECLINE. J. S. GrkA. F. Van Maren 2 , 

D. . egeIandJ.Gerufu i k 1 

USA-R, SaiaC FUSARIUM WILT OF TOMATO IN FLORIDA BEFORE AND AFTER AN OVERSEA- 

D.90 C. Stenger. CandiJ. Eoo. Dufus. USDA-ARE1 Salinas, CA 923 SONING PERIOD. John Paul Jones, J. W. Scott, and J. P. Grill, 

9390 & Univ Ex. Caif.Coop Srv.,El ento, C 9243.Gulf Coast Res. and Edu. Center, 5007 60th St. E., Bradenton, 

Tomato decline (TD) of fresh market tomatoes in the desert E 40 

areas of California has been observed since 1977. The disease A 2.5 month midwinter overseasoning period between cropsagreatly 

occurs only in fields with a history of previous tomato crops, reduced the incidence of Fusarium wilt or race 1-tolerant Rut- 

Although TO is known to be soil borne, the cause of the disease, gets (caused by race 1, 2, or 3), race 1-resistant Manapal 

until now, has not been determined. Tomatoes grown for 3 weeks (caused by race 2 or 3), race 1 and 2-resistant Walter (caused 

in a growth chamber at 14 C in soil collected from a field with by race 3). Disease incidence caused by race 1 (but not race 2 

a history of TO became infected with a Tombusvirus serologically or 3) was slightly decreased on susceptible Bonny Best. Yield 

indistinguishable from the BS-3 strain of tomato bushy stunt losses were sharply reduced by the overseasoning period on Rutvirus 

(TBSV). Field grown, symptomatic plants collected during gers (caused by race 1, 2, or 3), Manapal (caused by race 2 or 

1987 and 1988 were consistently found to be infected with TBSV, 3), Walter (caused by race 3), and Bonny Best (caused by race 1 

as determined by ELISA and bioassay. Eight tomato cultivars, or 2, but not race 3). Based on yields and disease incidence, 

which in field observations were considered to be very suscept- race 2 survived just as well as race 1, and race 3 survived just 

ible or resistant to TO, were mechanically inoculated with TBSV. as well as race 1 or 2. Manapal was less susceptible to race 2 

The symptoms were most severe on the ID susceptible plants, but or 3 than Bonny Best and no more susceptible than Rutgers. 

were very mild on TO resistant plants. These experiments im- Walter was less susceptible to race 3 than Bonny Best and no 

plicate TBSV as the etiological agent of TO. more susceptible than Rutgers or Manapal. 

208 

TOWARDS MANAGEMENT OF FUSARIUM YELLOWS OF CELERY. K. F. Ireland 21 

and N. L. Lacy. Department of Botany and Plant Pathology, SQUASH SILVERLEAF AND ISASSOCIATION WITH THE SWEETPoTATO 

Michigan State University, E. Lansing, MI 48824. WHITEFLY. R. K. Yokomi , L. S. Osborne- and K. A. Hoelmer. 

Vol. 79, No. 10, 1989 1161

USDA, ARS, 2120 Camden Road, Orlando, FL 32803 Univ. of Resistance levels of 20 muskmelon cultivars to squash mosaic 

Florida, IFAS, CFREC, Apopka, FL 32703 2 virus (SqMV) were determined by rating the number of days 

from inoculation to first symptom (DTS), disease severity, 

In 1988, a new disorder called squash silverleaf (SSL) appeared and number of plants infected. Significant differences 

in squash and other cucurbits across south and central Florida. (p=0.05) occurred among cultivars in DTS and disease severity 

SSL symptoms first appear as a veinal chlorosis followed by a ratings. 'Perlita', 'Sunshine', and 'Hiline' had 50, 42, and 

whitening of veins which can extend throughout a mature leaf. 33%, respectively, symptomless plants. Symptomless plants 

Sectored discoloration of fruit was also observed. Extracts were reinoculated and those remaining symptomless were selffrom 

affected tissue did not react with an array of cucurbit pollinated. The proportion of symptomless plants in the 

and whitefly-transmitted virus antisera. SSL was not graft or selfed progenies was greater than in the original 

mechanically transmitted. SSL symptoms appeared within 10 days populations. A double antibody sandwich enzyme-linked 

of plant colonization from all local populations of the immunosorbent assay (DAS-ELISA) was developed to provide a 

sweetpotato whitefly (SPWF) tested. Ten nymphs per plant more precise method for comparing plant responses to SqMV. 

induced symptoms, but severity increased with more SPWF. New Titer cut-off values were determined from titration curves of 

growth following elimination of the whitefly by insecticides clarified viral concentrates. Symptomless plants were 

was normal. These data suggest that an insect toxin may be associated with reduced virus titer. 

involved in SSL symptom expression. 

217 

213 EPIDEMIC WAVES OF RUST ON ALFALFA. R. D. Berger and D. A. Robers, 

EFFECT OF DOWNY MILDEW (PSEUDOPERONOSPORA CUBENSIS) ON SUGAR University of Florida, Gainesville, FL 32611. 

CONTENT OF WATERMELON. C. L. Patterson, Wes Watkins Agri. Res. 

and Ext. Center, Oklahoma State Univ., Lane, OK 74555. Waves of disease have been assumed in the early stages of epidemics 

caused by foliar pathogens, but such waves have never been detected and 

Sugar content of watermelon fruit decreased as the incidence of characterized in natural epidemics. Discrete waves of rust (Uromyces striatus 

downy mildew (Pseudoperonospora cubensis) increased to 25% or Schroet.) on alfalfa (Medicago sativa L.) were observed when disease 

greater. Bravo 720, Ridomil/Bravo 81W, and Ridomil/MZ58 intensity was estimated thrice weekly with a logistic disease-assessment 

reduced the rate of the downy mildew epidemic. The Ridomil scale. Pictorial keys were prepared for the mid-point of each of the ten 

formulations delayed the epidemic 2-wk and resulted in classes of severity in the range 0.00035

Spatial patterns of disease were studied in four central Leafspot epidemics on white clover and alfalfa caused by 

Florida nurseries infected with citrus bacterial spot. Both Leptosphaerulina spp. were studied during spring and fall 1987 

indices of dispersion and ordinary runs analysis indicated and spring 1988. Disease severity was assessed twice/wk on 

aggregation in all four nurseries. Spatial lag correlation plants of alfalfa (A) or white clover (C) in radial arms around 

analysis indicated within-row autocorrelation corresponded to disease foci. The plant combinations AA, AC, CC and CA (first 

mechanical spread of disease down nursery rows. Some across- letter = focus host, second letter = recipient host) were 

row autocorrelation corresponded to natural or mechanical arranged randomly with foyr replications in each season. Disease 

spread. High disease incidence on rootstock plants was directly progress was described (R =0.85-0.96) for all treatment combirelated 

to disease on new scion shoots in the same spatial area. nations in each season by the model y=a[l-exp(-bt)]/[l+exp(od)] 

Two nurseries had discrete areas of high disease incidence wich where y is the proportion disease at distance d from the focus 

were interpreted as potential foci of disease. Shallowest dis- at time of assessment t, and a, b, and c are parameters. The 

ease gradients corresponded to down-row mechanical spread from appropriateness of this new model for all combinations and 

hedging in one nursery, and across-row natural spread to the parameter estimates suggests that characteristics of 

southeast in a second. Natural vs. mechanical spread was Leptosphaerulina leafspot epidemics are similar. However, the 

directly related to citrus cultivars, bacterial strain rate of disease spread to and increase on white clover was 

aggressiveness, use of bactericides, and cultural management. slower than on alfalfa regardless of the disease focus host. 

221 

EFFECT OF SELECTED CONSERVATION-TILLAGE PRACTICES ON WETNESS SPATIAL PA TTERN OF CERCOSPORA LEAFSP(Y ON WHITE CLOVER IN TWQ 

DURATION OF WHEAT STRAW RESIDUE AND ASCOCARP PRODUCTION BY WHITE CLOVER/TALL FESCUE GRASS PASTURES. Scot C. Nelson and C. 

PYRENOPHORA TRITICI-REPENTIS. W. Zhang, W. F. Pfender, E. Lee Campbell. Department of Plant Pathology, North Carolina 

Adee, and A. Nus. Dept. of Plant Pathology, Kansas State State University, Raleigh 27695-7616. 

University, Manhattan, KS 66506. Severity of Cercospora leafspot of Ladino white clover was 

estimated on every leaf in each of four 0.09 m samples per 

Field plots of wheat straw infested with Pyrenophora tritici- quadrat within four blocks of 64 2.4 x 2.4 m contiguous quadrats 

repentis were subjected to no-till or two different levels of in two white clover/tall fescue grass pastures in September 

reduced tillage. Tillage type and microsite (location of straw 1988. Disease incidence and severity were correlated 

relative to soil surface) were studied as factors influencing significantly (r = 0.71-0.79) in all four blocks. Using 

ascocarp production and wetness duration of the straw, estimates of Taylor's b, aggregation was indicated from disease 

Ascocarps were counted on straws sampled in mid-winter. Straw severity data while spatial pattern based on incidence data 

wetness was monitored by electrodes connected to a micrologger. ranged from regular to aggregated. Significant autocorrelations 

Ascocarps/ha were greatest in no-till plots. Ascocarps/g were were found in all four blocks for disease incidence and in three 

not affected by tillage type, but were reduced in straws near of four blocks for disease severity, indicating that similar 

the soil surface and on the lower portion of standing stubble, levels of disease tended to occur in quadrats of close 

Cumulative duration of straw wetness differed by tillage type proximity. From results of the Greig-Smith blocked quadrat 

and, across tillage types, was longer for straws near the soil variance procedure and estimates of sampling costs, optimum 

surface. In two tillage types, number of ascocarps/g was quadrat size among those tested was 5.76 m with an optimum 

negatively correlated with number of wetness periods of >10 hr. number of samples per quadrat of one or two. 

225 

222 226 

A STOCHASTIC MODEL FOR THE INITIAL OCCURRENCE OF FUNGICIDE RAIN SPLASH DISPERSAL OF COLLETOTRICHUM ACUTATUM OF STRAWBERRY. 

RESISTANCE IN PATHOGEN POPULATIONS. M. G. Milgroom. X. Yang, L. L. Wilson, L. V. Madden, and M. A. Ellis, Dept. of 

Department of Plant Pathology, Cornell University, Ithaca, NY Plant Pathology, Ohio State Univ., Wooster, OH 44691. 

14853. 

A rain simulator developed at OSU/OARDC was used to investigate 

The probability that resistance will occur in a population and the influence of rain intensity (15 and 30 mm/hr), duration 

increase to unacceptable levels is estimated as a function of (15-60 min), and types of ground cover on splash dispersal of 

inital pathogen population size (N ), mutation rate (p), conidia of C. acutatum on strawberry fruit. Potted strawberry 

pathogen growth rates (r) and fungicide efficacy (1-a) using a plants were held in two concentric circles within a wood frame 

stochastic model. Model results support the intuitive and inoculated fruits with sporulating lesions were placed in 

prediction that there is a greater probability of resistance the center. Inner and outer circles, 15 and 30 cm from the 

occurring when Nn, p, and r are large. In contrast, higher inoculum source, were exposed to a uniform zone of generated 

levels of fungic de efficacy reduce the risk of resistance rain. Studies were conducted with bare soil, soil covered with 

occurring in some circumstances, depending on values of Nn, p, fresh straw (>6 cm deep) or plastic mulch. Disease incidence 

and r. When N 0 is small, high values of 1-a may result il generally was less in the outer circle, but increased over time 

lower risks of resistance occurring. These model results only with soil. With a plastic cover, 100% incidence was 

support the intensive use of selective fungicides as a obtained at both distances and all times. Horizontal airflow 

resistance management tactic under some conditions. also was tested and resulted in less disease up-wind compared 

to down-wind. 

223 

SPATIAL DISTRIBUTION OF SOYBEAN RHIZOCTONIA FOLIAR BLIGHT AND 27 

EFFECT OF AGGREGATION OF EARLY INFECTION ON DISEASE DEVELOPMENT. DENSITY OF OVERWINTERING POPULATIONS OF UNCINULA 

NECATOR ON BARK OF GRAPEVINES. David M. Gadourv and Roger C. 

X.B.Yang, J.P. Snow, and G.T. Berggren. Dept. of Plant Path. & and Crop Pearson, Department of Plant Pathology, Cornell University, New York State 

Physi. La. Ag. Expt. Sta., LSU Ag. Center, Baton Rouge, LA 70803. Agricultural Experiment Station, Geneva 14456. 

As they mature, cleistothecia of U. necator are washed by rain to bark of 

Soybean foliar blight caused by Rhizoctonia solani Kuhn is initiated by grapevines, where they overwinter. In 1988, we monitored the development of 

inoculum from soil and spreads by means of interplant mycelial growth. Six powdery mildew, then trapped cleistothecia in rainwater collected by funnels 

beneath vines, and finally determined the number of ascocarps on bark in 17 

soybean fields with a history of the disease were subdivided into 0.75 by commercial vineyards. Detayed disease development was associated with the 

0.75 m quadrats. Disease in each qudrat in each field was assessed early and subsequent delayed dispersal of ascocarps. The density of ascocarp populations 

ranged from 291,905 cleistothecia/kg bark on unsprayed vines of Vitis vinifera 

late in the growing season. Spatial pattern analysis showed a clustered 'Chardonnay' to 8,864 cleistothecialkg bark on V. labrusca 'Concord'. Areas 

distribution of the disease. Disease incidence late in the season was predicted under disease progress curves were correlated with subsequent ascocarp numbers 

by the amount and the degree of aggregation of diseased leaves early in the on bark. However, the most consistently accurate predictor of population density 

on bark was the number of cleistothecia trapped in funnels. Density of asococarp 

season. Incorporation of an aggregation factor, AF, into the logistic disease populations on bark is probably a function of both disease incidence and 

growth model, dx/dt=rx(1 -x)/AF, improved disease prediction ability, subsequent efficiency of dispersal. Trapping cleistothecia during dispersal 

provides more reliable assessments of mnoculum dose than esoimates derived from 

assessments of disease. Future studies will examine relationships between 

224 primary inoculum dose and development of epidemics. 

SPATIoTEMFORAL MODELS TO DESCRIBE EPIDEMICS OF LEPTOUSPHAERULINA 

LEAFSPOTS ON WHITE CLOVER AND ALFALFA. 0. Modesto Olanya and C. 228 

Lee Campbell, Department of Plant Pathology, North Carolina 

State University, Raleigh, NC 27695-7616. SEASONAL OCCURRENCE OF SPORES OF MONILINIA OXYCOCCI IN A 

Vol. 79, No. 10, 1989 1163

WISCONSIN CRANBERRY FIELD. P. G. Sanderson and S. N. Jeffers. 232 

Dept. of Plant Pathology, Univ. of Wisconsin, Madison 53706. 

VIRULENCE OF SINGLE AECIOSPORE ISOLATES OF CRONARTIUN QUERCUUN 

A Burkard 7-day recording volumetric spore trap was used to F. SP. FUSIFORME. E.G. Kuhlman and F.R. Natthews, USDA, 

determine when spores of Monilinia oxycocci, the causal agent Forest Service, SEFES, Athens, GA, 30602. 

of cranberry cottonball, were present in a commercial cranberry 

field. In 1987 and 1988, ascospores were first detected on 5 Single gall isolate LHNC-2 produced galls on twice as many 

May, ca. one week before bud break, and continued to be trapped seedlings of rust resistant loblolly pine family 10-5 as 

until one week before bloom. They occurred for 31 consecutive did standard composite isolate 1-73. Thirty-two single 

days in 1987 and 28 consecutive days in 1988. Conidia were aeciospore isolates from LHNC-2 were used to produce basidiospore 

first detected ca. 10 days before bloom in 1986, at the start inoculum for inoculating seedlings of family 10-5. Six 

of bloom in 1987, and at 507 bloom in 1988. Conidia were weeks after inoculation symptoms were unusually frequent. 

present for 34, 30, and 26 consecutive days beginning 7, 6, and Three months after inoculation with the single aeciospore 

17 June in 1986, 1987, and 1988, respectively. The two spore isolates, the percentage of seedlings with galls varied 

types never occurred concurrently. Both ascospores and conidia from 33-85. In the same experiment isolate LHNC-2 and 1-73 

exhibited a diurnal periodicity. Ascospores were trapped pre- produced galls on 60 and 44% of the seedlings, respectively. 

dominantly between 1000-2100 hr and were most abundant between Single aeciospore isolates from virulent single gall isolates 

1600-1800 hr. Most conidia were trapped during the daylight appear to have great potential for determining variations 

hours, particularly around midday. in virulence in the pathogen population and in resistance 

among pine families. 

229 

233 

THE INFLUENCE OF TEMPERATURE AND FRUIT AGE ON INFECTION 

OF APPLE FRUIT BY VENTURIA INAEQUALIS. K. L. Reynolds and R. C. 

Seem. Department of Plant Pathology, Comell University, New York State 

Agricultural Experiment Station, Geneva, NY 14456. 

COMPARATIVE VIRULENCE OF HETEROBASIDION ANNOSUM ISOLATES. F. 

Cobb, Jr., T. Chase, W. Otrosina, A. Ratcliff, and T. Popenuck. 

Dept. of Plant Pathology, Univ. of CA, Berkeley, 94720 and 

USDA-For. Serv. Pacific Southwest Forest Exp. Station, Berkeley 

Several studies have reported ontogenic resistance to V. inaequalis in apple fruit, 

but none has observed the effects of this resistance on germination, appressorium 

formation, or colonization. Attached and detached fruit were inoculated with 

ascospores or conidia every 3 weeks beginning at petal fall. Attached fruit were 

kept moist at ambient orchard temperatures for 1, 2, 4, 8 or 16 days. Detached 

fruit were kept moist and incubated at 13, 22, or 31 C for 94 hr. Epidermal 

sections were stained in cotton blue/lactophenol and examined microscopically. 

The proportions of spores that had germinated, developed appressoria, and 

successfully penetrated and formed a subcuticular stroma were recorded. 

Temperature had a pronounced effect at all stages of infection. Fruit age did not 

affect germination or appressorium formation, but the proportion of successful 

penetrations decreased significantly as fruit matured. These results suggest that 

ontogenic resistance is not due to inability of the pathogen to germinate or form 

appressoria, but rather to inability of the fungus to penetrate the cuticle or an 

interaction with the host that prevents formation of a subcuticular stroma. 

No differences in mortality of white fir or ponderosa pine 

seedlings inoculated with dikaryons and progeny homokaryons of 

the 'S' and 'P' groups, respectively, were apparent but results 

among homokaryons varied considerably. In another test, four 

dikaryon isolates each of 'P' and 'S' and one of H. araucariae 

from Australia were inoculated into seedlings of nine conifers. 

All 'P' isolates were highly virulent on ponderosa and sugar 

pines and less virulent on fir, Douglas-fir, Norway spruce, 

giant Sequoia and western hemlock. The 'S' isolates were most 

virulent on non-pine species and more variable than 'P' (one 

was avirulent). The Australian isolate was moderately virulent 

only on ponderosa pine, Sequoia, and Sitka spruce. Sitka 

spruce was highly susceptible to all isolates and incense-cedar 

was highly resistant to all isolates. 

230 

EFFECTS OF WATER STRESS ON WHITE FIR WOUND RESPONSE TO 

TRICHOSPORIUM SYMBIOTICUM, FUNGAL SYMBIONT OF THE FIR ENGRAVER 

SURVIVAL OF MUCOR PIRIFORMIS ON COLONIZED STONE FRUIT ENDOCARPS. 

T. J. Michailides, Univ. of Calif., Berkeley, Kearney Agric. 

Center, Parlier, 93648, and J. M. Ogawa, Dept. of Plant Pathology, 

Univ. of Calif., Davis, 95616. 

BEETLE. W. J. Otrosina and G. T. Ferrell, USDA Forest Service, 

Pacific Southwest Forest and Range Experiment Station, 

Berkeley, CA. 

Effects of water stress on white fir reaction to stem 

Sporangiospores of 1lucor piriformis attached to peach and 

nectarine endocarps an opartially buried survived better in dry 

(-1,300 1,300tbarn bars enari matric potend potential) partially soil buried than survve in wetd0.3bary 

wet (-0.3 

in 

bar 

mtests. 

matric 

potential) soil and longer at 0 and 10 C than at 27 and 33 C. 

Sporangiospores declined over time in a polynomial (quadratic) 

fashion in wet soil a all temperatures and in dry soil only at 

33 C. The fungus grew and sporulated on endocarps incubated at 

0, 10, and 21 C but not at 27 or 33 C. Washings from endocarps 

induced germination of sporangiospores of M. piriformis (up to 

50%) and increased its growth and sporulation. The decline of 

M. piriformis propagules on endocarps buried in soil in a peach 

orchard was exponential after the initial 5 mo. Chlamydosporelike 

structures developed in mycelia and sporangiophores were 

inoculation with the fungal symbiont of the fir engraver beetle 

(Scolytus ventralis LeC.) were assessed in greenhouse and field 

carried 

Inoculum 

by individual 

was selected 

beetles. 

from surveys of fungal 

Among 

populations 

the most prevalent fungi 

recovered from beetle washings were Trichosporium symbioticum, 

unidentified species of Cladosporium, Graphium, Paecilomyces, 

Penicillium, and yeasts. In both greenhouse and field 

inoculations with T. symbioticum, firs under high water stress 

(xylem pressure potentials under -2.0 mPa) produced lesions 

which were longer and less resinous than those of firs under 

lower stress. The differential host reactions to this fungus 

may be potentially useful in assessment of susceptibility 

drought stressed white fir to attack by the bark beetle-fungus of 

complex. 

found in decayed tissues. More than 75% of the colonies of M. 

piriformis recovered from propagules on endocarps originated 

from sporangiospores.23 

NUCLEI IN NATURE AECIOSPORES FRON TWO ZYNODENES OF PERIDERMIUM 

231 HARKNESSII IN CALIFORNIA. 0. R. Vogler, F. W. Cobb, Jr., and 

231 L. Epstein, Department of Plant Pathology, University of 

INTERFERTILITY OF '5' AND 'P' GROUPS OF HETEROBASIDION ANNOSUM California, Berkeley, CA 94720. 

IN NORTH AMERICA. T.E. Chase, W.J. Otrosina, and F.W. Cobb, Jr. 

USDA-Forest Service, Pacific Southwest Expt. Sta. P.O. Box 245, We used epifluorescent microscopy to count nuclei in aecio- 

Berkeley,CA 94701 and Department of Plant Pathology, University spores from two zymodemes (electrophoretic types) of Peniof 

California, Berkeley, CA 94720. dermium harknessii J. P. Noore. For each single-gall isolate, 

spores were fixed overnight at 4C in 3.7% formaldehyde, 

pelleted by centrifugation, resuspended in 0.1 ug/ml 4,6- 

H. annosum in North America consists of two biological species diamidino-2-phenylindole (DAPI) for 6 min, pelleted and rinsed 

designated '5' and 'P. In experiments to be discussed with water 6 times, and mounted in 75% glycerol in 25% phosinterfertility 

(% dikaryons formed) between groups in the phate buffer (pH 8). Nuclei were counted at 800X using filters 

western U.S. was quantified as cm. 18%. Interfertility is not recommended for DAPI by the microscope manufacturer(Zeiss). 

random but can be ascribed to presence of '+ intersterility Spores from zymodeme I (collected from several pine hosts 

alleles in both groups. Thne distribution of intersterility throughout California) were predominantly (>75%) binucleate, 

alleles, the nature of inter-group hybrids, their stability, while spores from zymodeme II (collected from lodgepole pine in 

and significance in the pathology and speciation of the fungus the central Sierra Nevada) were predominantly (>85%) uniwill 

be discussed. Isozymes provide additional markers for nucleate. Implications for the nuclear behavior of this 

monitoring heterokaryon and dikaryon formation and stability, endemic forest pathogen are discussed. 


234

236 240 

INDUCTION OF RESISTANCE TO BLIGHT IN CHINESE CHESTNUT. ROOT DAMAGE IN WHITE ASH INFECTED WITH MYCOPLASMALIKE 

Louis Shain and Joseph B. Miller. Dept. of Plant Pathology, ORGANISMS. A. T. Dyer and W. A. Sinclair, Department of Plant Pathology, 

University of Kentucky, Lexington, KY 40546. Cornell University, Ithaca, NY 14853. 

The mechanism of resistance to blight, caused by the Asian Pathological changes were monitored in roots of white ash (Fraxinus 

pathogen Cryphonectria (Endothia) parasitica, by Asian species americana) infected with the mycoplasmalike organisms (MLOs) associated 

of chestnut is unclear. Previous workers focused on preformed with ash yellows. Seedlings in their 4th year were potted in soil or installed 

compounds, i.e. those present prior to infection, to explain in hydroponic culture after grafting with MLO-infected shoots or (fo 

the resistance of Chinese chestnut (Castanea mollissima). Bark inhroponic ctre after gfn wiTh e shot or (for 

tannins, ether solubles, and proteins were implicated. Our hypo- controls) with strips of their own bark. The DAPI (4',6-diamidino-2thesis 

is that compounds induced after infection may play a phenylindole.2HCl) fluorescence test indicated MLO infection of roots of all 

definitive role in host resistance. Preformed inhibitors would inoculated but no control seedlings 33 days after grafting. Dead and dying 

be expected to protect the host during the growing as well as lateral roots were first noted at 34 days, and leaves on some plants began 

the dormant season. Our inoculation results indicate, however, wilting at 68 days, after grafting. In secondary phloem of living, diseased 

that while cankers expanded more quickly on American than on lateral roots, starch accumulation was reduced, hyperplasia developed, many 

Chinese chestnut during the growing season, canker expansion on sieve tubes and companion cells collapsed, and sclerenchyma was commonly 

dormant stem segments was comparable for both hosts. Prelimi- observed adjacent to the collapsed elements. Tap roots and stems remained 

nary bioassays of extracts from challenged and unchallenged alive. After vernalization, diseased plants grew feebly. Control plants showed 

bark from both species, furthermore, indicate that compound(s) none of the above symptoms. Root damage may be important in the decline 

inhibitory to C. parasitica were produced by bark of challenged of ash naturally infected with MLOs. 

Chinese chestnut only during the growing season. 

237 

SCREENING YOUNG SEEDLINGS OF CHESTNUT FOR BLIGHT RESISTANCE. FV 

Hebard and L. Shain. University of Kentucky, Lexington, KY 40546. 

241 

PRODUCTION OF MONOCLONAL ANTIBODIES 

AGAINST SPECIES OF THE ARMILLARIA MELLEA 

COMPLEX 

Greenhouse-grown chestnut seedlings, 1 and 2 years old, were tested for blight resistance by 

direct inoculation with Endothiaparasitica and by measuring ethylene production in stem 

segments exposed to mycelium for 2 days. Tests included highly blight-resistant Chinese 

chestnut and blight-susceptible American chestnut, their moderately blight-resistant first 

generation hybrids, and first backcrosses to American chestnut, as well as grafted scions of 

very highly blight-resistant Chinese chestnut, cv Nanking. The mean of blight resistance in a 

backcross population should be intermediate between the hybrids and American chestnut. In 

the direct inoculation test, there was little canker expansion on cv Nanking, confirming its 

very high resistance to blight. Rates of canker expansion were quite similar for the other 

populations. Seedling Chinese chestnut could be distinguished from the remaining 

populations due to superficiality of cankers and sparse fructification. Thus direct inoculation 

of young chestnut seedlings could distinguish very highly resistant plants from highly 

resistant plants, and these in turn from moderately resistant and susceptible plants, but it could 

not distinguish moderately resistant and susceptible plants. In contrast, the ethylene technique 

correctly ranked all classes of plants. However, we were unable to precisely distinguish 

resistance classes within the backcross population because of large variance associated with 

the technique. This problem may be solvable by increasing the number of replicates. 

T. A. B6rub6 and M. Dessureault. Centre de Recherche en 

Biologie Forestiere, Faculte de Foresterie, Universit6 Laval, Ste- 

Foy, Qu6bec, Canada, GIK 7P4. 

The root pathogen, Armillaria mellea in the broad sense is 

comprised of nine reproductively isolated groups or biological 

species in North America. Soluble proteins characteristic of 

species I and V were separated and purified by SDS-PAGE. These 

proteins were used to generate specific monoclonal antibodies 

against species I and V from immunized spleen cells of BALB/ 

against spe ie I Vomi cells oBLB/c 

strain and murine myolema cell line Sp 2/0. Immunoblotting 

analysis and ELISA confirmed specificity against species I and V. 

The usefulness of this technique to recognize haploid and diploid 

isolates of mycelia, rhizomorphs and fruiting bodies of all nine 

species from different geographical area will be discussed. 

242 

238 INCIDENCE AND SILVICULTURAL SIGNIFICANCE OF ENDOCRONARTIUM 

CELLULAR CHANGES IN SLASH PINES CAUSED BY COMPATIBLE AND 

INCOMPATIBLE REACTIONS TO CRONARTIUM QUERCUUM F. SP. 

FUSIFORME. C. H. Walkinshaw. USDA, Forest Service, 

Southern Forest Experiment Station, Gulfport, MS 39505. 

HARKNESSII ON JACK PINE IN ARTIFICIALLY REGENERATED AREAS OF 

NORTHWESTERN ONTARIO. J. Juzwik and N. Chong, Pest 

Management Section, Ont. Min. Nat. Res. , Sault Ste. 

Marie, Ontario, P6A 5N5. 

Fifty n ine slash pine (Pinus elliottii Engelm . var . 

elliottii) families ellitti) that tat vry vary failis greatly reaty in i rut rust resistance reistnceduring 

were inoculated with single-gall field isolates of Cronartium 

Pine-pine gall rust (Endocronartium harknessii [J. P. 

M o or e ac c o u st f o r 9 9 % o fa ll ru st s eJr 

Moore)) a accounted 19B7 survey f or of >99% jack of pine all (Pious rusts banksiana encountered Lamb.) 

in 71 artificially regenerated sites in the Northwestern 

quercuum (Berk.) Miyabe ex Shirai f. sp. fusiforme. Tissues Region of Ontario. The sites had been regenerated with 

were fixed 9 to 30 days after inoculation, processed, 

sectioned, and stained for light microscopy. Cortical cells planted stock (bareroot or container seedlings) or by 

invaded by the fungus acted as carbohydrate sinks. In cells direct seeding (ground or aerial) . In the six distric 

of susceptible plants this was evident by a high affinity of the Region, district averages for rust incidence 

for stains. Nuclei in these cells were normal in size and 

shape. In contrast, cells in resistant seedlings were 

swollen, had faded chromatin, and showed signs of hydrolytic 

enzyme activity. Deposition of ergastic substances in cells 

of resistant seedlings did not coincide with changes in 

cortical cells. 

mean ranged disease from 6.7incidence 

17.2%. Significant differences in 

among regeneration strategies 

were 1.%o not detected. h akpn Galls occurred bevd on hs the main anse stem of 

i4%o th jakpn obevd Tes min tm 

infections were associated with no damage except for 

the actual gall on 67% of the trees, with crooks or 

multiple leaders on 29%, and with mortality of 3.4%. 

239 243 

SURVEY OF TIlE NATIONAL MALL FOR ELM LEAF SCORCH ASSOCIATED CONE HARVESTING PRACTICES AFFECT THE INCIDENCE OF BLACK SEED 

WITH XVLELLA PASTIDIOSA. J_. L. Sherald and J. Lei, Center for ROT OF SLASH PINE CAUSED BY LASIODIPLODIA THEOBROMAE. S. W. 

Urban Ecology, National Park Service, Washington, DC, 20242 Fraedrich, I. Miller, USDA Forest Service, Olustee, FL 32072, 

and Twyford International, Inc., Santa Paula, CA 93060. F. J. Spirek, Nekoosa Packaging Co., Valdosta, GA 31603. 

Elm trees of the National Mall in Washington, D.C. were Cones were harvested from 4 ramets of 4 clones on 3 dates (B/31, 

examined for leaf scorch symptoms characteristic of those 9/14, 9/26/88) and divided among 3 treatments: no ground 

associated with Xy1ella fastidiosa. Examinations were made contact/no storage (NGC/NS), no ground contact/storage (NGC/S), 

of 580 trees, predominantly Ulmus americana, in Sept. of ground contact/storage (GC/S). Seeds from each cone were 

1986, 1987, and 1988. Approximately 25% of the trees were assayed for fungus damage and fungi. Specific gravity was 

affected each year. Trees were classified according to the determined for cones from each tree at each sample period. 

percentage of the canopy affected: (0%) 75%, (trace - 5%) Fungus-damaged seed were absent in the NGC/NS treatment, and 

13%, (6 - 25%) 4%, (26 - 50%) 3%, (51 - 75%) 1%, (76 - 99%), occurred at low frequency (0.25-2.25%) among cones in the NGC/S 

3%, (100%) 1%. Leaves were collected in June and Sept., 1987 treatment. Incidence of fungus-damaged seeds was high (12%) 

from 47 trees for detection of _X. fastidiosa with an ELISA among cones harvested early in the CC/S treatment; incidence 

test kit. Samples were collected from all symptom classes, decreased with later harvest dates. Analysis of data by clones 

Of 18 leaf scorch-affected trees 12 were detected in June provides additional evidence that degree of cone maturation at 

prior to symptom development and 17 in Sept. after symptom harvest may be critical for seed colonization and infection by 

development. In Sept., 2 of the 29 symptomless trees pathogenic fungi. Lasiodiplodia theobromae was the predominant 

produced weak positive ELISA reactions. fungus isolated from diseased seeds. 

Vol. 79, No. 10, 1989 1165

244 Factors were studied that limited take-all development on 

wheat following sorghum in doublecropping systems. Two 

CORRELATION OF ARMILLARIA ROOT ROT WITH DAMAGE TO TREES field studies with plots exhibiting decreased take-all 

DEFOLIATED BY THE JACK PINE BUDWORM. K.I. Mallett and W.J.A. severity following sorghum had lower soil pH than plots 

Volney. Forestry Canada, Northern Forestry Centre, 5320 - 122 where wheat followed soybean. Bioassays for microbial 

St. Edmonton, Alberta, Canada T6H 3S5 suppressiveness indicated wheat:sorghum doublecropped soils 

were suppressive to take-all at levels similar to soils with 

Pinus banksiana trees severely defoliated by the jack pine long histories of wheat monoculture. Growth chamber studies 

budworm (Choristoneura pinus) in 1985 and '86 were dissected to indicated no differences in disease development on wheat in 

determine the extent of root infections by Armillaria sp. The artificially infested soils after amending with various 

severity of damage to the tree crowns was correlated with the summer crop residues, including sorghum, or planting with 

incidence of root infection: 100 % of the dead trees were these summer crops, indicating no differences in pathogen 

infected with Armillaria sp., 60% of the trees whose upper survival. 

crowns had been killed were infected, but only 20% of the 

healthy trees were infected. The value of these observations 

in hazard-rating jack pine stands is discussed. 249 

RHIZOSPHERE COMPETENCY OF FIVE Fusarium SPECIES ON MAIZE PLANTED 

245 IN THE FIELD. C. M. Ocamb, T. Kommedahl, and P. M. Burnes, 


VARABILITY IN VM CE OF OOSPORE INOCUIUM OF 1HYflJORA Paul, MN 55108. 

CAPSICI AND THE RELATIONSHIP OF ME DENSITY OF OOSPORES IN 

SOIL TO PI I4JTAMFLI. J.H. Bowers and D.J. Mitchell. Dept. In 1988, maize seeds were coated with one of five species of 

of Plant Pathology, Univ. of Florida, Gainesville, FL 32611. Fusarium and planted in the field. Virtually the entire root 

systems of the seedlings were colonized by each of three 

Oospore progeny from 20 crosses of pathogenic isolates of species. Of these, F. moniliforme constituted 90%; E. 

Phytophthora capsici were found to vary in pathogenicity to proliferatum, 48%; and F. oxysporum, 39% of the colonies 

pepper. Only oospore inoculum from specific crosses caused isolated from roots. Seedling root systems were not extensively 

disease in pepper seedlings with an initial inoculum density colonized by F. solani (9%) or F. equiseti (11%). The seedling 

of 25 oospores per gram of soil. The highest mortality (30 to root systems of the dry control, water control, and the captan 

75%) resulted fra oospore inoculum produced from crosses in- treatment yielded primarily F. oxysporum and F. solani. 

volving certain isolates of A2 compatibility type. Oospore Rhizosphere competence differed in laboratory, greenhouse, and 

inoculum from other crosses caused little or no disease (0 to field tests. Field studies offer evidence that Fusarium species 

5%). Oospores produced from one cross resulted in 40 and 60% present in the seed differ as potential inoculum sources for 

plant mortality with inoculum densities of 10 and 15 oospores root infections. 

per gram of soil, respectively. The ID50 was calculated to be 

41 oospores per gram of soil. Inoculum efficiency was calculated 

using the multiple infection transformation as the 

estimated number of infections per propagule and was found to 250 

be 0.011. EFFECT OF INOCULUM DENSITY OF CEPHALOSPORIUM GRAMINEUM AND SOIL 

pH ON CEPHALOSPORIUM STRIPE OF WINTER WHEAT. L. P. Specht and 

246 

T. D. Murray. Department of Plant Pathology, Washington State 

University, Pullman, WA 99164-6430. 

SURVIVAL OF FUSARIUM OXYSPORUM F. SP. VASINFECTUM IN 

SOIL AS AFFECTED BY FOUR CROPPING SYSTEMS. J. P, McEntee, 

R. D. Martyn, and J. L. Starr. Department of Plant Pathology and 

Microbiology, Texas A&M University, College Station, 77843. 

Winter wheats (cvs. Stephens and Nugaines) were grown in soil 

adjusted to pH values of 4.5-7.6 and maintained at -0.01 MPa. 

Soil in pots was drenched with conidial suspensions of Cephalosporium 

gramineum when seedlings were in the four- to five-l 

Microplots infested with a sand:cornmeal inoculum of Fusarium 

oxysporum f. sp. vasinfectum (FOV) were planted with a FOVsusceptible 

cotton cultivar (Rowden), a resistant cultivar (Auburn 634), 

a nonhost (Sorghum), or left fallow. Populations of FOV were 

determined initially and again at mid- and end-of-season. At the end 

of the season, plants were examined for vascular browning (VB) and 

isolation frequency of FOV from root and stem tissue. Final FOV 

populations were highest in microplots planted to sorghum, while no 

difference was observed between FOV populations from Rowden and 

Auburn 634. The fallow treatment had the lowest FOV populations. 

Differences in plant numbers with VB and in the nature of the VB 

were observed between cotton cultivars. VB incidence and severity was 

greatest in Rowden; however, no significant difference in isolation 

frequency of FOV was observed between cultivars. Examination of the 

sand:cornmeal inoculum revealed that it was composed largely of 

microconidia, chlamydospores and mycelia. When dried under sterile 

conditions, the microconidial and mycelial populations decreased 

rapidly, leaving primarily chlamydospores. 

stage. Inoculum density of C. gramineum, soil pH, and cultivar 

h " 

' 

all had significant (P

y suppression of Mn oxidizers in the wheat rhizosphere. This study was 

initiated to determine if the pathogenicity of Ggt influenced rhizosphere 

populations of Mn oxidizing bacteria. Nine isolates of Ggt, differing in 

Phytophthora root rot of Eastern white pine, Pinus strobus (PS), 

occurred in field and container grown trees during August of 

1988. Both Phytophthora citricola (Pci) and P. cryptogea A 2 

pathogenicity, grown on PDA and Bromfield's media were evaluated on 

wheat plants grown for 5 weeks in a low-N field soil. The population of 

rhizosphere Mn oxidizing organisms was determined by dilution plating 

(Pcr) were isolated from dead roots of 6-year-old trees in a 

Bucks County field where 44% and 8% out of 360, were dead and 

yellowed, respectively. The disease occurred more in clay 

soils and lower slopes than in upper shale areas. Pci was isorhizosphere 

soil on Gerretsen's medium. The Ggt isolates strongly influ- lated from a shipment of 200 container grown 3-year-old Ps from 

enced population levels of Mn oxidizers (600 CFU/g soil- 63,000 CFU/g western PA. Both Pcr and Pci caused sudden wilting, greying, 

soil); however, virulence of Ggt was independent of population of Mn tip-curling, and browning of needles as a result of root rot 

oxidizers in the rhizosphere. when 2-year-old Ps were transplanted into a soil mix containing 

the inoculum grown in V8 juice-vermiculite medium; Pcr caused 

253 

a greater tree mortality and shoot stunting than Pci. 

SUGAR BEET STAND ESTABLISHMENT AND SOILBORNE PATHOGENS IN TARE 

SOIL. Carol E. Windels and Donna J. Nabben, Northwest Experiment 

Station, University of Minnesota, Crookston, 56716. 

When sugar beets are piled for storage in the Red River Valley, 

soil removed from beets during piling (tare soil) is returned to 

fields. In 1986-88, tare soils from 45 fields were evaluated for 

pathogens by a seedling assay. Aphanomyces cochlioides was 

isolated from dying seedlings in four of six tare soils where it 

had been verified during the growing season; no emergence 

occurred in the other two soils. Of 39 tare soils collected at 

random, Rhizoctonia solani was isolated from dying seedlings in 

22 sites; in 1987, 66% of the cultures were AG-4, 27% AG-2-2, 

and 7% were unidentified. Seedling emergence in paired tare and 

field soils was statistically less (55 vs 89%, respectively) for 

22 of 44 locations. At four of these sites, emergence averaged 

3% in tare soils, and was associated with high soil pH, organic 

matter, soluble salts, phosphorus, and potassium compared to 

most tare soils and all field soils. Since tare soils often 

become mixed, returning tare soil to beet fields can spread 

soilborne pathogens and affect beet emergence. 

254 

INOCULUM DENSITY OF PYTHIUM MYRIOTYLUM AND ITS RELATION TO 258 

PEANUT PLANT VIGOR. R. Garcia-Espinosa, R. Rodriguez-Kabana, 

and P. A. Backman, Alabama Agricultural Experiment Station, SUSCEPTIBILITY OF SUGAR BEET CULTIVARS TO AG-2-2 CULTURES OF 

Dept. of Plant Pathology, Auburn University, AL 36849-5409. RHIZOCTONIA SOLANI ISOLATED FROM LEGUMES AND SUGAR BEET. Cheryl 

A. Engelkes and Carol E. Windels, Department of Plant Pathology, 

On a quantitative basis, 'the damage caused by P. myriotylum University of Minnesota, St. Paul, 55108 and Northwest 

to the peanut host has been documented mainly for seedling Experiment Station, Crookston, 56716. 

disease and pod rot. The effect of increasing inoculum 

levels of P. myriotylum on peanut plants allowed to germinate Crown and root rot of sugar beet (Beta vulgaris L.) caused by 

free of, but immediately exposed to the pathogen was AG-2-2 of Rhizoctonia solani occurs in Minnesota and North 

evaluated. Significant negative correlations were observed Dakota. Cultures of AG-2-2 isolated from fababean, pinto bean, 

between retrieved populations of the pathogen and plant vigor and soybean (crops rotated with beet) and from sugar beet were 

parameters. Based on exponential correlation analysis, a 50% field-tested for pathogenicity on the roots of three sugar beet 

reduction in leaf index can be expected with 30 to 40 cultivars. Roots were rated on a 0-7 scale (O=healthy, 7=100% 

propagules/gram (ppg) of soil, and a 50% reduction in fresh rotted). 'Maribo Ultramono' gave a higher root rot index (3.3) 

weight can be expected with 50 ppg of soil. On average, compared to 'ACH 184' (2.1) and 'Fc712' (1.8). Four cultures 

populations >100 ppg of soil caused a profound proportional from pinto bean gave an average root rot index of 4.4; tWO 

stunting of all the aerial parts of the plant. The cultures from soybean averaged 3.7; ten cultures from sugar beet 

importance of this pathogen to peanut production in the averaged 1.8; and three cultures from fababean averaged 1.6. In 

southeastern U.S.A. is discussed. fields where AG-2-2 is present, rotation of legume crops can 

provide inoculum that causes crown and root rot of sugar beet. 

255 259 

SYMPTOM DEVELOPMENT OF SOYBEAN SUDDEN DEATH SYNDROME IN RELATION TO T-2 

TOXIN PRODUCTION BY FUSARIUM SOLANI. B.S. Corwin, T.D. Wyllie, G.E. 

Rottinghaui, and F.P. Roii, Department of Plant Pathology and Vet. Med. Diag. 

Lab., University of Missouri, Colunbia, MO 65211, and USDA/APHIS/NVSL, 

SHOOT TIP PROPAGATION USED TO PRODUCE AGROBACTERIUM-FREE MUSCA- 

DINE PLANTS. D. E. Griffin, C. H. Graves, Jr., and K. L. Thies, 

Department of Plant Pathology and Weed Science, Miss. State, MS 

Amen, IA 50010. 39762. 

We report the possible involvenent of a tosin with soybean sidden death 

syndrome (SDS). Fusarium solani isolate M01247 was obtained from field Because of the incidence of crown gall caused by A robacterium 

grown soybean plants with symptoms of SDS. Pathogenicity of this isolate tumefaciens (AT) in muscadine (Vitis rotundifolia) plantings in 

was confirmed in the greenhouse. The isolate was grown in Czapek-Dox broth Mississippi and widespread systemic presence of Agrobacterium 

in stationary culture at 22 + 2 C. Culture filtrates were estracted with spp. in symptomless cultivated muscadine, there is need to 

ethyl acetate, concentrated, and fractionated by thin layer chromatography. determine pathogenicity of the vascular isolates from such 

One component of the fraction was identified as T-2 tosin and the plants. Muscadine is the only known host for muscadine isolates 

identification was confirmed by tandem mass spectrometry. Soybean plants, of AT. Failure of repeated screening techniques to free rooted 

cv. 'Lee 74', in growth stage V3 were root dipped in 200 l of 100, 500, softwood cuttings of AT led to production of muscadine plants 

or 1000 ppm T-2 toxin in 20 ml of water for 45 min. Plants esposed to all through tip culture. Muscadine plants for determining pathoconcentrations 

of T-2 eshibited marginal necrosis and chlorosis of leaflets, genicity of AT isolates have been successfully produced from 

as well as cupping within 2 days posttreatment. No symptoms developed on shoot tips on variations of Murashige-Skoog and Woody Plant Methe 

controls. The similarities in foliar symptoms between plants inoculated dium amended with benzylaminopurine. The effectiveness of 

with F. solani isolate M01247 and plants treated with T-2 tosin suggest different growth regulators on shoot proliferation will be disthat 

the tosin may be involved in soybean SDS. cussed. 

256 260 

EVALUATION OF 'BULGARIA 12' TOMATO SOMACLONE PROGENY FOR 

PHYTOPHTHORA ROOT ROT OF WHITE PINE IN PENNSYLVANIA. S. H. Kim, RESISTANCE TO CLAVIBAcTER MICHIGANENSIS SUBSP. MICHIGANENSIS. 

T. N. Olson, and P. Keller. Pennsylvania Department of Agricul- R.M. De Vries-Paterson and C.T. Stephens. Department of Botany 

ture, Harrisburg, 17110 and Messiah College, Grantham, Penusyl- an--d Pl-ant Pathology, Michigan State University, East Lansing, 

vania 17027. MI 48824. 

Vol. 79, No. 10, 1989 1167

A total of 191 somaclones were generated from leaf explants ID 83843 and 3 Kimberly, ID 83341; 2 of 'Bulgaria 12', a tomato cultivar with partial resistance 

to Clavibacter michiganensis subsp. michiganensis (CMM), 

causal organism of bacterial canker of tomato. Thirty-two 

percent of the somaclones were discarded due to abnormal 

phenotype or poor growth. One aberrant Rl line produced 

currant-type fruit on a vine. Fifty-five R1 progeny were 

screened for resistance to CMM in a growth chamber. There 

were some variations in reaction to CMM among progeny regenerated 

from the same individual callus. A few R1 lines showed 

an increased level of resistance to CMM and could prove useful 

to plant breeders. 

261 

Epidemiology and spread of Clavibacter michiganensis subsp. 

michiganensis on tomato. R. J. Chang, S. M. Ries and J. K. 

Pataky. Dept. of Plant Path., Univ. of Illinois, Urbana. 

Harris-Moran Seed Co., San 

Juan Bautista, CA 95045. 

Black chaff of gramineacious crops, caused by Xanthomonas cam- 

pestris pv. translucens (Xct) develops from seedborne inoculum. 

A program was established in Idaho to produce Xct-free seed. 

In 1987 and 1988, black chaff occurred in spring wheat fields 

grown from Xct-free seed at Tetonia and Aberdeen. Epiphytic 

populations of Xct were found on leaves of known hosts (Bromus 

inermis, Agropyron repens) and on previously undescribed hoata 

(Poa pratensis, Festuca arundinaceae, F. rubra, Hordeum lepori- 

num, Medicago sativa) near these fields. Black chaff symptoms 

were not observed, but they contained populations of 13 cfu to 

7.5 x 106 cfu per gram fresh tissue. Pathogenicity tests 

indicated a broad and variable host range, with high levels of 

virulence on wheat and barley. Xct was not isolated from plants 

in southwest ID where black chaff does not occur frequently. 

Ten rifampin-resistant mutants of Clavibacter michiganensis 

subsp. michiganensis (CMM) were used to study the epidemiology 

and spread of bacterial canker. CMM spread from infected to 

healthy plants in beds of 10,000 direct seeded plants clipped 

six times with a rotary mower. Initial disease incidences of 

0.01, 0.05, 0.1 and 0.5% resulted in 4, 8, 12 and 83% infected 

plants, respectively, when clipped plants were transplanted. 

CMM also was spread during transplanting when healthy and 

diseased plants were pulled, shaken, and mixed together in 

shipping crates. Twelve, forty-two and fifty-two per cent of 

the transplanted plants showed canker symptoms when initial 

proportions of diseased plants placed in crates were 1, 5, and 

10%, respectively. A susceptible and a tolerant cultivar 

supported epiphytic populations of CMM of 107-109 CFU/g fresh 

weight, although symptoms of secondary infection, marginal 

scorch of leaflets, were more severe on the susceptible 

cultivar. 

265 

PSEUDOMONAS VIRIDIFLAVA, THE CAUSE OF A STEM CANKER OF POINSET- 

TIA PLANTS. Arthur W. Engelhard and Jeffrey B. Jones. Univ. 

of Florida, IFAS, Gulf Coast Research & Education Center, 

Bradenton, FL 34203 

A stem canker occurred in December 1984 on potted poinsettia 

plants grown outdoors near Bradenton, Florida. A fluorescent 

bacterium was isolated that caused a positive reaction for 

tobacco hypersensitivity, DL-lactate, D(-) tartrate, erythritol 

and a negative one for arginine dihydrolase, oxidase and levan. 

It, thus, was identified as Pseudomonas viridiflava. Two 

strains of the bacterium tested'were patho"geni-con all seven 

cultivars of poinsettia plants evaluated, although the culti- 

vars varied in susceptibility. The disease was most severe at 

10 and 15*C, mild at 27.70C. and no disease occurred at 32.2°C 

These Florida strains of P. viridiflava are not expected to 

262 

become a serious problem in poinsettia plant production in 

Florida because of their low optimum temperature requirement 

for their pathogenicity. 

Survival of wildtype and gentically-altered Erwinia carotovora 

subsp. carotovora (ECC) strains in soil. H.K. Austin, G.H. Lacy 

qnd J. Cairns, Jr., UCE&HMS and the Laboratory for Molecular 266 

Biology of Plant Stress, VPI&SU, Blacksburg, VA 24061-0331. 

CYPROCONAZOLE: A NEW SYSTEMIC FUNGICIDE FOR TURF DISEASE 

We assessed survival of genetically-altered, kanamycin-resistant MANAGEMENT. G. G. Thomas, W. B. O'Neal, and P. Schmid. 

L-864 and wildtype, rifampin-resistant L-863 strains of ECC in Sandoz Crop Protection Corporation, 1300 East Touhy Avenue, 

non-amended soil; in soil amended with germinating seeds of rad- Des Plaines, IL 60018. 

ish, carrot, tomato (all hosts), or grass (non-host); and in 

soil amended with potato tuber pieces or enrichment medium (EM; Cyproconazole, a-(4-chlorophenyl)-(l cyclopropylethyl l)-l 1,2, 

Phytopathology 66:367-370, 1976). Survival of a genetically- 4-triazole-l-ethanol, is a new broad spectrum fungicide with 

altered, pectate lyase-deficient ECC strain L-872 in soil excellent systemic activity. Cyproconazole has rapid plant 

amended with potato tuber pieces or EM was also studied. L-863 tissue penetration and is translocated primarily acropetally. 

and L-864 did not differ significantly (P > 0.05) in response to At low dosages(O.1 to 0.4 kg a.i./ha) cyproconazole has 

seedlings, potato pieces, or EM; their densities increased > demonstrated control of a wide range of fungi important in 

500-fold with potato pieces (P < 0.01). Indigenous bacteria turf grass disease management. Common diseases, brown patch 

increased 30-fold in soil amended with potato pieces or the caused by Rhizoctonia solani and dollar spot caused by 

polygalacturonic acid EM. Survival and detection of the geneti- Sclerotinia homoeocarpa have been effectively controlled for 

cally-altered ECC was enhanced in soil amended with potato tuber a period of three to five weeks following application. In 

pieces or EM. addition to foliar disease control, cyproconazole has shown 

potential to control turf grass root diseases; necrotic ring 

spot caused by Leptosphaeria korrae and summer patch caused by 

263 Magnaporthe poae. 

COLLOIDAL GOLD/PROTEIN A IMMUNOBLOT ASSAY FOR Clavibacter xyli 

subsp, xyli CAUSE OF RATOON STUNTING DISEASE OF SUGARCANE. 267 

K. E. Damann. Jr. and W. J. Todd. Dept. of Plant Path. & Crop 

Physiol., and Dept. of Vet. Science, LABS, LSU Agricultural CONTROL OF IRIS RUST WITH FUNGICIDES. Albert 0. Paulus and 

Center, Baton Rouge, LA 70803. Robert 0. Raabe. Departments of Plant Pathology, University of 

A one-step assay for 

California, 

detection of 

Riverside 

Clavibacter 

92521 

xyli 

and 

subsp, 

Berkeley 

xyli 

94720. 

was developed. Application of 1 ul of vascular extract or Rust of iris, resulting from infection by 

sugarcane 

Puccinia 

sap 

irndia, 

from ratoon 

is 

stunt-diseased or healthy cultivar comimon in coastal California. 

L 62-96 

Bearded 

to 

iris 

a nitrocellulose 

(Iris germanica), 

strip gave a positive red-purple Dutch iris (I. xiphium and I. filifolia x I. xiphium) and a 

color in response to infected plants. This occurred when 

strips were incubated 

Pacific 

for 

coast 

1 hr 

native, 

with 1 ml 

I. 

of 

munzii, 

protein A/gold 

are particularly 

and In 

susceptible. 

experiments to control rust 

10 

on Dutch 

ul of a 

iris 

100-fold 

cv. Blue 

dilution 

Ribbon 

of 

in 

antiserum to either C. x. southern California, plants were sprayed 

subap, 

3 

xyli 

times 

or 

at 

C. 

2-week 

x. subsp, cynodontis. The technique was intervals. Diniconazole gave excellent 

also used 

control, 

in tissue 

Mobay 

blots 

1608 

by centrifuging vascular contents gave very good control and flusilazole 

from 1 

and 

cm stalk 

myclobutanil 

sections 

gave 

onto a nitrocellulose filter. The moderately good control. In northern 

distribution 

California, 

of colored 

sprays 

spots 

were 

on the filter was compared with applied 3 times at 3-week intervals 

the 

to seedlings 

distribution 

of bearded 

of vasucular bundles exhibiting alkaline- iris. Best control resulted with Ciba Geigy 453 

induced 

and myclobutanil. 

metaxylem autofluorescence in the stalk section. Mdrtl odcnrlrsle ihoyabxn eoai 

Theresltswil bedisussd.and Nor Am SN 596. Control with Nor Am SN 39865, penconazole, 

triadimefon, prochloraz, and benomyl was not satisfactory. 

264 268 

NEW PERENNIAL HOSTS OF EPIPHYTIC POPULATIONS OF XANTHOMONAS 

CAMPESTRIS PV. TRANSLUCENS. 0. C. ThompsonI, N. W.~Schaad, FLOWERING OF FLORIBuNDA ROSES IN RESPONSE TO WEEKLY APPLICATIONS 

and R.C. Forster 3 . IDept. PSES, University of Idaho, Moscow, OF A COMBINATION AND SINGLY APPLIED FUNGICIDE SPRAY. P. F. 

1168 PHYTOPATHOLOGY

Colbaugh. Texas Agricultural Experiment Station, Texas A&M Univ. 

Res. and Ext. Center, 17360 Coit Rd., Dallas, TX 75252. 

Research Station, Agriculture Canada, 

Winnipeg, Manitoba, R3T 2M9 

195 Dafoe Road., 

Replicated field plantings of four varieties of floribunda rose 

Carboxin, at concentrations of from 0.01 to 1000 iig/mL, was 

applied to 24-h sporelings of three isolates of Ustilago nuda 

were sprayed weekly with a combination benomyl 50W + mancozeb growing in liquid shake culture. The cultures were examined 

80W or triforine 16.5 EC protective fungicide spray to determine by light microscopy after further incubation. The number of 

their influence on flowering. Both fungicide spray programs are 

in use by commercial Texas rose growers for foliar disease conapical 

cells per sporeling was used as a measure of growth. 

Death of apical cells of sporelings was determined with Evans 

trol. Counts of flower blossoms and opened flower buds were made blue. Sporelings were not affected by 24 h of exposure to 

on treated plants each week prior to the application of fungi- carboxin concentrations below 0.03 ug/mL. Carboxin 

cides. The flowering of treated roses during August 1985 varied concentrations of 1 ug/mL or higher stopped growth of 

by rose variety; however, the mean weekly flowering of all var- sporelings without causing apical cell death. However, 

ieties was increased on roses receiving triforine sprays. The carboxin concentrations between 0.03 and 1 jug/mL reduced 

increased flowering response of triforine treated rose varieties growth rate and killed apical cells. An agar growth test 

over the four week period was as follows: Charisma (65%), Angel showed that most sporelings were dead after 5 days at 0.125 

Face (55%), Bahia (62%), and Red Gold (74%). Observations of ug/mL while few sporelings were killed by 10 ug/mL for 5 days. 

disease activity during the study indicated both fungicide sprays Carboxin has a fungicidal effect on U. nuda at concentrations 

were effective for controlling powdery mildew and blackspot 

diseases. 

below those at which it has a fungistatic effect. 

269 

CHEMICAL CONTROL OF PHOMOPSIS VITICOLA ON VITIS VINIFERA IN 

CALIFORNIA. G. M. Leavitt, W. J. Moller (deceased), University 

of California Cooperative Extension, 328 Madera Avenue, Madera, 

CA 93637. 

Post bud break spring rains are necessary for the spread of 

Phomopsis viticola. All materials registered for control were 

tested on Vitis vinifera cultivars Thompson seedless and 

Grenache. All were effective in reducing disease incidence. 

Two well timed foliar applications on Grenache at bud break to 

1 inch and 8 days later at shoots 4-6 inches tended to be superior 

in control. Application of one foliar captan at bud break 

equaled dormant treatment control levels but was not as effective 

as the two treatments. On Thompson seedless, one foliar 

application at shoots 2-4 inches after a post bud break rain 

reduced further infections. Significant crop loss on Grenache 

to Phomopsis viticola was measured. 

270 

EFFECT OF FOLIAR IRRIGATION ON THE ACCUMULATION OF METALAXYL 

IN POTATO TUBERS. R. J. Young and Thomas Basden. Dept. of 

Plant Pathology, West Virginia University, Morgantown, WV 

274 

SUPPRESSION OF SOUTHERN STEM ROT OF PEANUT WITH GRANULAR 

26506-6057. 

Below normal rainfall and soil moisture in 1987-88 were related 

to non-detection of metalaxyl (met) activity in tubers. 

Rainfall, May through August (87-88), averaged 38% below 

SOIL INSECTICIDES. A. K. Hagan, and J. R. Weeks. Auburn 

University, Al 36849. 

Peanuts treated with Chlorpyrifos 15G (2.2 kg a.i./ha), 

ethoprop 15G (3.3 ka a.i.Iha). or fonofos 1OG (2.2 kg 

normal. In irrigated plots (1988) soil moisture was kept at a.i./ha) were compared with PCNB 1OG (11.2 kg a.i./ha) for 

20% and plants received a short intensive watering 8 and 24 southern stem rot suppression (ScLerotium rolfsii) in onhours 

after met (one or two) treatments. Metalaxyl activity farm trials, 1985-1987. Stem rot counts compared to the 

was determined by measuring growth of Phytophthora infestans non-treated 198tr987.e semreouc cunesccoparedby the 

on tuber slices inoculated with zoospores, and incubated for noreated corpyrifos cntro and two wr of three ree years ea r by by ethoprop the u and of 

4 and 7 days at 19 C. No met activity was detected in tubers 

from met/non-irrigated plots. Tubers from met/irrigated 

fonofos. Ethoprop and chlorpyrifos suppressed disease as 

well as PCNB each year, and fonofos two of three years. only 

plots showed activity beginning 7 days after treatment, and 

continued through five weekly harvest. Both 8 and 24 hr. 

in 1986 did the insecticide-treated plots outyietd the 

untreated plots while PCNR treatments increased yields in 

irrigations were effective, and 2 met treatments were more 

effective than a single tubes sowe treatment. nometlaxlativtyt At harvest #5, 23% of the 

tubers showed no metalaxyl activity. 

a h o se y ear tr e a ted eds w i it tCN-tee h fo no f os o r ots e th ere op r op hae b ut thn no t 

chtorpyriofos. Pooled results show that all insecticides 

and PCNB suppressed stem rot but only PCNB and 

271 chlorpyriofos increased yield. Yield differences between 

PCNR and chlorpyrifos over 3 years were significant 

EVALUATION OF NATURALLY-OCCURRING TOLERANCE AND RESISTANCE TO 

METALAXYL IN PHYTOPHTHORA MEGASPERMA F.SP. GLYCINEA. K. M. 

Howard and A. F. Schmitthenner, Dept. of Plant Pathology, Ohio 

State Univ., Wooster, OH 44691. 275 

Sensitive, tolerant and resistant isolates of P. meciasperma FUNGICIDAL CONTROL OF TAKE-ALL PATCH. GarX A Chasta ner and 

f.sp. glycinea (Ping) which can grow at 0, 1 and 10 ppm John M. Staley, Wash. State Univ., Puyallup, WA 98371. 

metalaxyl, respectively, were evaluated for growth rate in 

vitro and virulence in vivo in the presence of metalaxyl. During 1984-85, fenarimol and triadimefon (0.29 gm ai/m 2 Resistant isolates were significantly different (£=0.05) from 

) and 

propiconazole (0.63 gm ai/m 2 sensitive or tolerant isolates in all tests and able to grow 

) were applied to 'Highland' bent- 

grass turf in 79.9 ml of water/in 2 and cause disease at concentrations up to 100 5g/ml. Tolerant 

during December or December 

and April. During 1985-86, fenarimol (0.15 - 0.59 gm ai/m 2 isolates varied in response to metalaxyl and were similar to 

) and 

triadimefon (0.07 - 0.29 gm ai/m 2 sensitive isolates in both assays. Qospore progeny from 

) were applied in December or 

April. Applications of propiconazole (0.29 and 0.59 gm ai/m 2 ) 

tolerant isolates segregated for resistance, tolerance and were only made in.-December. In June 1985, 28% of the turf area 

sensitivity to metalaxyl whereas resistant and sensitive in the check plots had take-all patch (TAP), caused by Gaeumanisolates 

appeared to be homozygous. Results suggest that nomyces graminis var. avenae. The level of TAP in the treated 

metalaxyl resistance in Pin is homozygous dominant as reported plots ranged from 0-6% and there were no significant differences 

for P. infestans. between December or December and April applications. Data col- 

lected during June 1986 indicated that December applications 

significantly reduced the level of TAP. The level of reduction 

272 was similar for all three fungicides and was dependent upon the 

rate of active ingredient applied. Applications in April were 

FUNGICIDAL AND FUNGIsTATIC EFFECTS OF CARBOXIN ON MYCELIAI. not as effective as the applications in December. Some phyto- 

GROWTH OF USTILAGO NUDA. 0. Mewcombe and P.L. Thomas. toxicity was observed on the turf treated with propiconazole. 

Vol. 79, No. 10.,1989 1169

276 BORNE AND FOLIAR PATHOGENS IN PEANUT. H. S. McLean and 

P. Schmid. Sandoz Crop Protection, 1300 East Touhy Avenue, 

LY211795 - A Novel Foliar Applied Fungicide for Control of Des Plaines, IL 60018. 

Powdery Mildew in Cereals and Grapes. W. R. Arnold, M. J. 

Coghlan, H. R. Hall, E. V. Krumkalns, Lilly Research Cyproconazole, a-(4-chlorophyenyl)-(I cyclopropylethyl 1)-i 1, 

Laboratories, A Division of Eli Lilly and Company, P. 0. Box 2,4-triazole-lethanol, is a new broad spectrum fungicide with 

708, Greenfield, IN. C. Longhurst, Lilly Research Centre Ltd., excellent systemic activity. Cyproconazole systemic activity 

Erl Wood Manor, Windlesham, Surrey GU20 6PH, UK. is characterized by rapid plant tissue penetration and acropetal 

translocation. In field trials conducted 1984-1988, cypro- 

Lilly 211795 is highly effective as a foliar fungicide on wheat conazole has consistently provided control of important foliar 

and barley powdery mildew (Erysiphe graminis; f. spp. tritici and soil borne diseases in peanut when applied at relatively 

and hordei, respectively) and grape powdery mildew (Uncinula low rates (0.061-0.098 kg ai/ha) on a 14-21 day spray schedule. 

necator). The compound penetrates into the plant tissue very Early and Late leafspot (Cercospora arachidicola and Cercosporirapidly 

and is translocated acropetally. Compound 211795 shows dium personatum) are easily controlled with cyproconazole. 

excellent protectant activity against mildew resistant to DMI Southern Blight (Sclerotium rolfsii) and Rhizoctonia limb rot 

chemistry. When used in mixtures with other fungicides, a wide (Rhizoctonia solani) are also controlled by foliar applications 

range of diseases can be controlled, of cyproconazole within the same rate range as listed for leafspot. 

Peanuts have excellent tolerance to Cyproconazole and 

display virtually no plant growth regulation effects. 

277 

THE EFFECTS OF PENCONAZOLE AND PROPICONAZOLE ON THE 281 

ULTRASTRUCTURE OF PYTHIUM ULTIMUM AND 

PHYMATOTRICHUM OMNIVORUM. J. R. Anciso and S. D. Lyda. Production of hydroxyl radical in photodynamDic biocidal reaction 

Department of Plant Pathology and Microbiology, Texas Agricultural of methionine riboflavin mixture. 0. 0. Tzeng, and N. H. Lee. 

Experiment Station, College Station, TX 77843. Department of Plant Pathology, National Chung Hsing University, 

The accepted mode of action of penconazole (Topasr) and propiconazole 

(Tiltr) is the inhibition of ergosterol biosynthesis. A time course study Production of hydroxyl radical (-OH) was detected from the 

on effects of the two chemicals on Pythium ultimum and methionine riboflavin mixture (MR) under continuous illumination 

Phymatotrichum omnivorum was investigated. The ED 5 0 for P. ultimum by the method developed by Baker and Gebicki. The rate of "OH 

is 25 ppm with penconazole and 98 ppm with propiconazole, while the production from MR was much greater at pHs 4.0-5.0 than at pHI 

ED 5 0 for P. omnivorum is 9 ppm with penconazole and .01 ppm with 6,0-8.0, which indicated the possible involvement of iron conpropiconazole. 

The ED 5 0 (minimum inhibitory concentration) was tamiinant in the reaction. The addition of exogenous iron was 

chosen as the concentration to observe ultrastructural changes. The 

primary observation was the loss of normal-appearing endoplasmic stirulatory to the -OH formation of nR. However, the presence 

reticulum and the abundant appearance of circular endoplasmic of iron chelators like desferal, or phenanthroline, and free 

reticulum-like material in the treated for both species. P. ultimum radical scavengers like thiourea, all greatly reduced the radiwhich 

does not synthesize or require sterols has shown "moderate cal forming activity. At pH 4.0, it was also noted that addisensitivity" 

to penconazole in greenhouse and in vitro tests. These tion of ascorbic acid or H 2 0 2 at certain concentrations greatly 

similar ultrastructural changes found in the nonsterol-synthesizing P. enhanced the generation of the test radical. The rapid increaultimum 

and the sterol-synthesizing P. omnivorum suggest that another ment of -OH formation via the iron catalyzed Haber-Weiss reacmode 

of action exists for both of these triazoles. tion was apparently a major factor which contributed to the 

278 

RESPONSE OF CYST-NEMATODE RESISTANT AND SUSCEPTIBLE 

SOYBEAN CULTIVARS TO IN-FURROW APPLICATIONS OF 282 

photodynamic biocidal activity of MR. 

ALDICARB. P. M. Phipps, Tidewater Agr. Exp. Sta., VPI&SU, Suffolk, VA 

23437-0099. THE EXPRESSION OF RESISTANCE OF USTILAGO AVENAE TO 

TRIADIMENOL IS AN INDUCED RESPONSE. Wolfram Kdller and 

Franzine 0. Smith, Department of Plant Pathology, N Y State 

Soybean cultivars were evaluated with and without applications of aldicarb Agricultural Experiment Station, Geneva, NY, 14456. 

(Temik 15G) in fields naturally infested with soybean cyst nematode (SCN) in 

1987 and 1988. Cultivars were main plots consisting of six 10.7-m rows spaced A strain of U. avenae sensitive to triadimenol (sen) and a 

0.9-m apart. Treatments were applied to the seed furrow of two-row subplots menol resistant (2mg/L) laboratory after 15 mutant h of growth (rl) were in treated liquid culture. with triadi- 

at planting. The experimental design employed 9Initially, four randomized complete Inil rerodcto reproduction of 

of both 

in was 

strains 

altcme t 

was almost completel 

blocks. The effect of cultivars on nematode populations and yield was blocked; however, the inhibitory phase was transient for rl, 

significant (P=0.05), however, aldicarb only had a significant effect on yield. and full growth resumed after 10 h. This pattern of initial 

The overall yield of SCN-resistant cultivars (Centennial, Forrest, Pioneer Brand growth inhibition and subsequent recovery was correlated with 

P9581) averaged 2576 kg/ha (38.3 bu/A) compared to 1917 kg/ha (28.5 bu/A) a decline of sterol precursors, as analyzed by GC-MS. Al- 

for susccptible cultivars (Essex, York). Aldicarb at 0.56 and 1.12 kg/ha though precursors (pre-dominantly 24-methylenedihydrolanosterol) 

accumulated during the phase of growth inhibition, 

improved yield of susceptible cultivars 276 and 390 kg/ha (4.1 and 5.8 uA) and also were still prominent at the onset of renewed growth, 

and yield of resistant cultivars 363 and 356 kg/ha (5.4 and 5.3 bu/A), they were absent after 24 h of treatment with triadimenol. 

respectively. Pulse-labeling of sterols at various time intervals after 

treatment with the inhibitor revealed that the continuous 

disappearance of precursor sterols is not explained by a 

dilution of the inhibitor from the target site. 

279 

EFFECTS OF FUNGICIDE RH3486 ON SCLEROTINIA BLIGHT OF 

PEANUT IN HIELD AND MICROPLOT TESTS. F. D. Smith, P.M. Phipps 283 

and R. J. Stipes, Tidewater Agr. Exp. Sta., VPI & SU, Suffolk, VA 23437. NTRDSESBEGAUE EHOOY .. Lro 

~and The fungicide, RH3486, and several other 

J.M. 

fungicides 

Denis. 

were 

UCB 

evaluated 

Chemicals 

for control 

Corp., 5365-A 

Hood 

Robin 

Rd., Norfolk, VA 23513 and UCB Chemical Sector, 

of Sclerotinia blight of peanut, caused by Sclerotinia minor. Two applications Ave. Louise 326, B-1050 Brussels, Belgium. 

of RH3486 at 0.56 or 1.12 kg a.i./ha significantly (P=0.05) suppressed disease 

69 and 81% and increased yields 2191 and 2243 kg/ha, respectively. Similar UCB is one of the world's leading producers of 

applications of iprodione (1.12 kg/ha), vinclozolin (0.84 kg/ha), dicloran (3.36 methylamines and their derivatives such as Thiram 

kg/ha) or PCNB at (5.60 kg/ha) suppressed disease only 42, 47, 21 and 18%, and Ziram fungicides. In efforts to improve worker 

respectively. Microplots, 76-cm-dia., were infested with S. minor sclerotia from safety, 

strinS- 

UCB 

-8-T 

evaluated 

(wldtye)or (icaboimderesstnt 

other methods for 

ad 

formulating 

panedto Thiram and Ziram and concluded that the water- 

strinS-2(wld-yp) 

r B83T2 diaroxiid-reisan) ad lanedto dispersible granule (WG) formulation was the most 

Florigiant peanut. Plots treated three times with RH3486 at 1.12 kg/ha 

averaged 0 and 1.0 lesions/plot with strain S-2 and B-83-T2, respectively, 

Untreated plots likewise averaged 27.0 and 31.5 lesions/plot. Three applications 

of iprodione at 1.12 kg/ha did not significantly suppress disease caused by either 

strain. Two years of similar field data have shown RH3486 to be an extremely 

active fungicide against wild-type and dicarboximide-resistant strains of S. minor, 

apr rit. TeW tchogydvledbUC hs 

been registered under the trade name "GRANUFLO." 

The high quality granules readily disperse in water 

and offer many advantages over traditional formula- 

tions including being virtually dust-free, easy to 

package, easy to handle, insensitive to temperature 

280 

CYPROCONAZOLE: A NEW SYSTEMIC FUNGICIDE FOR CONTROL OF SOIL 

variations and having improved product efficacy. 

The Granuflo technology allows opportunities for 

formulating associations of fungicides which are 

useful in developing anti-resistance strategies. 

1170 PHYTOPATHOLOGY

284 

BASELINE-SENSITIVITY OF THREE POPULATIONS OF VENTURIA 

INAEQUALIS TO FLUSILAZOLE. Franzine D. Smith, Wolfram Khl1er and 

Diana M. Parker, Department of Plant Pathology, Cornell University, New York 

State Agricultural Experiment Station, Geneva 14456. 

CULTIVARS FOR EXPORT. B. T. Manji, J. M. Ogawa, J. E. 

Adaskaveg, and J. M. Osorio. Department of Plant Pathology, 

University of California, Davis, CA 95616. 

Nectarine fruit in bins were preconditioned to a pit (inner mesocarp) 

temperature of 21 C and fumigated with methyl bromide, 48 g/m 3 One hundred monoconidial isolates of V. inaequalis were collected from each of 

two abandoned orchards (orchards 1 and2), where no steroldemethylation 

inhibitors (DMd ) had been used, and from a research orchard where DM1 

fungicides had been used for 12 years (orchard 3). Themean EDso values based 

on colony diameter were 0.0083 jtg flusilazole/ml, 0.0072 jtg/ml, and 0.0105 

jtg/ml for orchards 1, 2, and 3, respectively. ED5o values for individual isolates 

ranged from 0.002 to 0.0654 jtg/ml, 0.0001 to 0.0469 jtg/ml, and 0.0011 to 

0.1108 jig/ml, in orchards 1, 2, and 3, respectively. There was no significant 

difference between the mean of the log10 transformed ED50 values of any orchard. 

Our results indicate that the three populations examined had similar mean EDs0 

values, and that highly tolerant isolates occurred in natural populations prior to 

for 

2 hr at 21 C. After fumigation, non-treated and fungicide-treated 

fruit were packed into boxes and stored for 12-14 days at 1 C to 

simulate transit by ship to oversea markets. In non-fungicide 

treatments, early-maturing cultivars (May Glo, May Grand, and Spring 

Red) generally developed less than 5% brown rot (Monilinia 

fructicola), whereas, later-maturing cultivars (Red Diamond and 

Firebrite) developed as high as 80% brown rot during the 5 day 

ripening period at 21 C. Generally, fruit washed with 100 pg/mi 

chlorine had less brown rot than non-chlorinated fruit. Postharve 

treatment with iprodione in a water soluble wax further extended the 

shelf-life of the fruit by 3-5 days. 

flusilazole exposure. The range and variance of ED 50 values observed in our study 

also indicates that small sample sizes are unlikely to represent accurately the 

sensitivity of populations of V. inaequalis to DMI fungicides. 

289 

EFFICACY OF COPPER RESINATE AND TRI-BASIC COPPER SULFATE IN 

PEANUT DISEASE CONTROL. W.W. Osborne and J. D. Taylor. IAI, 

285 

Inc., South Boston, VA 24592; and J 

Skippers, VA 23879. 

& S Consultants, Inc., 

SULFUR DIOXIDE RESIDUES IN TABLE GRAPES AFTER S0 2 FUMIGATION. 

J. L. Smilanick, J. M. Harvey, P. L. Hartsell, D. J. Henson, 

C. M. Harris, D. C. Fouse, and M. Assemi. USDA, ARS, HCRL, 

2021 South Peach Avenue, Fresno, CA 93727 

Grapes were fumigated to evaluate factors that influence sulfur 

dioxide (SO2 ) residues. Residues with SO2 decreased rapidly 

after fumigation; the S02 half-life was 24-48 hr. Grapes 

fumigated at warmer temperatures accumulated higher but less 

persistent residues than those fumigated at low temperatures. 

Immature grapes accumulated more S02 than mature grapes. S02 

residues were located on or near the berry surface. Grapes 

infected with Botrytis cinerea or injured by cuts accumulated 

more S02 than sound grapes. Sulfur dust applied before harvest 

left no S02 residues. The decay suppression with low (312-1250 

ppm) SO2 doses was only slightly inferior to that obtained by 

standard doses (2500 ppm). S02 residues after low doses did 

not exceed 10 ppm. High residues can be avoided by low-dose 

fumigations, good temperature management, and use of grapes 

free from rot or injuries for storage, 

286 

EVALUATION OF FUNGICIDES FOR CONTROL OF LEAF RUST AND 

SEPTORIA BLOTCH OF WHEAT. R. T. Gudauskas, A. K. Hagan, 

E. L. Carden, and N. R. McDaniel, Dept. of Plant Pathology 

and Gulf Coast Substation, Auburn University, AL 36849 

During 1983-1988, 24 fungicide treatments were evaluated 

for control of leaf rust and Septoria blotch on highly 

susceptible cultivars 'Blueboy' and 'McNair 1003' and a less 

susceptible cultivar 'Fla. 301'. One or two applications of 

each fungicide were made between growth stages 8 and 10.2. 

Most of the fungicides tested reduced the severity of both 

diseases as compared to the unsprayed control. Yield 

increases associated with fungicide treatments in the highly 

susceptible cultivars ranged from 268 to 2016 kg/ha; yield 

increases in 'Fla. 301' ranged from 0 to 806 kg/ha. 

Fungicides giving the highest levels of disease control and 

yield increases included diniconazole, propiconazole, 

mancozeb, terbutrazole, and triadimefon. 

287 

USES OF FUNGICIDE COMBINATIONS CONTAINING 

TETRAMETHYLTHIURAM DISULPHIDE OR THIRAM ON APPLES, 

PEARS AND STRAWBERRIES. J.M. Denis and P. Creemers. 

UCB Chemical Sector, Ave. Louise 326, 8-1050 

Laboratory and field studies conducted during the past four 

years with copper resinate and tri-basic copper sulfate show 

an inverse relationship between chemical rates and control of 

major peanut diseases. Copper resinate was the most 

economical and effective treatment when compared with tri- 

basic copper sulfate and other fungicides currently being 

used for the control of major peanut diseases. 

290 

EVALUATION OF AGRI-CHEMICALS ON SPORES AND MYCELIUM OF 

STIGMINA CARPOPHILA AND MONILINIA FRUCTICOLA. A.J. Feliciano, 

J.E. Adaskaveg, and J.M. Ogawa. Univ. of California, Davis, 95616. 

Spores of S. carpophila (Sc) and M. fructicola (Mf) were exposed to 

(chemical/concentrations-pg/mi al.): NaOC1/400; Hg/D2; Cu/1800; ziram 

/1800, captan/50, 500, 1200; iprodione/1200; and captafol/10, 100,500, 

using the cellophane-transfer technique. Spores were exposed for 2 or 

24 hr, rinsed for 0, 24, 48, 72 or 96 hr, and plated on PDA. Colony 

formation (cf) was determined after incubation for 10 da at 25C. 

Inhibition of cf for each fungus at chemical concentrations tested was: 

Sc - Hg, NaOCi, or captafol/2 hr (chemicals/exposure); captan/24 hr; Mf 

- Hg or NaOCI/2 hr; and captan or captafol/24 hr. Ziram, Cu, or 

iprodione did not inhibit cf of either fungus at both exposures. Rinsing 

increased the rate of growth but not % cf. Increasing concentrations of 

captan decreased the time of exposure that inhibited cf: Mf - 16 hr 

exposure/50 pg/ml, 12/500, 8/1200; and Sc - 4/10, 4/100, and 2/500. In 

a 24-hr, direct exposure study using mycelium growing on cellophane, 

1200 pg/ml of captan or 500 pg/ml of captafol did not inhibit mycelial 

growth of either fungus. Current concepts of defining chemicals as 

fungicides may need re-evaluation based on fungal tissue, chemical 

concentration, exposure time, and duration of incubation on a medium. 

291 

MITOCHONDRIAL PLASMIDS IN ENDOTHIA PARASITICA. N. Mahanti 

and D.W. Fulbright. Dept. Botany and Plant Pathology, Michigan 

State University, East Lansing, MI 48824. 

Hypovirulent strains of Endothia parasitica found in Europe 

and North America are associated with dsRNA. One strain of 

E. parasitica (CL25) from Michigan has all the characteris- 

-tics of dsRNA-associated transmissible hypovirulence but harbors 

no detectable dsRNA. Studies with CL25 show plasmids are present 

in mitochondria. These plasmids are in low titer and have 

been difficult to isolate consistently. The plasmid DNA was 

isolated by electrophoresing total mitochondrial DNA in an 

Brussels, Belgium and Opzoekingsstation van Gorsem, agarose gel, cutting the plasmid band from this gel, followed 

Brede Akker 3, 8-3000 Sint-Truiden, Belgium. by electroelution. The restriction enzyme Saulll-A was used 

Field trials with Thiram alone or in combination with to clone the plasmid into vector PRL 498. Southern hybridizaother 

fungicides were conducted at the Fruit Research tion studies show that this plasmid is present in some virulent 

Station of Gorsem. The use of the compound Thiram in and other hypovirulent strains even though the plasmid bands 

fungicide combinations showed an increase of are not visible in gels after ethidium bromide staining. Therefore 

these plasmids may not have a direct effect on hypovirupreventive 

activity, a broadening of the spectrum of lence but may be affecting the mitochondrial genome. 

activity, and a decrease of the selection pressure 

of the fungicide with which it was combined. It 

is concluded that Thiram is an effective fungicide 

partner when used on apples (scab), pears (scab) and 

strawberries (grey mould). 

288 

VARIABILITY OF DISEASE DEVELOPMENT IN NECTARINE 

Voj. 79, No. 10, 1989 1171

The nit-2 gene of N. crassa encodes an activator protein that 

governs overall nitrogen metabolism in the cell. We have used 

the cloned gene as a heterologous probe to survey various 

fungal phytopathogens for homologues. A wide variety of genera 

showed homology as determined by Southern blotting. To further 

study nitrogen regulation, we have chosen Fusarium app. 

Genomic libraries of Fusarium moniliforme and Fusarium sacchari 

have been constructed. Following screening, we have isolated a 

6kb fragment from both fungi with homology to the nit-2 gene 

from N. crassa. We also used the nit-2 gene to complement a 

mutant of Fusarium graminearum that is defective in overall 

nitrogen metabolism. Following transformation colonies were 

obtained which now could utilize nitrogen sources similar to 

the wildtype. This suggests that this heterologous regulatory 

gene is expressed in Fusarium and functions to activate 

expression of nitrogen regulated genes in this organism. 

297 

STRUCTURAL AND FUNCTIONAL ANALYSES OF TWO Q-TUBULIN GENES IN 

COLLETOTRICHUN GRAMINICOLA. D. G. Panaccione and R. M. Hanau. 

Dept. of Botany and Plant Pathology, Pudue University, West 

Lafayette, IN 47907. 

In organisms with multiple tubulin genes, expression of 

individual tubulin genes is often developmentally regulated. 

Two S-tubulin genes, TUBI and TUB2, were cloned from 

Colletotrichum graminicola with the interest of studying their 

involvement in conidial development. Southern hybridization 

and DNA sequencing demonstrated that although the two genes 

are considerably divergent, they both have the capacity to 

encode 3-tubulin. RNA blots indicated that the level of TUB2 

message relative to TUBI message increased in conidiating 

cultures. To study the functional significance of this, the 

TUB2 gene was replaced with a truncated copy of the gene by 

site-specific integrative transformation. Transformants 

carrying the truncated TUB2 allele did not display any 

294 abnormalities in conidial development. 

IDENTIFICATION OF SEQUENCES WITH PROMOTER ACTIVITY FROM 298 

GIBBERE1IXA PULICARIS (FUSARIUM 

Yangkyo 

SAMBUCINUM) 

P. Salch and 

TRANSFORMANTS. 

Marian N. Beremand, USDA, Agricultural 

Research Service, 

TRANSFORMATION 

Northern Regional 

OF TRICHODERMR 

Research 

SPP. 

Center, 

TO HYGROMYCIN 

Peoria, 

B RESISTANCE. 

A. Siva, TE. Stasz, G.E. Harman and M. Hemmat, 

IL 61604 

Dept. of Horticult 

Sciences, Cornell Univ., Geneva, NY, 14456. 

G. pulicaris (GP) is a heterothallic 

rot on potato 

ascomycete 

tubers. 

and 

GP 

causes 

protoplasts 

dry 

were transformed with a 

cosmid 

r 

cosHygl containing 

protoplasts 

hygromycin 

were obtained 

B phosphotransferase 

by digesting mycelium with 

Novozyme 

(hygB) fused 

234. 

to 

Protoplasts 

promoter 

were 

1 from 

treated 

Cochliobolous 

with the plasmid 

heterostrophus. 

pH11B (obtained 

Transformation 

from O.C. 

occurred 

Yoder) containing 

by random an 

integration 

Escherichia 

of the 

col 

cosmid 

hygromycin B 

phosphotransferase gene, which was used 

into 

with 

the GP 

the 

genome. 

permission 

Based on restriction of 

digestion 

Eli 

and 

Lilly & 

Southern 

Co., fused to a Cochliobulus 

hybridization 

heterostrophus promoter. Treated 

analyses, 

protopla 

one of the transformants with 

a single 

Co., 

copy 

fused 

insertion, 

to a m 

63C3, had the recombination promoter 

event 

Tred prtopasts 

were 

occurring 

plated in 

between 

a molten 

the 

agar 

5' 

medium 

end of 

which 

hygB 

later 

coding 

was 

sequence 

covered with 

and 

a second 

the 

end 

3' 

of promoter 1. 

medium 

Expression 

layer 

of 

containing 

hygB resistance 

the antibiotic. 

most 

The 

likely 

application time 

antibiotic 

of the 

resulted from endogenous GP 

was 

promoter-like 

critical and differed 

sequences. 

between 

Cloning 

strains. Southern analysis 

putative 

of 

and analysis of 

transformants 

the GP promoter-like 

showed integration 

sequences 

into 

will 

the 

be 

genomic DNA. The 

frequency of transformed nuclei in hygromycin B resistant isolates was 

discussed. 

usually lower than 1%. Therefore, 

were 

nuclei 

allowed 

from 

to segregate 

all putative 

through conidiation. 

transformants 

Single spore isolates were 

295 obtained that were mitotically stable on selective and non selective media. 

CAROTENOID-OVERPRODUCING TRANSFORMANTS OF =]RQýBRQFAQM5 ARE 

NOT RESISTANT TO CERCOSPORIN. _ C og_Qpperman, M. E. Daub, R 299 

G. Carolina Upchurch, State and University, G. A. Payne. Raleigh, Dept. of NC Plant 27695-7616. Pathology, North 

HEAT SHOCK-INDUCED DEVELOPMENT OF INFECTION STRUCTURES BY THE 

The photoactivated toxin cercosporin 

RUST FUNGI: 

produces 

EXPRESSION 

singlet 

OF 

oxygen 

THE INF GENES. 

Staples, 

S. Bhairi 

Boyce 

and R.C.__ 

Thompson Institute, 

and 

Cornell 

superoxide. University, 

Resistance Tower 

of roso~~ spp. to cercosporin Road, Ithaca, NY 14853. 

appears to act 

singlet 

at several 

oxygen 

levels. 

quenchers, 

Carotenoids, 

are associated 

which 

with 

are 

resistance 

potent 

to 

cercosporin. 

Germlings of 

A 

Uromvces 

Cercospora 

aoDoendiculatus 

nicoti__anae genomic 

induced 

library 

by exposure to 

28.5°C 

constructed 

heat for 

in 

1.5 

the 

hr 

cosmid 

developed 

vector 

infection 

pSV50 was 

structures 

used to transform 

similar 

cercosporin-sensitive 

to those induced thigmotropically 

Ljjosoo Capa. 

and 

A 

at 

clone 

about 

(B5) 

the 

was 

same rate. 

identified 

After heat 

that 

shock, 

conferred 

appressorium 

increased 

development 

carotenoid 

was 

production 

accompanied 

to 

by 

the 

the 

transforsants. 

appearance of at 

Eight 

least 

additional 

six heat-shock 

B5 transformants 

proteins, 

also 

but 

synthesis of 

overproduced 

the thigmotropic-specific 

carotenoids. Southern 

proteins 

blot 

which 

analysis 

occurs 

indicated 

insertion 

the 

of 

during 

9 ico__tia.nae 

contact-induced 

DNA into the 

development 

genome. The 

was 

carotenoidnot 

observed. 

Thigmotropically-induced 

overproducing transformants 

appressorium 

did not 

development 

show increased 

is 

resistance 

to cercosporin. 

accompanied by 

These 

an 

results 

upshift 

support 

in the expression 

the previous 

of a 

hypothesis 

small group 

(Phytopathology 

of INF genes, 

79:180) 

and we 

that 

have 

carotenoids 

identified 

are 

six 

not 

of 

the 

these 

sole 

by now. 

Here 

mechanism 

we have 

of 

examined 

cercosporin 

the expression 

resistance, 

of four ds-genes after 

inducing infection structure 

' 

development by heat shock. Genes 

IN___FFli 56 and INF24 were induced only moderately. I__Fl F64 was 

induced in germlings heat shocked for 4 hr but not when heat 

shock was applied for only 2 hr. INF88 was not induced. 

296 

REGULATION OF NITROGEN METABOLISM IN~ FUSARIUM BY A HOMOJLOGOUS 300 

NEUOSOR GEE.Mati B.Dikmn~ an 

of 

JhnF.Leslie, 

Plant Path., Univ. 

Dept. 

of Nebraska, 

RESTRICTION 

Lincoln 

FRAGMENT 

68583, 

LENGTH 

and 

POLYMORPHISMS 

Dept. of 

BETWEEN 

AND ADZUKIBEAN 

SOYBEAN 

Plant Path., Kansas 

PHIALOPHoRA 

State Univ., 

GREGATA 

Manhattan 

ISOLATES. 

66506. 

L. E. Gray, 

and A. Hepburn, USDA, Agricultural Research Service, and 

1172 PHYTO PATH OLOGY

Department of Agronomy, University of Illinois, Urbana, IL 

61801. 

304 

Phialophora gregata causes a vascular disease of soybean and 

adzukibean. We have been using RFLP analysis to study 

genome relationships between soybean and adzukibean strains 

of the fungus. A Library of BamHi fragments from a soybean 

isolate was made in puC8. Selected clones were then used to 

probe BamH1 and EcoRi digested DNA of both soybean and 

adzukibean strains of the fungus. In general all soybean 

isolates show the same hybridization pattern or a band 

deletion. With adzukibean isolates, with a given soybean 

isolate probe, different adzukibean isolates show different 

hybridization patterns. In some cases no hybridization is 

observed. These results indicate that the adzukibean 

Phialophora isolates are not closely related to the soybean 

isolates. 

APPLICATION OF RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS 

AS A TAXONOMIC TOOL TO DIFFERENTIATE PYRENOPHORA SPECIES. B. M. 

Baltazar, A.L. Scharen, and V. Raboy, USDA, ARS, Plant Pathology 

Dept, Montana State University, Bozeman, MT 59717-0002. 

Pyrenophora species cause economically important diseases of 

barley, wheat, rice and other crop species. Little is known 

concerning the phylogenetic relationships among species in this 

genus. Restriction fragment length polymorphism (RFLP) analysis 

provides one way to estimate the evolutionary distance between 

species. A genomic library was made from P. teres f. sp. 

maculata DNA in pUC12. Individual clones from this library have 

been screened to identify those which hybridize to polymorphic 

sequences among the various species. As an example, a 2.4. kb 

genomic clone hybridized to a 3.7 kb band in EcoRI digested 

genomic DNA of R. teres f. sp. maculata, and to 5.0 and 5.9 kb 

301 

bands in EcoRI digested genomic DNA of P. teres f. sp. teres. 

This clone did not hybridize to P. araminea or barley genomic 

DNAs. We plan to employ PCR (polymerase chain reaction) to 

amplify fungal DNA in infected plant tissue, which might be used 

TRANSFORMATION OF COLLETOTRICHUM GLOEOSPORIOIDES F. SP. to detect and identify Pyrenophora species or subspecies. 

AESCHYNOMENE. D.O. TeBeest, and M.B. Dickman. Dept. of Plant 

Path., Univ. of Arkansas, Fayetteville, AR 72701 and Dept. of 

Plant Path., Univ. of Nebraska, Lincoln, NE 68583. 

305 

Colletotrichu gloeosporioides np. f. eaechynomene, 

an anthracnose 

incitant 

on 

of 

Aeschyrnomene virginica, was transformed to 

resistance to MBC (methyl-2-benzimidazole carbamate). 

Protoplasts were exposed, in the presence of PEG, to the 

A FAMILY OF CONSERVED REPETITIVE DNA ELEMENTS FROM THE FUNGAL 

PLANT PATHOGEN GLOMERELLA CINGULATA (TELEOMORPH 

LINDEMUTHIANUM). 

OF COLLETOTRICHUM 

R. .. .Rodrig~u~ez. Dept. of Plant Pathology, 

Univ. of California, Riverside, CA 92521. 

vector, pSV50, encoding a gene for B-tubulin from Neurospora 

crassa The gene confers resistance to MBC. Transformants were 

isolated by overlaying regeneration media with nutrient media 

containing MBC (1 ug/ml). Southern blot hybridizations of 

genomic DNA from resistant strains confirmed integrative 

transformation. Resistance to MBC was maintained in all 

isolates after passage through plants and after serial transfer 

to non-amended media and thus was mitotically stable, 

Comparison of a wild-type isolate to transformed isolates 

suggested transformation reduced aggressiveness and fitness on 

A. virginica. 

302 

Glomerella cingulata (Colletotrichwm lindemuthianum), transformed 

with the hygromycin B phosphotransferase gene, and the DNA flank- 

ing the site of integration was isolated for analysis. This 

flanking DNA contained a sequence which was repeated many times in 

the genome. The repetitive DNA was dispersed in the genome and 

showed a high level of conservation of genomic locations among 

different isolates of this fungus. The sequence of the repeated 

element consists of tandemly arranged CAX triplets, in which X is 

most commonly G or A. Sequences of three independently isolated 

copies of the repeated element indicated that it represents a 

family of repeats, each varying in length and in the nucleotide 

representing X in the CAX triplet. A probe representing this 

family of repeats was found to hybridize with genomic DNAs of 

several different fungal genera in a species-specific manner. 

The potential for using this repetitive element in disease 

diagnoses and identification of fungal species will be discussed. 

A RESTRICTION FRAGMENT LENGTH POLYMORPHISM MAP OF COCHLIOBOLUS 

HETEROSTROPHUS. Tzeng. [., C. R. Bronson and C. Ford, 

Departments of Plant Pathology and Genetics, Iowa State 

University, Ames, IA 50011. 

C. heterostrophus, the causal agent of southern leaf blight of 

maize, is a model for reseach on how fungi infect plants. We 

are developing a genetic map of this pathogen to facilitate the 

cloning of pathogenicity genes and to characterize a chromosome 

rearrangement hypothesized to be associated with the virulence 

locus Toxl. Restriction fragment length polymorphisms (RFLPs) 

and phenotypic markers are being used to construct the map. To 

date, of 99 markers analyzed, 89 RFLP markers and 4 phenotypic 

markers have shown significant linkage, indicating the coverage 

of about 94% of the genome. The map length at present is about 

1200 cM. Several differences in chromosome arrangement between 

the two parents of our cross have been identified. Markers 

have been found tightly linked to Toxl. These markers should 

be useful for cloning this pathogenicity gene by chromosome 

walking. 

306 

QUANTIFICATION OF BLUEBERRY SHOESTRING VIRUS RNA AND ANTIGEN 

IN ITS APHID VECTOR, Illinois pepperi, DURING ACQUISITION, 

RETENTION AND TRANSMISSION. B.T. Terhune, D.C. Ramsdell, 

and K.L. Klomparens. MI State Univ. E.L., MI 48824. 

Blueberry shoestring virus (BBSSV) was monitored in late 

instars of Illinoia pepperi by dot-ELISA, a silver enhanced- 

colloidal gold-immunosorbent assay, and dot-hybridization. 

Aphids acquired BBSSV-antigen at a rapid rate during a 12 

hr acquisition access period (AAP) from sachets containing 

BBSSV or BBSSV-infected blueberry plants, but the acquisition 

rate declined between AAPs of 12 and 96 hr. BBSSV-RNA was 

acquired at higher levels, and the acquisition rate did not 

decline over a 4 day AAP. Levels of BBSSV-antigen and RNA 

retained by aphids declined rapidly 1 day after acquisition, 

but remained constant during the next 3 to 4 days. BBSSV 

303 

antigen and RNA were retained after a molt, and both were 

detected in aphid hemolymph after 1 to 4 day AAPs. Aphids 

were able to transmit BBSSV to blueberry plants 8 to 10O days 

after a 24 hr AAP on sachets or BBSSV-infected plants. 

CHARACTERIZATION OF DOUBLE-STRANDED RNA IN MEXICAN, EUROPEAN, 307 

AND PERUVIAN ISOLATES OF PHYTOPHTHORA INFESTANS. J. R. 

Newbouse and P. W. Tooley, USDA-ARS, Frederick, MD 21701. DFIECTIfNI OF TOBACC2O MODSAIC AND TOBACCOJ NECIUSIS VIRUSES IN 

1'ATER USING POSITIVELY CHAIYGED ME4BRANE FILTERS. V. Jacobi, 

Double-stranded RN~A (ds-RNA) recently was found in Mexican and J.D. Castello. Faculty of Environmental and Forest Biology, 

isolates of P. infestans. Additionsal isolates of the fungus State University of New York, College of Environmental Science 

from diverse populations were evaluated for ds-RNA. Isolates and Forestry, Syracuse, NY 13210. 

from Mexico (15, 83%), Europe (6, 29%), and Peru (1, 3%) were Ten liters of distilled water were seeded with tobacco mosaic 

positive for ds-RNA, while those from the United States (35) virus at 50 ng/ml and passed through Zeta Plus 50 S filter disks 

were negative. Both Al and A2 mating type Mexican isolates (90 rmn). Followring elution percent virus recovery determined by 

contained ds-RNA, but among European isolates, only A2 types local lesion assay was 75%. Reducing elution time to 5 mai. inwere 

positive. Ten banding patterns were distributed among creased recovery to 90%. Prefiltration through 5, 1, or 0.5 urn 

the isolates tested. Sizes of the bands in kilobase pairs depth filters reduced recovery to 0-10%. Optimu virus binding 

(kbp) were determined by denaturing and electrophoresing the to the filter occurs at pH 5.5. 100% recovery of IT4V and tods-RNA 

on formaldehyde gels along with known standards. The bacco necrosis virus (TNV) was demronstrated by ELISA when 10 1 

bands ranged in size from 1. 35 to 11. 35 kbp, and some Mexican of distilled water was seeded with these viruses at 0.1 ng/rnl 

and European isolates were found to have ds-RNA bands of the and eluted with 0.$ M NaC]. or 0.1 N NaCI (pH 8.0), respectively. 

same size. This preliminary evidence suggests that European This corresponds to a virus detection sensitivity of 1 pg/rn] 

A2 mating type isolates of P. infestans may have originated in and 2 pg/ml for these two viruses, respectively. This system 

Mexico. will be used to recover plant viruses from natural waters. 

Vol. 79, No. 10, 1989 1173

308 312 

SYSTEMIC RESISTANCE TO TOBACCO MOSAIC VIRUS INDUCED IN SUSCEP- INHIBITION OF TOMATO BUSHY STUNT VIRUS BY DEFECTIVE INTERFERING 

TIBLE PLANTS AFTER LOCALIZED INFECTIONS BY TOBACCO NECROSIS PARTICLES IN TOBACCO PROTOPLASTS. R. W. Jones, A. 0. Jackson and 

VIRUS. D. A. Roberts, Univ. of Florida, Gainesville, FL 32611. T. J. Morris, Dept. of Plant Pathology, Univ. of California, 

Berkeley, CA 94720. 

The lowest three expanded leaves of plants of Turkish tobacco 

(Nicotiana tabacum L. 'Samsun'), susceptible to tobacco mosaic Defective interfering (DI) particles of tomato bushy stunt virus 

virus (TMV) but hypersensitive to tobacco necrosis virus (TNV), (TBSV) are small RNAs generated from genomic RNA, which attenuate 

were inoculated with TNV. Leaves of comparable control plants disease symptoms. The effect of DI presence on TBSV replication 

were mock inoculated with juice from healthy plants. One week was determined by PEG-mediated protoplast inoculation of TBSV 

later, two expanded leaves above the TNV-inoculated and mock- genomic RNA, with or without DI RNA. Protoplasts were derived 

inoculated ones were challenge inoculated with TMV. TMV-inocu- from Nicotiana edwardsonii, N. benthamiana and N. tabacum. In 

lated leaves and those systemically infected were harvested and each tobacco, DI replication, based upon tritiated uridine 

frozen, respectively, one and two weeks after the challenge in- incorporation, was directly correlated with the quantity of DI 

oculation. Infectivity of TMV in the juice from thawed leaves inoculum added. Inoculation of equal amounts (1:5 molar ratio) 

was assayed by the half-leaf method in Samsun NN Turkish tobac- of genomic and DI RNA markedly inhibited TBSV genomic RNA 

co. In six experiments, infectivity of TMV in TMV-inoculated accumulation within 18 hr. In contrast, DI RNA continued to 

leaves was significantly (P = 0.01) reduced below that in the accumulate between 18 and 39 hr and reached a 20-fold greater 

controls by an average of 19%, in the systemically infected molar ratio over that of genomic RNA. Data indicates that the 

leaves, by 32%. Thus, replication of TMV was slowed in plants disease attenuation found in the presence of DI RNA may be 

of Turkish tobacco previously inoculated with TNV. directly related to a suppression of genomic RNA synthesis. 

309 313 

PURIFICATION OF THE RMV AND SGV ISOLATES OF BARLEY 

YELLOW DWARF VIRUS FOR ANTISERUM PRODUCTION. G.N. ANTISERA TO CYTOPLASMIC INCLUSION PROTEINS OF POTYVIRUSES 

Webby and R. M. Lister, Purdue Univ., W. Lafayette, IN 47907, and S. M. CONTAIN CROSS-REACTIVE ANTIBODIES. John Hammond. USDA-ARS, 

Gray, Cornell Univ., Ithaca, NY 14853. Florist and Nursery Crops Laboratory, Beltsville, Md. 20705. 

Past attempts to purify the RMV and SGV isolates of barley yellow dwarf Antisera against the cytoplasmic inclusion proteins (CIPs) of 

virus (typifying those transmitted specifically by Rhopalosiphum maidis and sweet potato feathery mottle (SPFMV), iris severe mosaic (ISMV), 

Schizaphis graminis, respectively) have resulted in very poor yields (e.g. less bean yellow mosaic (BYMV), clover yellow vein (CYVV), and turnip 

than 50 .lg.Kg-1 plant tissue for SGV). Moreover, production of useful mosaic (TuMV) viruses were used to probe Western blots and dotvirus-specific 

antisera has proved difficult due to contaminating plant blots of potyvirus CIPs. On Western blots antisera to SPFMV, 

antigens. We have investigated enhancing yields and purity of these isolates BYMV, CYVV and TuMV CIPs each reacted with 17 potyvi rus CIPs; 

for the production of specific antisera of high titer. Propagation factors ISMV-CIP antiserum reacted with fewer. Cross-reactivity was 

examined included host, environmental conditions, harvest time, and plant much reduced on dot-blots; specific reactions above background 

parts used. For purification, a procedure previously developed at Purdue for were not observed with all sera. The cross-reactivity is 

other isolates was evaluated and modified for use with RMV and SGV. presumably due to antibodies reactive with conserved sequences 

Improved yields (2-400 ýtg.Kg-l) of each were obtained using oat shoots, not exposed on the native subunit but accessible on denatured 

ground in liquid nitrogen, and extracted by repeated blending in 0.5 M proteins; this would explain the superior activity on Western 

phosphate, pH 6.0. Incorporating macerating enzymes during extraction blots compared to dot-blots, and the greater virus-specificity 

reduced yields slightly. When injected into rabbits the preparations yielded previously observed by others in gel-diffusion tests with 

polyclonal antisera readily capable of discriminating RMV and SGV from antisera against potyvirus CIPs. Thus potyvirus CIPs and coat 

other isolates by DAS-ELISA. proteins may both have virus-specific exterior epitopes, and 

conserved interior sequences. 

310 

EPITOPE DIVERTITY AMONG CITRUS TRISTEZj VIRUS ISOLATES. 314 

S. M GarH. C e T. Kano T A. Permar . M. Cambra 2 COMPARISON OF ELISA AND DOT BLOT HYBRIDIZATION FOR DETECTING 

M. Koizumi , and C. Vela . USDA, ARS 3Orlando, FL, Fruit Tree TOMATO RINGSPOT VIRUS IN NECTARINE TISSUE. C.A. Powell, A. 

Research Station, MAFF, Okitsu, Japan, Instit~to Valenciano 

Investigaciones Agrarias, Moncada, Spain, and Ingenasa, 41 

Hermanos Garcia Nobelas, Madrid, Spain. 

tladidi, and J.M. Halbrendt. AREC, Univ. of Florida, Fort Pierce, 

FL 34954; USDA. ARS, Beltsville, MD 207G5; and Fruit Research 

Lab, Penn State Univ., Biglerville, PA 17307. 

4 ~1 

At least five distinct epitopes were detected in citrus tristeza Approximately I g of leaf, bark, or root tissue from 20-yearvirus 

(CTV) when 28 selected CTV isolates from Japan, Florida, old nectarine trees with symptoms of the Prunus stem pitting 

and Brazil were tested against a polyclonal antiserum (PCAS) (PSP) disease was frozen in liquid nitrogen, triturated with a 

and four monoclonal antibodies (MCA). IgG from the PCAS was mortar and pestle, and thawed in PBS. One-half of each sample 

used as trapping antibody, and the Spanish MCAs, 3DFI and 3CA5, was analyzed for TmRSV antigen by DAS ELISA. Total nucleic 

and the Florida MCAs, MCA13 and MCA14, were used as intermediate acid was extracted from the remainder of each sample using 

antibodies in double-sandwich indirect ELISA. Binding specificity 

of each MCA among the isolates was distinct. None of the 

phenol/chloroform and analyzed for TmRSV-specific RNA by dot 

blot hybridization with a 3 2 p-labeled cRNA probe. DAS ELISA dofour 

MCAs reacted with all isolates tested, but all isolates 

reacted to the PCAS in double-antibody sandwich direct ELISA. 

tected TmRSV in 0 of 17 leaf samples, 8 of 17 bark samples, and 

12 of 17 root samples. Uot blot hybridization detected TmRSV 

Many field sources of CTV apparently contain complexes of CTV RaNA in U of 17 leaf samples, 17 of 17 bark samples, and 3 of 17 

serotypes. root samples. Total nucleic acid was extracted from bark collected 

from various locations on two young nectarine trees with 

'SF. TmRSV RNA was detected by dot blot hybridization from bark 

where PSP) symptoms were visible. 

311 

CROSS PROTECTION AND RELATIONSHIPS AMONG BARLEY 315 

YELLOW DWARF VIRUSES. F. Wen and R. M. Lister. Department of THEORY 

Botany and Plant Pathology, Purdue Univ., W. Lafayette, IN 47907.ELS.PN.Brosad0W. 

OF TESTING FOR SEROLOGICAL IDENTITY IN QUANTITATIVE 

anet ClmnUivsty 

Studies on cross protection in barley yellow dwarf viruses (BYDV) using Clemson, South Carolina 29634. 

ELISA and cDNA probes (Phytopathology 78: 1587) were extended to 

include additional isolates representing different serological groups and Curwes of optical density response to dilutions of antigen 

serotypes. The serotypes were: PAV (non-specifically transmitted by preparations are characterized by parametric functions which 

Sitobion avenae and Rhopalosiphum apidl~dd); MAV (specifically transmitted by provide a definition of serological identity for virus isolates 

S. avenae), and SGV (specifically transmitted by Schizaphis graminum), 

regarded as Group 1 serotypes, and RPV (specifically transmitted by R. padJi) 

in terms of hypothesized parametric identities that can be 

tested for each antiserum. The necessity of allowing for 

and RMV (specifically transmitted by R_. maidis), regarded as Group 2 unknown and different antigen concentrations, in virus 

serotypes, together with two closely related serotypes derived by preparations before dilution, reduces the number of vulnerable 

subculturing from an MAV source. Cross protection was most efficient and parametric identities by one. Combined analysis of response 

persistent between the two serotypes derived from MAV, and undetectable curwes from different antisera changes the definition of 

between RPV and either MAV or PAV. Other combinations also showed that serological identity and increases the sensitivity of the test. 

the degrees of cross protection obtained were consistent with serological This method can be validated by 'blind' testing of different 

relationships as indicated by ELISA, and with genomic relationships as preparations of the same virus isolate for which the hypothesis 

indicated by eDNA hybridizations and sequencing information, of serological identity should not be rejected. 

1174 PHYTOPATHOLOGY

316 

SEROLOGICAL NONIDENTITY OF IRIS SEVERE MOSAIC VIRUS AND ITS DEMONSTRATION OF THE SATELLITE NATURE OF VIRUS-LIKE 

BEARDED IRIS MOSAIC STRAIN BY QUANTITATIVE ELISA. 0. W. PARTICLES ASSOCIATED WITH MAIZE WHITE LINE MOSAIC 

Barnett and P. M. Burrows, Clemson University, Clemson, South VIRUS. L. Zhang, T. A. Zitter, and P. F. Palukaitis, Department of 

Carolina 29634. Plant Pathology, Cornell University, Ithaca, NY 14853. 

Bearded iris mosaic virus, once considered a separate virus, 

now is considered a strain of iris severe mosaic virus. The 

bulbous iris and bearded iris strains are serologically Virus-like particles (17 nm diam.) associated with maize white line mosaic 

indistinguishable by enzyme-linked immunosorbent assay (ELISA) virus (MWLMV, 35 nm diam.) were separated from MWLMV by two 

in their homologous and heterologous antisera. Serological cycles of 10-40% sucrose gradient centrifugation. A dilution series of 

identity of the strains was tested by comparisons of curves of both virus-like particles and MWLMV preparations was made and used 

optical density responses to dilutions of antigen preparations separately to inoculate sweet corn. No plants inoculated with the virusin 

indirect, double antibody sandwich ELISA. When undiluted l p 

and half strength preparations of virus were compared in like particles developed symptoms and no virus-like particles could be 

bulbous iris and bearded iris antisera, the hypothesis of detected. In almost every case the virus-like particle .RNA was detected 

serological identity was not rejected as required for a valid in plants inoculated with MWLMV. These results show that the virustest. 

When preparations of the two strains were compared in like particles associated with MWLMV cannot infect plants without 

the two antisera in four experiments, the hypothesis of MWLMV and hence are a satellite virus of MWLMV (SV-MWLMV). 

identity was always rejected. Thus by quantitative ELISA 

comparisons the bulbous iris and bearded iris strains are 

closely related but serologically nonidentical. 

321 

317 WHY UREDINIOSPORE GERM TUBES OF PUCCINIA SORGHI DO NOT ADHERE 

EFFECTS OF PLANT SAP ON ANTIGEN CONCENTRATIONS CALIBRATED BY TO MAIZE LEAVES WITHOUT EPICUTICULAR WAX. R. Chaubal, V. A. 

ELISA. S. W. Scott, P. M. Burrows, and 0. W. Barnett, Clemson Wilmot, and W. K. Wynn, Plant Pathology Department, University 

University, Clemson, South Carolina 29634. 

of Georgia, Athens 30602. 

Calibration of optical density response to standard antigen Germinating urediniospores of most cereal rusts are appressed 

concentrations enables estimation of unknown antigen to leaves with epicuticular wax but not to those without wax. 

concentrations and formulation of detection rules for future To understand this phenomenon, the extracellular mucilage 

samples tested in a quantitative ELISA system. But produced by germ tubes of Puccinia sorghi was visualized 

calibrations performed with purified virus preparations (bean ultrastructurally after adding cationic compounds 

yellow mosaic, clover yellow vein, peanut stunt, red clover (cetylpyridinium chloride, ruthenium red) to conventional 

mosaic and southern bean mosaic viruses) are not reliable for fixation solutions. On waxy leaves the mucilage flowed from the 

estimation of concentrations in preparations that include plant lower surface of the germ tubes into the spaces around the wax 

sap. Associations of saps with virus can have disruptive or projections, anchoring the fungus to the jagged cuticle. On 

cooperative effects on optical density responses depending on leaves without epicuticular wax, the adhesive material spread 

the sources of plant sap. Disruptive associations lead to to the outside of the germ tubes but was not present beneath the 

underestimation of virus concentration while cooperative fungus to attach it to the smooth cuticle. The role of the 

associations lead to overestimation. Therefore it is necessary mucilage in adherence was confirmed by treatments with dilute 

to perform ELISA calibrations in sap of the plant species for alkalies, pronase E, and laminarinase which removed the mucilage 

which the assay is intended subsequently. and also detached germ tubes from artificial surfaces. 

318 

DETECTION OF APPLE SCAR SKIN AND DAPPLE APPLE VIROIDS WITH 322 

SP6-GENERATED cRNA PROBES. A. Hadidi, ARS-USDA, Beltsville, INFECTION PROCESS OF CERCOSPORA ARACHIDICOLA ON PEANUT LEAVES. 

Maryland 20705. H. A. Melouk, and S. S. Aboshosha. USDA-ARS, Dept. of Plant 

Pathology, Oklahoma State Univ., Stillwater, OK 74078-9947, 

Scar skin and dapple diseases are among the most damaging fruit and Dept. of Plant Pathology, College of Agriculture, 

blemishing apple diseases in Japan and China. The occurence of Alexandria, Egypt. 

these two viroid diseases in North America or Europe is rare. 

Recombinant plasmids composed of an pSP 65 vector containing Leaflets of peanut Tamnut 74 (susceptible) and PI 276235 

sequences derived from apple scar skin viroid (ASSV) were used (resistant) were inoculated with C_. rachidicola (CA) by 

to generate high specific activity 3 2 P labeled ASSV cRNA applying 0.1 ml conidial suspension (2X104 /ml) on adaxial 

probes by SP6 RNA polymerase. In Northern blot and dot blot surface. Leaflets were incubated on moist filter paper in 

hybridization assays, probes hybridized with RNA from ASSV or petri plates at 25+IC in darkness. Samples were collected at 

dapple apple viroid (DAV)-infected, but not uninfected apple various times after inoculation, and processed for SEN. CA 

tissue. DAV or ASSV was detected from apple seed, fruit, leaf, conidia germinated after 24 hr on both genotypes. Germ tubes 

bark, or root tissue. These assays are rapid, accurate, and did not enter open stomata of Tamnut 74. Smooth surfaced, round 

sensitive. Testing periods for either viroid can now beenterlub enaped mapp 

reduced from several years by observing the fruit symptoms on genotypes within 3 days. Infection pegs emerged from the 

grafted woody indicators to a few days by recombinant DNA appressoria at 4 to 5 days on Tamnut 74. Most appressoria on 

assay. This advancement will make monitoring ASSV and DAV PI 276235 collapsed and disintegrated at 4 to 6 days 

world-wide possible. incubation. Infection of peanut leaves by CA appears to occur 

319 

320 

by inter-stomatal penetration. 

PARTIAL CHARACTERIZATION OF A VIRUS-LIKE PARTICLE 

ASSOCIATED WITH A HYPOVIRULENCE IN Leucostoma sp. 

C. J. P. Jensen and G. C. Adams, Department of Botany and Plant Pathol- 32 

ogy, Michigan State University, East Lansing, MI, 48824 

Abundant isometric virus-like particles were visible in the hyphae of isolate 

14.4a, a hypovirulent isolate of Leucostoma sp. Our objective was to purify 

FIELD TEST FOR PHYSIOLOGIC SPECIALIZATION AMONG ISOLATES OF THE 

SOYBEAN STEM CANKER DISEASE PATHOGEN. B. L. Keeling, USDA-ARS, 

Jamie Whitten Delta States Research Center, Stoneville, MS 

and characterize these particles. Mycelia from 14 to 21 day old liquid 38776 

cultures were homogenized in 0.1 M phosphate buffer pH 7.0, and 

centrifuged to remove cellular debris. The particles were precipitated from 

the supernatant with 8% polyethylene glycol and clarified by differential 

Tevrlneo 

Tevrlneo 

wny 

wny 

fu 

fu 

sltso 

sltso 

iprh 

iprh 

centrifugation. Spectrophotometric analysis of sucrose and CsCI density 

gradients revealed two peaks with an absorbance at 254 nm. Only the top 

Phaseolorum var. caulivora isolated from soybean plants 

symptomatic of the stem canker disease in 1979, 1983, 1984, and 

peak consistently yielded both protein and nucleic acid. The top peak 1986 was measured and compared. Virulence of the isolates was 

contained isometric particles with diam.eters of 40 nm. The particles had a accessed by measuring lesion development 30 days after 

buoyant density in CsCl of 1.313 g/cm- and had a sedimentation iouaig6-a l il rw lnso h utvr 

coefficient of 104S as determined from linear-log sucrose gradients. 

Extraction of protein from the top peak yielded one major coat protein in 

SDS-PAGE with a molecular weight of 32,000. Extraction of nucleic acid 

incuating, 60-day, Akold finenld gonpan ts7733 ofin tectivarse 

TayN -OAkoCnenaadJ739uigifse 

toothpicks. Results demonstrate a wide difference in isolate 

from the top peak yielded one major segment of dsRNA that is slightly virulence from very weakly pathogenic to very virulent. The 

larger than the three dsRNA segments normally associated with the virulence of isolates to different cultivars do not support a 

presence of the particles in hyphae, hypothesis of physiologic specialization among isolates. 

Vol. 79, No. 10, 1989 1175

324 

NUCLEAR NUMBER AND BEHAVIOR IN SCLEROTINIA SCLEROTIORUM. E. J. 

Ford, D. C. Sands, and K. Adkisson, Dept. of Plant Pathology, 

Montana State University, Bozeman, MT 59717. 

was only partially effective on strawberry. Both mixtures 

increased dicarboximide resistance. Thiram or chlorothalonil 

useo alone did not provide satisfactory crey mould control hut 

did decrease the incidence of dicarboximide resistant strains. 

Fluorescent microscopy was used to study nuclear number and 

behavior in asci, ascospores, germinating ascospores, hyphae, 

protoplasts and microconidia of S. sclerotiorum. Specimens were 

stained with hydroethidine, DAPI, propidium iodide, Hoerst, and 

calcofluor either singly or in various combinations to reveal 

details of nuclear number, meiosis, mitosis, and cell wall 

development. Classical patterns of meiosis were observed in 

asci with 8 nuclei being formed followed by wall formation and 

then a final nuclear division to yield two nuclei per mature 

ascospore. Nuclear division in ascospores was evident after 2 

hrs incubation in a nutrient broth at room temperature, which 

was prior to germination of the ascospores. Young sporelings 

had up to 16 nuclei prior to cross wall formation. A nuclear 

generation time of ca. 2.4 hrs was determined for sporelings. 

Hyphae from young shake cultures had 20-30 nuclei/cell, 

Spheroplasts formed from active hyphae averaged 3.5 nuclei per 

spheroplast. Microconidia were uninucleate. 

328 

HYBRID PERFORMANCE AND YIELD LOSSES ASSOCIATED WITH GRAY LEAF 

SPOT DISEASE OF CORN IN VIRGINIA. P. J. Donahue and E. L. 

Stromberg, Departments of Agronomy and Plant Pathology, Phy 

Strog, departments of Alant PA Phy- 

siology and Weed Science, VPI&SU, Blacksburg, VA 24061-0331. 

Commercial corn hybrids were evaluated for 7 years at Wythe and 

Shenandoah Counties and 1 year at Montgomery Co. under field 

conditions for response to gray leaf spot disease, caused by 

Cercospora zeae-maydis. Plants were scored 3 times during 

the growing season. Disease severity indices were regressed 

against grain yield and harvest moisture, and lodging by location 

and year. Significant grain loss due to disease occurred in 2 

of 6 years at the Shenandoah Co. site and 1 of 6 years at the 

Wythe Co. site. A significant increase in lodging occurred in 

325 

ON HOST FAMILIES AND GENERA OF PERONOSPORA AND PLASBMOPBA 

DOWNY MILDEWS AND ON GEOGRAPHIC PROCLIVITIES OF THE PATHOGENS. 

R.Kenneth, Hebrew Univ. Fac. of Agric., Rehovot 76100, Israel 

2 of 6 years at the Shenandoah Co. site and 5 of 6 years 

at the Wythe Co. site. These data represent the effects of C 

zeae-maydis 

tial. 

on a range of genotypes 

The 

differing in yield poten- 

fact that significant associations occurred indicate 

the strong effect gray leaf spot can have when environmental 

conditions are favorable. 

Data from host-downy mildew (dm) check-lists provided information 

on the two most common genera of Peronosporaceae. Of the 

ca 51 host families with Peronospora, 8 are stricken in single 

countries only; of the 22 with Plasmopara, at least 4. Despite 

a dearth of dms of these genera in the tropics, a few thrive 

e.g. Plasmopara on Vitaceae and Peronospora on Euphorbiaceae, 

the latter unrecorded in cold regions. Australia and New 

Zealand have few dms: 3 and 10 host families respectively with 

PeronosPora and 1 and 2 with Plasmopara (vs 34 in Romania with 

Persnoanora and ca 17 in USA and 9 in Canada with Plasmopara). 

Although Plasmopara is recorded on only 7 families in Britain 

and 8 in France, there are 20 and 26 host genera of Umbelliferae 

vs only 4 and 2 in USA and Canada (and none in the east 

Mediterranean Basin). For Plasmopara on Asteraceae, however, 

there are ca 20 host genera in USA vs none in Britain and 4 in 

France. Of the 33 legume genera with Peronosl.ora, 8 are in 

single countries only e.g. Cicer in Israel, yigna in USA. 

326 

SCLEROTINIA BLIGHT OF PEANUT IN TEXAS: OCCURRENCE AND DETECTION 

OF THE PATHOGEN IN SEED. R.A. Taber, D.H. Smith, J. S. Neck, 

S.L. Segner, D.M. Porter, D.H. Lewis, and T.M. Omran. Dept. 329 

Plant Path. & Micro., Unierity tatonandYokum Tx. Cllee Agri. Exp. Sta., T;; Texas SD, AS, A&M EVALU]ATION OF MEIHDSFOR SAMPIG, RIECOVERY 

University, 

AND 

College Station 

ENUMERATION 

and 

OF 

Yoakum, Tx.;-USDA, ARS, BAC IA APPLIED TO THE PHYLLOSPHERE. Q. A. MatyacI, K. 

Suffolk, Va.; and Vet. Micro., TAMIU, College Station, Tx. 77843 Donegan , R. Seidler 2 , V. Prince 1 and A. Porteus2. N.S.I. 

Peanut Sclerotinia blight was first observed in Texas in Mason Technology ServicesI and E.P.A.2, Corvallis Environ.ental 

County in 1981 on cv. 'Florunner'. Since 1981 it has been Research Laboratory, Corvallis, OR. 97333. 

observed in other counties in Texas. The role of seed in Erwini herbicola or Enterobacter cloacae were sprayed on oa 

propagule dispersal is currently under study. Sclerotia were orwin le a Bacteri ac te r oaca e le af a nd bulk 

observed in infested seed; however, the incidence was less than or bean leaves. Bacterial Counts from single leaf and bulk 

1%. In spite of seed shriveling and discoloration, leaf samples were similar 1-7 days after application, bit after 

germinability and emergence from infested seed exceeded 90%. 14-35 days Counts from bilk samples were significantly larger. 

Presence of the pathogen was successfully detected in seed Butlk sample values could be adjusted to those of mingle leaves 

using the Agri-Diagnostics Inmunoassay Kit and serial using estimates of the single leaf varianc. More bacteria 

sectioning. The pathogen was most prevalent in the peanut were remo~ved by stomacher blending than sonication, blending, 

testa. Fluorescein isothiocyanate-labeled monoclonal antibodies or washing leaves in biffer. Stomacher blendling was reliable 

developed from an isolate of Sclerotinia from Texas peanuts ovrawdrngofbceilppatnsndhn2-0%f 

permitted localization and verification of specific hyphae of t)he Leaf sample carried bacteria. The surface drop technique 

the pathogen in the seed with the aid of immunofluorescence, f~r enumerzation of bacteria uses four 10 ul aliquots and showed 

nO cifferene from the 100 ul aliquot spread plate method. 

,These methods proved to be rapid, efficient and precise in 

327 estimating parametera of bacterial populations. 

INFLUENCE OF SPRAY SCHEDULES O)N RESIST/GIT POPULATIONiS OF BOTRY- 

TIS CINEREA. M.L. GULLINC, C. ALDI and A. GARIBALDI Istituto di 330 

Patologia vegetale, Via Giuria 15, 10126 Torino, Italy. 

In the presence of benziinidazole and dicarboximide resistant 

strans cnera f Btryts Pes. nd f hih dseae inidece, 

treatm~ents with di carboximides provided only partial control of 

grey mould on grape, strawberry and tomato and increased the 

percent of dicarboximide resistant strains. The combination of 

a benzimidazole with diethofencarb (alternated or not with a 

dicaooxinie) dicaboxmid) stisactry 

atifactry povied 

oovied ontol 

ontol o 

f 

gry 

gry 

muldand 

muldand 

decreased the percent of benzimidazole resistant strains. The 

mixture of procysidone with thiran controlled grey mould of 

grape, while the combination of orocymidone and chlorothalonil 

DIFFERENTIATION OF TOMATO RACES 1 AND 2 OF VERTICILLIUM DAHLIAE 

USING VEGETATIVE COMPATIBILITY ANALYSIS. T. R. Joaauim and R. 

C. Rowe, Dept. of Plant Pathology and W. A. Erb, Dept. of 

HriutrOi tt nvWotr H461 

Sixteen strains of Verticillium dahliae, designated as either 

tomato races 1 or 2, were tested for vegetative compatibility. 

Compatibility was assessed by pairing complementary, nitrate- 

mutants 

nonutilizing 

of tester 

(nit) 

strains 

mutants 

representing 

derived from each 

several 

strain with 

vegetative 

nit 

compatibility groups (VCGs) (Phytopathology 73:1305-1308). Nit 

mutants were isolated from each wild-type strain by selecting 

for chlorate resistance on corn meal agar with dextrose (Difco) 

1176 PHYTOPATHOLOGY

amended with 25 g/L of KC1O Preliminary results indicate the root gall index from these crops were reduced (92 - 98%) 

that all 12 race 1 strains from Ohio, North Carolina, Japan, by soil solarization. Growth and yield of these crops were 

Canada and Australia belong to the same VCG. The remaining enhanced in solarized soil. The beneficial effects of 

four race 2 strains from North Carolina and Australia were in a solarization was observed in the second year following two 

distinctly separate VCG. Vegetative compatibility analysis may additional cropping cycles of collard greens and sweet 

be a useful tool for rapid differentiation of these races of V. potatoes. 

dahliae in place of pathogenicity tests. 

335 

331 EFFECT OF THE BASIDIOMYCETE ATHELIA BOMBACINA ON DEVELOPMENT 

OF VENTURIA INAEOUALIS PSEUDOTHECIA. C. S. Young and J. H. 

PARTIALLY AUTOMATED BIOASSAY TO DETECT SPORANGIUM GERMINATION Andrews. Department of Plant Pathology, University of 

AND GROWTH IN PYTHIUM ULTIMUM AND GROWTH IN RHIZOCTONIA SOLANI. Wisconsin-Madison, 1630 Linden Drive, Madison, WI 53706 

K. K. Kim, R. D. Lumsden, and S. Mischke. USDA-ARS, Biocontrol 

of Plant Diseases Laboratory, Beltsville, MD 20705. McIntosh apple leaves, naturally infected with V. 

inaequalis, were inoculated with the antagonist A. bombacina, 

In vitro assays for sporangium germination and growth of Pythium incubated in an orchard from November 1986 to May 1987 and 

ultimum and growth of Rhizoctonia solani following exposure to sampled monthly. An immunochemical stain based on polyclonal 

metabolites of Gliocladium virens in fermentation extracts were antibodies to A. bombacina was sufficiently specific to 

developed. The assays used a Bio-Tek model EL-307 ELISA plate detect hyphae of the fungus in situ. A. bombacina grew 

reader or a Titertek Fluoroskan II. The bioassays were carried endophytically and epiphytically. It did not prevent growth 

out in liquid medium (100-200pi) in each well of 96 well plates of V. inaeoualis hyphae into the interior of leaves, or 

after incubation for 24 hr. Sample density was measured with initiation of pseudothecia. There was no spatial association 

the EL-307. Also, the samples were incubated for additional between hyphae of the two fungi, nor any sign of direct 

time periods with treatments. Aliquots of fluorescein diacetate parasitism of hyphae or pseudothecia of V. inaequalis. 

were added and fluorescence was determined with the Fluoroskan Pseudothecia in leaves inoculated with the antagonist did not 

II. These bioassays results correlated well with those mature further than to produce pseudoparaphyses. 

obtained from microscopic counts of P. ultimum germination and Pseudothecia in leaves without the antagonist developed asci 

with visual observation of growth 2 days later for P. ultimum and enlarged normally. 

and R. solani. 

332 

COMPARISON, USE AND MANUFACTURE OF A CYLINDRICAL IMPEDANCE 

LEAF WETNESS SENSOR. R.E. Pitblado, and T.J. Gillespie, RCAT, 

Ridgetown, NOP 2C0 and University of Guelph, NiG 2W1, Ontario, 

Canada. 

Development of weather-timed fungicide spray programs in 

vegetable crops depend on the accuracy and ease of recording 

critical weather parameters. Leaf wetness duration has been 

identified as one of those essential elements. Commercially 

manufactured instruments for recording surface wetness are 

available ranging from the mechanical types to electircalresistance 

sensors. The need for rapid recovery and use of 

timely data has shifted interest towards the electricalresistance 

sensors which can be connected to electronic recording 

devices for real time data analysis. In the development of 

TOM-CAST a weather-timed fungicide spray program for field 

tomatoes a comparison was made between several flat-plate and 

cylindrical impedance sensors. The method of manufacturing the 

cylindrical impedance leaf wetness used for TOM-CAST will be 

described. 

337 

333 A PUTATIVE NULL GENE OF PSEUDOMONAS SYRINGAE PV. SYRINGAE BLOCKS 

AN IMPROVED SELECTIVE MEDIUM FOR ISOLATION OF 

GAEUMANNOMYCES-LIKE FUNGI. M. L. Elliott, University of 

Florida, Fort Lauderdale Research and Education Center, 

Fort Lauderdale, Fl. 33314 

PATHOGENICITY ON TOMATO. D. L. McCoy and J. V. Leary. Dept. 

of Plant Pathology, Univ. of California, Riverside, CA 92521. 

A cosmid clone containing an approximately 22 kb insert of 

chromosomal DNA from a strain of Pseudomonas syringae pv. 

A medium has been developed for the isolation and syringae triparental which mating gives into a null a strain response which on is tomato strongly was pathogenic moved by 

differentiation of Gaeumannomyces-like fungi from plant 

diffe+ýrentIaTion of Gaema^"nnom sikCM funik I from plant 

root tissue. It is based on medium SM-GGT3 (Juhnke et al. 

rpenamtignoasrinwchssrnlyahgr 

on tomato. When the transconjugant was inoculated 

variety Pakmor, 

to tomato 

the pathogenicity was greatly reduced or 

1984. Plant Disease 68:233-236) and is composed of 500 mg 

L-s-3,4-dihydroxyphenylalanine (L-DOPA), 100 mg streptomycin 

sulfate, 10 mg metalaxyl, 10 mg dicloran, 10 mg 

flutolanil, 10 mg vinclozolin and 1 mg CGA-449 in 1 liter 

potato dextrose agar. Thus far, the medium has been used 

absent. The in planta growth of the transconjugant was significantly 

reduced when assayed over 5 days post-inoculation. 

Two restriction fragments of 12.4 kb and 8.6 kb were ligated 

into pLAFR5, transformed into E. coil HB101 and mobilized into 

the pathogenic tomato strain. Each of the subclones blocked 

to isolate G. incrustans and Magnaporthe poae from pathogenesis and restricted the growth of the transconjugants 

bermudagrass roots. Based on pure culture studies, the 

medium should also be useful in the isolation of 

in Pakmor tomato. The intact cosmid clone and the 12.4 kb sub- 

clone did not inhibit the pathogenicity of the transconjugants 

Phialophora spp., the G. ciraminis group and other relatedonFbbeahstucpileobthtrn. 

fungi associated with cereal grains or turfgrasses. 

334 338 

LONG-TERM EFFECT OF SOIL SOLARIZATION ON CONTROLLING MOLECULAR BASIS OF IAA P'RODUCTION IN 2 ERWINIA HEBICOLA pv.1 

ROOT-KNOT NEMATODES IN VEGETABLES. C. Stevens, V. A. Khan, GYPSOPHILAE. S. Manulis , E.N. Clark , Y. Ophir , I. Barash 

A. Y. Tang, C. Bonsi and M. A. Wilson, Dept. of Agricultural end Y. Gafni 2 

fPatPtooy 1et n ln Gnis2, 

Sciences, Tuskegee University, Tuskegee, AL 36088. 

ARO, The Volcani Center, Bet Dagan 50250, Israel. 

A three year study involving solar heating of soil (soill 

solarization) with polyethylene mulch demonstrated for two Erwinia herbicola pv. gyvpsophilae (Ehg) is a bacterial 

years control of root knot nematodes (Meloidogyne 

incognita). The population of M. incognita was reduced >90% 

in the 0-30 cm depth of solarized soil from 1985 vegetables 

phytopathogen which generates galls on the ornamental 

Gypsophila paniculata. A correlation between IAA production in 

culture and gall formation was found. Twenty two isolates of 

crops i.e Vates collard greens, Market Topper cabbages, Ehg were examined for their plasmids content and found to 

Early Sprouting Calabbrese broccoli and Georgia Jet sweet contain between 1 to 4 plasmids. A DNA fragment that exhibits 

potatoes. The number of eggs per gram root recovered and homology to the IAA genes of Pseudomonas savastanoi was cloned 

Vol. 79, No. 10, 1989 1177

from a lambda library of Ehg (isolate 713) plasmid DNA. A the plasmid pBE6. When conjugated into strain K60, pBE6 

7.5 kb fragment was further subcloned into pUCl18 plasmid in caused loss of both virulence and EPS production. 

both orientations. Both plasmids direct IAA biosynthesis in Mutagenesis of pBE6 with Tn3-GUS indicated a functional DNA 

transformed E. coli cells, as can be seen on TLC. Two new region of approximately 1.0 kb. The direction of 

proteins were shown to be made by these plasmids in a minicell transcription of the gene was determined; the 1.0 kb 

system. fragment encoded a protein of about 25.5 kDa in maxicell 

assays. The results suggest that over-expression of this 

gene in P. solanacearum has a negative regulatory effect on 

339 both virulence and EPS production. 

GENETIC ANALYSIS OF SYRINGOTOXIN. (ST), PRODUCTION IN PSEUDOMONAS 

SYRINGAE PV. SYRINGAE (PSS) STRAIN B457. R.O. Nordeen, G. Somlyai 343 

and A.K. Chatterjee, Department of Plant Pathology, University of 

Missouri, Columbia, MO 65211, U.S.A. NONOCLONAL ANTIBODIES (NAS) AGAINST XANTHONONAS CANPESTRIS PVS. 

BEGONIAE (XCB) AND PELARGONII (XCP). J. B. Jones, GCREC, 5007 

Previous analysis of 11 ST- Tn5 mutants of B457 suggested 60th St. E., Bradenton, FL, 34203, J.W.L. Van Vuurde, IPO, The 

that the insertions mapped to adjacent 21.8 and 10.1 kb EcoRI Netherlands, and A. Karu, Univ. of California, Berkeley. 

fragments. The wild-type alleles were isolated by complementing 

the ST- mutants with a cosmid (pSF6) library of the Pss (B452) In an attempt to produce highly specific rAS, different antigen 

genome. Clone pNCl021 complemented 10 of the 11 mutants. This preparations (whole cell or two membrane fractions) from XCB 

observation and homology between restriction sites in the pNCI021 and XCP were injected into two Balb/c and Bo0.Q mice. The N2 

insert and flanking regions of the Tn5 insertions associate a fraction, a mixture of inner and outer membranes, responded 

contiguous stretch of about 32 kb DNA in ST production. Tn3 HoHo better serologically than the outer membrane fraction and was 

mutagenesis of pNCI021 resulted in the isolation of over 200 lacZ compared with the whole cell antigen for production of NAS. 

fusions. B-galactosidase activity specified by 21 of these in Balb/c and BrO.Q mice responded beat to whole cell and N2 anti- 

B457, and the position of the insertions in pNCI021, suggest the gens, respectively, and were used in the fusion. Resulting 

presence of two promoters separated by approximately 15 kb DNA. hybridoma supernates were screened against the antigen prepar- 

8-galactosidase assays of these promoter fusions in potato ations using ELISA. Supernates with high specificity were 

dextrose broth, syringomycin minimal (SRM) and minimal salts observed from the XCB whole cell preparation, but not from 

media indicated maximum activity in SRM. either N2 or XCP whole cell preparations. ELISA was more sensitive 

than Indirect Immunofluorescence for detecting positive 

supernates in secondary screening. Whole cell preparations 

340 produced more specific MAS than the M2 fraction. 

CO-REGULATION OF pTIC58 CONJUGAL TRANSFER AND OPINE 

CATABOLIC FUNCTIONS. S. Beck von Bodman, G. T. Hayman, and S. 

K. Farrand. Department of Plant Pathology, University of Illinois, 344 

Urbana, I1. 61801. CHARACTERIZATION AND CLONING OF ZINC RESISTANCE FROM PSEUDOMONAS 

FLUORESCENS. D. Kobayashi, A. Moayeri and T. Suslow. DNA Plant 

Conjugal transfer of Agrobacterium tumnefaciens Ti plasmid pTiC58 is Technology, 6701 San Pablo Avenue, Oakland, CA 94608. 

induced by agrocinopines A and B, two opines produced by crown gall 

tumors incited by strain C58. These compounds also induce the acc Foliar applications of agrichemicals containing Zn or Cu ions 

locus which encodes their catabolism and sensitivity to agrocin 84. A reduce populations of epiphytic bacteria. Strains of Pseudomonas 

fragment of pTiC58 was subcloned that represses both functions when fluorescens originally isolated from almonds were shown to be 

in trans to an acc-constitutive, Tra-constitutive mutant of pTiC58. resistant to ZnSO 4 and CuSO 4 at concentrations up to 2.5 mM in 

The gene encoding this activity was mapped to a 2 kb fragment casitone-yeast extract media (CYE). Pre-exposure of strains to 

located within the 3 kb region separating Tra region I from acc. subinhibitory concentrations of Zn or Cu induced growth in CYE 

Trans-repression of the two phenotypes is relieved by addition of the broth at normally inhibitory levels of these heavy metals. 

conjugal opines. A cis-acting region within Tral was identified, Strain 484AL was selected for subsequent studies to determine the 

which, when mutated, also leads to constitutive conjugal transfer. In genetic basis of the resistant phenotype. A genomic library was 

these mutants acc remains inducible. Genetic analysis shows this constructed in the cosmid vector pLAFR3 and maintained in E. 

mutation to be cis-dominant. These results show that the opine plant coli. Several cosmid clones were identified which conferred 

signals regulate Ti plasmid conjugal transfer through a single novel resistance to ZnSO 4 in t~e E. coli host. These cosmids, 

repressor that acts on Tra and agrocinopine catabolic operons. when mobilized into various Zn P. flu-oescens and P. syringae 

conferred resistance to ZnSO4 at concentrations up to 1.0mM. One 

cosmid was further subcloned to a 4 kb EcoRl-Kpnl fragment. 

Cloned ZnR from the original or suboloned fragments did not 

341 confer CuR in E. coli nor Pseudomonas. 

CHARACTERIZATION OF avrlO, AN AVIRULENCE GENE ISOLATED FROM 

XANTHOMONAS CAMPESTRIS PV. ORYZAE. S. Kelemu, F.F. White, M. 

L. Ryba-White, and J. E. Leach. Department of Plant Pathology, 345 

Kansas State University, Manhattan, KS 66506 U.S .A. A LOCUS REQUIRED FOR LESION FORMATION BY PSEUDOMONAS 

An avirulence gene from a race 2 isolate of Xanthomonas SYRINGAE PV. SYRINGAE ON BEAN AFFECTS SYRINGOMYCIN 

campestris pv. oryzae altered the phenotype of race 1 and race PRODUCTION IN VITRO. E. M. Hrab1, J. J. Rich1, q. J. KennedyI & 

6 transconjugants in rice cultivar Cas 209 (Xa-10 resistance D. K. Willisl, 2 , Dept. of Plant Pathology' & ARS-USDAL, Univ. of 

gene) from compatible to incompatible. The gene was located to Wisconsin-Madison, 53706. 

a 2.5 kb fragment. Southern analysis with the 2.5 kb fragment Pedmnssrna v yigcB2aiaaslaeto attia rw 

revealed sequence similarity to all races of X. cc. pv. o rvzae Pedmnsyigev yigeB2asacslaeto atra rw 

and other pathovars of _X. campestris. Other species of spot of bean (Phaseolus vulgaris). We are analyzing a genetic locus, designated 

Xanthomonas and Pseudomonas did not show sequence similarity by as lemA, required for lesion formation on pods and leaves of bean. A mutation 

DNA hybridization. A second clone (pSKll-33) from the race 2 in the /emA locus does not affect colonization of bean leaves or the ability to 

strain conferred incompatibility to Cas 209 when present in the incite the hypersensitive response on the non-host tobacco (Phytopathology 

race 1 stains. The cloned DNA did not hybridize with the avrl0 7.5:1320). In an effort to identify the functional product(s) affected by lemA, 

clone. Preliminary data indicate that a near iso genic rice the wild-type strain, B728a, and mutant derivative strain, NUJVS1 

line containing Xa-10 is susceptible to the race 1 strain with (lemAl::Tn5), were bioassayed for syringomycin (SR) production in vitro. 

pSKII- 33. This data suggest that Gas 209 contains a previously B728a produced SR invitro, but NUVSI did not. Additional Tn5-inducedSRunidentified 

resistance gene. The sequence data for avrlO will mutants of B728a fell into three classes: pathogenic, non-pathogenic, and 

also be discussed. intermediate. The fact that some SR-mutants were capable of causing brown 

spot symptoms indistinguishable from those induced by the wild-type suggests 

that SR production is not required for lesion formation. 

342 

M!OLECIJLAR CHARACTERIZATION OF A GENE THAT REktJJLATE VIRULENCE 346 

AND EXTRACELLULAR POLYSACCNARIDE (EPS) SYNTHESIS IN 

PSEUDOKONAS SOLANACEARUMH. Y. Nuan._.n and L.. Eequeira, Dept. 

Plant Path., 1630 Linden Dr., U.W.-adison, Hadison, WI 53706. 

GENETIC AND DNA SEQUENCE 

DIVERGENTLY TRANSCRIBED 

ANALYSIS OF THE INTERVAL BETWEEN TWO 

PHYTOTOXIN BIOSYNTHESIS GENES FROM 

PSEUDOMONAS SYRINGAE PV. SYRINGAE. N. B. Ouiglev, Y.-Y. Mo, and 

An 8 kb DNA fragment from P_. solanacearum that specifies B. C. Gross, Department of Plant Pathology, Washington State 

both virulence and EPS biosynthesis was identified from University, Pullman, WA 99164-6430. 

genomic libraries of the wild-type strain K(60 (race 1) 

(Vir+, EPS+) and the spontaneous mutant Bl (Vir-, P.sa. syringae genes involved in syringomycin biosynthesis have 

EPS-). The 8 kb fragment was cloned into pLAFR3 to yield been subcloned on cosmids and sapped by Tn3HoHol mutagenesis. 

1178 PHYTOPATHOLOGY

lacZ gene fusions have been used to determine transcriptional orum (Psm) from cherry orchards in Michigan were examined 

direction and to demonstrate that certain plant extracts induce for plasmid diversity. Psm isolates contained 4-8 plasmids 

expression of some of these genes. Tn5 insertions in one gene and Pss isolates contained 0-2 plasmids. Plasmid DNA ranged 

(syrD) were found to prevent transcriptional induction of lacZ from 25-121 kilo basepairs among the isolates. Plasmid profusions 

in an adjacent gene (syrB). It is thought that the syrD files of Psm isolates were found to vary both within and 

product positively regulates expression of the syrB gene. The among orchard locations, and up to five distinct profiles 

syrB and syrD genes map approximately 1 kb apart and are tran- were identified among Psm isolates from one orchard. Enumerascribed 

divergently. A fragment carrying the 5' ends of both tion and molecular weight estimates were used to further 

genes and the DNA between them has been subcloned and sequenced. characterize plasmid DNA content. Sequence homology among 

The important features of this intergenic interval and the plasmids from Psm and Pss isolates were demonstrated with 

regulatory interaction between the syrB and syrD genes will be DNA/DNA hybridizations. 

discussed. 

351 Withdrawn 

347 

CONSTRUCTION OF LACZ FUSIONS WITH COPPER RESISTANCE GENES OF 352 

PSEUDOMONAS SYRINGAE PV. TOMATO AND CELLULAR LOCALIZATION OF ASSESSING LATE LEAFSPOT DISEASE OF PEANUT WITH A MULTISPECTRAL 

PROTEIN PRODUCTS. J.-S. Cha and D. A. Cooksey. Dept. of Plant RADIOMETER. F. M. Shokes, D. W. Gorbet, and F. W. Nutter. N. 

Pathology, University of California, Riverside, CA 92521. Fla. Res. and Educ. Ctr., University of FL, Quincy, FL; Dept. 

of Plant Pathology, University of Georgia, Athens, GA 30602. 

LacZ fusions were constructed with each of the four open 

reading frames (ORFs) of the copper resistance operon of pPT23D In 1986 and 1987 a multispectral radiometer was tested for 

by subcloning into the lacZ translational fusion vectors assessment of the severity of late leafspot on peanut (Arachis 

pURl90-192. Antibodies were raised to the lacZ fusion proteins hypogaea) cultivars and breeding lines. A total of 19 tests 

obtained from Escherichia coli clones. Western blot analysis 

of cytoplasmic, periplasmic, and membrane fractions of P. s 

tomato and P. s. svringae containing the cloned copper 

were compared with varying levels of disease within tests. At 

least four assessments were made using visual ratings (1-10 

scale) and radiometer ratings (800 nm band). Correlation 

resistance genes indicated that the products of the first two 

ORFs of the operon are periplasmic proteins. These proteins 

coefficients were calculated for visual vs radiometer ratings, 

ratings vs defoliation, and ratings vs yield. Correlations 

were only detected when cells were grown in media supplemented increased with disease severity. Visual ratings were highly 

with cupric sulfate. An additional coppcr-inducible correlated with radiometer ratings (-0.85 to -0.94) by late 

periplasmic protein encoded by chromosomal genes was detected 

in P. s. tomato using antiserum raised to the lacZ-ORFA fusion 

September. From results of these tests the radiometer appears 

to be as effective as visual ratings for determining severity 

product, but this protein was not detected in P. s. syringae. of late leafspot disease of peanut. It has an added advantage 

of greater precision of measurement as was evidenced by lower 

coefficients of variation than were obtained with visual 

assessments. 

348 

HOMOLOGY BETWEEN THE COPPER RESISTANCE OPERON OF PSEUDOMONAS 

SYRINGAE PV. TOMATO AND PLASMIDS IN COPPER-RESISTANT STRAINS OF 353 

XANTHOMONAS CAMPESTRIS PV. VESICATORIA and ERWINIA HERBICOLA. RELATIONSHIP BETWEEN PEANUT LEAFSPOT-INDUCED YIELD LOSS AND 

D. A. Cooksey and H. R. Azad. Department of Plant Pathology, HEALTHY LEAF AREA DURATION (HAD). V. M. Aquino, R. D. Berger, 

University of California, Riverside, CA 92521. F. M. Shokes and D. W. Gorbet. Department of Plant Pathology, 

University of Florida, Gainesville, 32611; N. Florida Res. and 

Copper-resistant strains of Xanthomonas campestris pv. Educ. Center, Quincy 32351. 

vesicatoria and Erwinia herbicola were isolated from a tomato 

leaf sample with bacterial spot disease. The X. c. vesicatoria A field trial was conducted in Marianna, Florida to test the 

strain grew on media supplemented with up to 1.5 mM cupric hypothesis that healthy leaf area duration (HAD) predicts 

sulfate, and the E. herbicola isolate grew on media with 2.6 mM yield loss due to late leafspot disease. The peanut cultivar 

cupric sulfate. Southern blot experiments showed homology Florunner was used with spray treatments of chlorothalonil to 

between the copper resistance operon of Pseudomonas syringae establish different levels of disease. Treatments (14-day 

pv. tomato and a 100 kilobase plasmid in the X. c. vesicatoria interval sprays were; i) beginning 35 days after planting, ii) 

strain. A larger plasmid of about 200 kilobases in the E. an unsprayed control, and iii) four treatments corresponding 

herbicola strain hybridized with the P. s. tomato copper to 0.13X, 0.25x, 0.50X, and 1.OOX times the rate of 1.5 pts/resistance 

operon. No homology was detected between the P. s. acre of Bravo 720 initiated when late leafspot incidence 

tomato copper resistance operon and DNA of copper-sensitive reached 1%. Disease severity, defoliation, and leaf area 

strains of either X. c. vesicatoria or E. herbicola. index (LAI) were measured eight times during the season. 

Healthy leaf area duration was calculated from LAI and total 

disease. Pod yields for all treatments increased with HAD. 

Results support the concept that HAD can be used to predict 

349 yield loss due to late leafspot disease. 

REGULATION OF AVIRULENCE GENE D (avrD) FROM PSEUDOMONAS 

SYRINGAE PV. TOMATO STUDIED USING A NOVEL Tn7-LUX SYSTEM. 

H. Shen and N. T. Keen. Dept. of Plant Pathology, University 354 

of California, Riverside, CA 92521. EFFECT OF FUNGICIDES ONTHANATEPHORUS CUCUMERIS (FRANK) 

An expression vector was constructed for investigating the DONK IN BEAN CULTIVARS. B.Mora; F.Villalobos,and G.Galvez. Dpto, 

regulation of avirulence genes from Pseudomnonas syringae. A Fitopatologia. MAG & CNP San Jose, Costa Rica. CIAT, Cali, Colombia. 

promoterless bacterial luciferase (Lux) operon was made as a 

reporter system and flanked by Tn7 border sequences which permit The effect of fungicide was studied in the bean varieties, Porrillo 70, 

its stable, single copy insertion into the bacterial chromosome Huetar, Ica Pijao and Diacol Calima to control T. cucumeris. Four 

[Barry, Gene 71 (1988)]. The promoter regions of avrD from treatements were evaluated: pentachloronitrobenzene(PCNB) at 15 kg 

P.s, pv. tomato and its homolog from P~s. pv. glycinea (P~sg) R4 a.i/ha; PCNB at 15 kg i~a/ha + benomyl 0.6 g a.i/I; benomyl 0.6 g a.i/l 

were transcriptionally fused to the Lux operono These con- and control. PCNB was applied to soil 7 days after planting, and 

structs were then inserted into the PsgR4 chromosome. Little benomyl was sprayed onto foliage after 20, 30, and 45 days. Porrillo 70 

or no light was produced when the cells were grown in rich or and Huetar averaged 323 kg/ha and 308 kg/ha respectively; whereas 

minimal culture media, but high level expression was induced DaoClmadIaPjoyedd4 n 3K/a lnswt 

from both promoters when the cells were inoculated into soybean DaoClmadIaPjoyedd4 n 3K/a lnswt 

leaves. Preliminary results have shown that addition of soybean benomyl yielded 365 kg/ha, and plants treated with PCNB produced 49 

l eaf intercell1ul ar fluids to mi nimal culture medi um al so i nduces kg/ha. The interaction of cultivars with fungicides showed that Porrillo 70, 

Lux expression. These results indicate that the avrD gene is yielded 753 kg/ha, had low disease severity, and an apparent infection 

plant inducible, possibly by specific plant constituents, rate (r) of 0 14; in contrast, Diacol Calima, had a yield of 1.7 kg/ha, 

higher disease severity, and an r value of 0 26. The disease progress 

curve fit a logistic transformation, in which the epidemic began slowly, but 

350 rapidly increased 30-50 days after planting. 

CHARACTERIZATION OF INDIGENOUS PLASMIDS IN PSEUDOMONAS SYRINGAE 

PV. MORSPRUNORUM AND P. 5. PV. SYRINGAE FROM CHERRY. J.M. 355 

Paterson and A.L. Jones. Department of Botany and Plant RELATIONSHIP OF IRRIGATION REGINE TO POPULATIONS OF 

Pathology. Mlichigan State University, East Lansing, MI 48824. NACROPHONIMA PHASEOLINA NICROScLEROTIA IN ROOT TISSUE AND 

YIELD OF SOYBEAN. S.R. Kendig and J.C. Rupe, University of 

Pseudomonas syringae pv. syringae (Pss) and P. s. pv. morsprun, Arkansas, Fayetteville, Arkansas 72701. 

Vol. 79, No. 10, 1989 1179

The relationship of populations of M. phaseolina and A. Karr. Department of Plant Pathology, University of Missouri, Columbis, 

microsclerotia (ms) in root tissue to final yield was deter- MO 65211. 

mined for the soybean cvs., Davis and Lloyd, grown under This study reports development of a rapid, sensitive, toxin bioassay with 

various irrigation regimes. Irrigation treatments included: E. coli and a selection by bioasssays with root cap, cotyledonary and leaf 

none, full-season, until flowering, and after flowering, cells and whole soybean plants for identification of resistant genotypes 

Biweekly, ma populations were determined in all roots to Macrophomina phaseolina toxin. Toxin is located by measuring the ability 

collected in a randomly selected 0.61 m section of row (my. of fractions to inhibit growth of E. coli, strain HB101, in soft agar 

12 plants/section). Recovery of ms began as early as 4 overlays. The samples to be tested [5 ul aliquots] in 95% (v/v) ethanol 

weeks after planting. Final soybean yields were positively are applied to the surface of the overlay, where toxic fractions cause a 

correlated (P=O.01) with irrigation and negatively corre- concentration dependent formation of clear plaques in the bacterial lawn. 

lated (P=0.01) with populations of ma from root tissue at For genotype selection, toxin-treated root cap, cotyledonary, or leaf cells 

weeks 16 (R4, pod development) and 18 (R5, early seed are tested for viability with Evans blue dye. Whole plants are scored for 

development). Microsclerotia were negatively correlated symptoms of leaf tissue collapse. With these techniques, the soybean 

with irrigation regimes (P0.01). There were no cultivar cultivars Chamberlain, Stine 3790, Resnik, Asgrow 4393 and Williams 82 

differences in populations of ma or yield. respectively, were ranked [least sensitive to most sensitive] in their 

sensitivity to the M. phaseolina toxin. Identification of the toxin raises 

the possibility for development of a selection program for resistance to 

356 charcoal rot. 

EFFECT OF RUST ON FORAGE YIELD AND DIGESTIBILITY OF DWARF AND 

TALL PEARL MILLET CULTIVARS. J.P. Wilson, R.N. Gates, and W.W. 

Hanna, USDA-ARS, Coastal Plain Expt. Stn., Tifton, GA, 31793. 360 

Effects of infection by Puccinia substriata var. indica on EFFECT OF CITRUS ROOTSTOCK ON POPULATIONS OF PHYTOPHTHORA 

yield and digestibility of pearl millet forage were examined PARASITICA IN FLORIDA. J. P. Agostini,* L. W. Timmer,** 

in 1988. Dwarf hybrids, Tifleaf 1 (susc) and Tifleaf 2 (res), W. S. Castle,** and D. J. Mitchell.* *Plant Pathology Dept., 

and tall hybrids, Gahi 3 (susc) and Gahi 4 (res) were treated Univ. of Florida, Gainesville, FL 32611; **Citrus Research and 

with chlorothalonil or inoculated after the first harvest to Education Center, 700 Expt. Station Rd., Lake Alfred, FL 31 

establish different levels of disease. No differences in yield The effect of rootstock on the population of P. parasitica was 

or digestibility were detected between disease-free plots of determined by plating soil from the root zone-from two Valencia 

susceptible and resistant cultivars for either dwarf or tall sweet orange rootstock trials and one pot test with seedlings 

types. Rust severities of inoculated plots of Gahi 3 were 54% on selective media periodically from Dec. 1987 to Feb. 1989. 

of those of Tifleaf 1. Green yield, dry matter yield, and in The average number of propagules per cm of soil over 4 to 5 

vitro digestibility were negatively correlated with final sampling dates was: (St. Cloud, Avon Park, and pot test): 

disease severity and area under the disease progress curve of Palestine sweet lime (29,10,...), C. volkameriana (30,... ,. 

both cultivars. Dry matter concentration was unaffected by Ridge Pineapple sweet orange (... ,13,102), Cleopatra mandarin 

disease. Digestible dry matter yield (Y, percent of healthy (17,10,87), trifoliate orange (14,8,14), and Swingle citrumelo 

control) as a function of percent disease severity 10 days (4,5,14). Populations were significantly higher in the sYmmer 

before harvest (X), could be expressed as Y=97.6-0.75X, and (32 propagules/cm 3 ) than in the winter (3.4 propagules/cm ) due 

Y=01.6-1.O1X for dwarf and tall cultivars, respectively, to the greater root biomass in summer. Soil populations 

generally reflected the susceptibility of the rootstocks to 

P. parasitica with the lowest population on trifoliate orange 

and Swingle citrumelo and the highest on sweet orange. 

357 

A POSSIBLE TOBAMOVIRUS ASSOCIATED WITH RINGSPOT AND OAKLEAF 

PATTERN IN EPIMEDIUM X YOUNGIANUM CV. NIVEUIM. M. L. Putnam and 361 

S. T. Nameth. Maryland Dept. of Agriculture, Annapolis, MD EFFECT OF TEMPERATURE ON DISEASE SEVERITY OF CORKY ROOT OF 

21401 and Dept. of Plant Path., The Ohio State Univ., Ohio Agr. TOMATO CAUSED BY PYR.ENOCHAETA LYCOPERSICI. N. Shishkoff, 

Res. and Dev. Center, Columbus, OH 43210. USDA-ARS, Frederick, MD 21701 and R. N. Campbell, Dept. Plant 

Pathology, Univ. Calif., Davis, CA 95616 

Sap extracts from Epimedium leaves taken from a nursery in 

Maryland, showing chlorotic ringspots and oak leaf patterns were Tomatoes were sown in infested soil in microplots at monthly 

rub inoculated onto the leaves of indicator hosts. Symptoms on intervals from Feb. to May 1988 to provide varying temperature 

indicator hosts included local lesions on the inoculated leaves regimes. The disease progress curves were linear and 

of Nicotiana tabaccum cv. Samsun and Gomphrena globosa and a significantly lower only for the last planting date. Disease 

systemic infection and eventual death of Nicotiana benthamiana. severity was greater at 16 or 21 C than at 27 C when tests 

Chenopodium quinoa did not react. Sap extracts from infected were done in infested vermiculite in controlled environment 

'Samsun' leaves tested negative for TV (common strain), TSWV, chambers. In other experiments seedlings were grown in 

TSRV and TomRSV using an indirect-sandwich ELISA but positive infested vermiculite at 16 or 27 C and switched to sterile 

for viral-associated dsRNA. Electrophoresis of dsRNA in vermiculite and grown at 16 or 27 C for a lesion development 

infected 'Samsun' extracts revealed the presence of a replica- period. Disease was more severe at 16 C than at 27 C for the 

tive form of dsRNA with an estimated molecular weight of 4.2 x lesion development period, regardless of the infection 

106 and 4 subgenomic dsRNA's with molecular weights ranging from temperature. 

2.3 - 0.4 x 106. This appears to be the first report of virus 

associated with Epimedium. 

358 

PREDISPOSITION OF LATE EMERGING CORN PLANWTSTO EAR ROThING 

FUNGI. D. M. Huber, H. L. Warren, T. S. Roseman, and C. Y. Tsai. Botany & Plant 

Pathology Dept., Purdue University, W. Lafayette, IN 47907. 

362 

PATHOGENICITY OF ISOLATES OP RHIZOCTONIA SOLAIII AG-3 COLLECTED 

FROM POTATO PLANTS AND SOIL. D.E. Carling and R.H. Leiner. 

University of Alaska, 533 E. Fireweed, Palmer, AK 99645. 

Sclerotial, hymenial and lesion isolates of Rhizoctonia solani 

The effect of delayed emergence on growth, yield, and disease of four maize 

hybrids (A632xLH39, Pioneer Brand 3165, B73xMo17, and Callahan Brand C773) 

was evaluated under high fertility conditions on a Brookston silt loam soil. 

Treatments consisted of all plants emerging uniformly early, uniformyly 0days 

late, or a continuous mix of two early and one plant 10 days late. Final plant 

populations were 52,000/ha. No differences in growth, barrenness, biomass, or 

yield were observed for uniformly early, uniformly late, or early emerging plants 

in the mixed emergence treatments. Delayed emerging plants, adjacent to early 

emerging plants in the row, were severely stunted, excessively barren (70-90%), 

and produced only 20-40% of the biomass and 2-10% of the grain yield of early 

emerging plants. Ear rotting fungi were assayed by plating kernels on PDA and 

incubating at room temperature. Delayed emerging plants had 40-100% more 

kernels infected with Fusarium moniliforme, F. roseum, and Colletotrichumgraminicola 

than early or late uniform emerging plants, 

were collected from 20 plants from each of three fields in 

southcentral Alaska. Also, isolates were collected from soil, 

directly from pegs placed on KHP media, and indirectly via beet 

seed baiting. Additionally, isolates were collected (directly) 

from soils associated with several crops other than potato. All 

isolates were characterized as to anastomosis group (AG). Path- 

ogenicity testing was done on all isolates of AG-3 in soil on 

developing sprouts at 50 F. Nearly 95% of sclerotial, hymenial 

and lesion isolates but only 52% of soil isolates belonged to 

AG-3. Most isolates of AG-3 were moderately to highly patho- 

genic on potato sprouts. Pathogenicities of isolates collected 

from lesions, sclerotia, hymenia and soil were the same, P = 

0.05. Pachogenicicies of isolates collected from soils associ- 

aced with potato, carrot, barley, bluegrass or fallow were the 

same, P = 0.05. These data suggest that source and associated 

plant are not indicative of pachogenicicy of K. solani AG-3. 

359 363 

DEVELOPMENT OF BIOASSAYS FOR IDENTIFICATION OF SOYBEAN GENOTYPES RESISTANT INFLUENCE OF INOCULUM LEVEL AND IRRIGATION ON PHYTOPHTHORA ROOT 

TO MACROPHOMINA PHASEOLINA. F. Beleid-El Moshaty, A. Novacky, T.D. Wyllie, ROT OF PROCESSING TOMATO. 0. A. Neher and J. N. Duniway. 

1180 PHYTOPATHOLOGY

Department of Plant Pathology, University of California, Davis, 

CA 95616. 

are caused by Xanthomonas campestris pv. translucens, a seed 

transmitted pathogen. This bacterium has apparently spread via 

seed to a number of countries in the past decade, as evidenced 

Effects of initial Phytophthora parasitica populations and duration 

of furrow irrigation on root rot development and yield 

by appearance of the pathogen first in breeder's seed and later 

in commercial fields. A dry heat treatment (72"C for 7 days) of 

loss in processing tomato variety 6203 were quantified. Field 

plots in which P. parasitica was not detected initially were inoculated 

to give 0, 1, 4 and 12 propagules per gram soil. Disease 

incidence and severity increased significantly with inseverely 

infested barley is sufficient to reduce the infestation 

rate by seven orders of magnitude to undetectable levels. 

Germination of wheat and barley is reduced by dry heat, but 

still remains above 80% after seven days. 

creased inoculum levels in both 1987 and 1988. However, there 

was significant yield loss at the highest inoculum level in one 

of two years. Moderately severe symptoms on roots and shoots 368 

were required before yield loss was observed. Extending irrigation 

episodes from 4 to 24 hours significantly increased root 

and shoot symptoms in one year at one intermediate level of in- 

GROWTH OF BACTERIA ON PLANTS: DETECTION AND QUANTIFICATION. 

Joe J. Shaw, Department of Botany and Microbiology, Auburn 

oculum. The results suggest that early season inoculum levels University, Auburn AL, 36849 

must exceed a threshold for disease to develop and that a higher 

threshold is necessary for significant yield losses to occur. Bacterial pathogens of plants are ubiquitous in nature. Plant 

364 

pathogenic bacteria are often found living in the soil, on nonhost 

plants and as epiphytes on host plants,and in these cases 

do not cause disease, although these sources may serve as 

primary sources of disease inoculum. Thus, it is important 

FACTORS INFLUENCING THE DEVELOPMENT OF FUSARIAL WILT OF BANANA to know where bacteria are what they are doing when they grow 

(PANAMA DISEASE). R.C. Ploetz, University of Florida, IFAS, upon plants without causing disease. Bacteria which have been 

18905 SW 280th Street, Homestead 33031. genetically engineered to bioluminesce may be easily detected 

Conidial suspensions of Fusarium oxysporum f. sp. cubense (incitant 

of Fusarial wilt or Panama disease), and tissue-culture-deupon 

plants and enumerated. A bioluminescent isolate of 

Xanthomonas campestris pv. campestris is studied during stages 

of plant colonization on crucifers. Factors such as humidity, 

rived plantlets of race differentials of banana were used to temperature and host are examined as they affect host 

characterize virulence and resistance/susceptibility in this colonization. 

pathosystem. Experiments were conducted in autoclaved silica 

sand which was recolonized by aerial microflora for at least two 

wk prior to use; calcareous soil from south Florida (autoclaved 

or nonautoclaved) or freshly autoclaved or nonautoclaved sand 369 

were responsible for less consistent disease development. Light 

intensity was positively correlated with disease severity in 

growth chamber (photosynthetic photon fluxes < 130 pmols sec- 

UNUSUAL CHARACTERISTICS OF Agrobacterium tumefacen_ STRAINS ISOLATED 

1 

FROM WILD BLACKBERRY. M.L Canfield and L.W. Moore. Department of Botanyand 

m- 2 ) and in greenhouse environments (< 1,200 pmols sec-I m- 2 ). Plant Pathology, Oregon State University, Corvallis, Oregon 97331-2902 

Root size (displacement volume) and inoculum density (conidia 

ml-i) were also positively correlated with disease severity. Interest in the distribution and ecology ofA. tumefacien$ led toa study of naturally 

When these factors were optimized, differential responses were occurring galls in agriculturally undisturbed areas. As part of this investigation, an 

obtained for compatible and incompatible combinations of races unusual group of agrobacteria was isolated from aerial galls of wild blackberry. Thiry- 

1, 2, and 4 and race differentials. one of the 43 isolates induced galls on tomato plants and 16 of these were also highly 

virulent on Emla 7 apple rootstock, a host which has been difficult to infect with A. 

tumefaciens strains. The bacteria grow well only on a mannitol glutamate medium 

365 Withdrawn supplemented with a large variety of salts, trace elements and yeast extract. Although 

the strains formed a homogeneous group, attempts to identify them using classical 

biochemical tests failed to place them into _A. tumefaciens biovars 1, 2, or 3, nor did they 

fit the description for A. rubi strains. The strains were, however, presumed to beA. 

tumefacien because of their ability to induce tumors and the strong homology between 

their DNA and 32 labeled probes for vir region genes of the Ti plasmid. These 

characteristics illustrate again the diversity found within the genus Aprobacterium. 

370 

A toothpick assay to determine the ice nucleation activity of bacteria. D.E. 

Legard and J. E. Hunter. Cornell University, New York State Agricultural 

Experiment Station, Geneva, NY 14456 

366 

A rapid assay was developed for the routine determination of ice nucleation 

activity (INA) of Pseudomonas syringae. The method involves placing a mass of 

cells from a 24 hr culture that have been prechilled at 4C on the end of a sterile 

toothpick and inserting the bacteria-laden end into a prechilled 1 ml glass culture 

tube containing 650 ul of sterile distilled water. After incubation at the desired 

RELEASE WALLS BY OF ENZYMES HYDROXYPROLINE FROM ERWINIA..CAROTOVORArSUBSP. RICH PROTEINS FROM POTATO CAROTOVORA CELL temperature for 20 min, the tubes can easily be evaluated for INA by determining 

if the protruding toothpick is still mobile. The effect of growth temperature, 

(Ecc). J. Lewosz, A. Kelman and L. Sequeira. University of media and, prechilling of cultures and tubes were evaluated with 93 strains of 

Wicni-aio,1630 Linden Drive, Madison, WI 53706 Pseudomonas syringae pv. syringae from numerous hosts. Several other 

WiscnsinMadionpathovars of P. syringae were also evaluated. Strains of P. syringae known to 

Cellwals ) wre cyoplsmi feedof proein bybe INA at -3.0C when grown at 22C for 24 hr would occassionaly fail to be INA 

Cellwals f aI wee ctoplsmi feed proein byat 

repetitive extraction with deoxycholate and b) noncovalently 

-3.OC when grown at 25C, and very few strains were INA when grown at 28C 

or 31C. Prechilling of the cultures and testing apparatus at 4C for 30 mmi before 

bound proteins by extraction with 2 N NaCl and phenol :ace tic transferring bacteria to culture tubes restored INA of strains grown at 25C, 28C, 

acid:water 12:1:11. Additional proteins and pectic materials or31C. Growth of strains on three media (KB, NA, KBC) did not affect lNA at 

were released by extracting with 0 .5% EDTA, hot ammonium -3.OC. The toothpick assay used with the prechilling protocol provides a reliable 

oxalate or 5 M, ammonia. Most hydroxyprol ine-r ichanraimenofeautglrenmbsofacraltansorIA 

glycoproteins were released by boiling the cell walls at pH adaimasfvlaigagnmesfatrasrisolA 

1.0. Extensins were partially released after splitting 

isodityrosine linkages with Na-chlorite. Solubilized 

extensins were not degraded significantly by bacterial 371 

proteases freed of pectinolytic enzymes by affinity 

chromatography on cross-linked polygalacturonate. Release of DIFFERENCES TM FATTY ACID PROFILES OF PSEUDOMONAS SYRINGAE 

hydroxyproline-containing compounds by culture filtrates of PV. TOMATO AND PSEUDOMONAS SYRINGAB PV. SYRINGAE DUE TO 

Ecc is attributed to pectin degradation that results in PHYSIOLOGICAL AGE AND CULTURE MEDIUM. A. E. Voloudakis, 

liberation of tightly-linked proteins. R.D0. Gitaitis, and R. W. Beaver. Univ. of Georgia, Tifton, 

GA 31793-0748. 

367 Strains of Pseudomonas syringae_ pv. tomato and P_. a. pv. 

DRY HEAT SEED TREATMENT FOR XANTHOMONAS CAMPESTRIS PV. 

TRANSLUCENS. D. C. Sands, E. Fourrest, and L. Rehms. Dept. of 

syringae were isolated from tomato transplants; grown on KMB 

and NA for 2, 4, and 6 days; and analyzed for total cellular 

fatty acids by gas-liquid chromatography. The percentage of 

Plant Pathology, Montana State University, Bozeman, MT 59717. 9-octadecenoic acid (18:1w9) was significantly lower in 

Bacterial black chaff and bacterial leaf streak of cereal grains 

cultures grown on KMIB. Thne relative levels of dodecanoic 

(12:0) and 2-hydroxydodecanoic (2-0H-12:0) acids increased 

Vol. 79, No. 10, 1989 1181

over time, whereas hexadecanoic (16:0) acid decreased in both of CBS; only the highly aggressive strains were spread natupathovars 

regardless of growth medium. The ratio of rally whereas less aggressive strains were spread mechanically. 

saturated to unsaturated fatty acids was higher in cultures Of 25 independent outbreaks since 1984, aggressive strains have 

grown on KMB. The ratio initially increased, but decreased appeared in only 4 nurseries. 

between days four and six for both pathovars and media. 

376 

372 INOCULUM PRODUCTION FROM ASIATIC CITRUS CANKER LESIONS AND 

MOLECULAR CLONING OF XANTHOMONADIN PIGMENT SYNTHESIS GENES EPIPHYTIC SURVIVAL OF XANTHOMONAS CAMPESTRIS PV. CITRI IN 

FROM XANTHOMONAS CAMPESTRI$ FOR IDENTIFICATION OF ARGENTINA. L. W. Timmer, Univ. of Florida, Citrus Research and 

XANTHOMONADS. Kawalek. MD. , Poplawsky, A.R., and Schaad, N.W. 2 . Dept. Education Center, 700 Expt. Station Rd., Lake Alfred, FL 33850. 

PSES, Division of Plant Pathology, Yniversit of Idaho, Moscow, ID 83843. Present 

address: 1Oregon State University; HarrisMoranSeedCo. Inoculum production was studied by placing water in plastic 

reservoirs over canker lesions and measuring bacterial 

Xanthomonads are the cause of many seed transmitted diseases. However, proper 

identification of these bacteria requires lengthy pathogenicity testing. Because the 

xanthomonadin pigment is unique to Xanthomonas species, genes for pigment synthesis 

could be very useful for identification purposes. Eleven pigment minus mutants of 

Xanthomona gampestri pv.gampestrij B-24 were produced by exposure to ethyl 

methanesulfonate. All mutants were typical in colony morphology on YDC agar, positive 

for starch hydrolysis, and virulent when tested in cabbage seedlings. Nine of the mutants 

were prototrophic when tested on basal M9 glucose agar. A genomic library of B-24 DNA 

was constructed in cosmid vector pLAFR3 and mobilized into one of the mutants via the 

helper plasmid pRK2013. Two different cosmids with B-24 DNA inserts of 27 and 32 kb 

complemented this mutation for pigment production. In subsequent matings of these 

cosmids, all eleven of the mutants were complemented. Experiments are underway to 

further subclone these inserts and determine their specificity to various xanthomonads. 

populations in exudates by plating on selective media. Upon 

wetting new, fully developed lesions, 0 to 1 bacteria/al 

were detected immediately and populations rose slightly a 

with further wetting to 24 hr. Old lesions produced 10 

bacteria/ml or less and extended wetting did not increase 

populations. A leaf-swab technique was used to assay epiphytic 

populations on symptomatic leaves and on asymptomatic leaves 

trfp 

of 

10 to seedlings. 106 per leaf Populations in the early on symptomatic morning and leaves declined varied 

to 12from 

105 per leaf respectively, by mid-afternoon The bacteri 

was consistently 3detected ot trap plants, but populationsw 

always low (< 10 per leaf). Most inoculum for spread of 

citrus canker is produced by exudation of bacteria from new 

lesions. Epiphytic bacteria on asymptomatic leaves are of 

little epidemiological significance. 

373 

LETTUCE CORKY ROOT CAUSED BY STRAINS OF A GRAuM-NEGATIVE 377 

BACTERIUM FROM MUCK SOILS OF FLORIDA, NEW YORK, AND WISCONSIN. 

A. H. C. van Bruggen, P. R. Brown, and K. N. Jochimsen. Dept. FRQUENOCY, DISTRIBUTION AND CHARACTERISTICS OF 

of 

EidOPaYTIC 

Plant Pathology, University of California, Davis, CA 95616. ?SEUDO4Oreg. SYRInA IN PEAR TREES. 

Spotts. 

S. L 

Oreg. 

Whitesidea 

St. Univ., 

and 

Mid-Columbia 

R A. 

Agric Research and 

Slow-growing bacteria similar to a Californian strain (CAI) Extension Center, Hood River, OR 97031. 

causing lettuce corky root (CR) were isolated from muck soils 

of Florida, New York, and Wisconsin, using lettuce seedlings Internal stem and root tissues of pear trees were sampled 

as bait. 

for 

All isolates were tested for reaction with poly- presence of Pseudomona syringes (Pa) at 7 orchard 

clonal 

sites 

antibodies 

in 

produced against strain CAI, and for patho- Oregon. Endophytic Pa were found in 76 (45/59) r of tries 

genicity on a CR susceptible (Salinas) and resistant (Green sampled with 140 of 195 isolates found in root 

Lake) 

tissues. 

lettuce cultivar in a greenhouse. Five strains from Electrophoretic banding profiles of total 

Florida, 

bacterial 

three 

DN iwere 

from New York, and three from Wisconsin induced used to compete isolates. Visual evaluation shoved identical 

severe CR symptoms on Salinas and mild symptoms on Green Lake. tr eei and between isolates fo mn d je t tre es owever, 

All strains were gram-negative, aerobic, oxidase, positive, tree and between isolates from adjacent trees. 

catalase 

However, 

positive, and reduced nitrate to ammonia. Whole-cell different profiles were observed between root 

fatty 

and 

acid 

stem 

compositions 

isolates 

were similar for all isolates, in- in one tree. Ps inoculations into internal tissues 

dicating 

of potted 

that CR 

bacterium 

of lettuce 

in 

is 

Florida, 

caused 

New 

by strains 

York, Wisconsin, 

of the same 

and California. trees 

stems 

resulted 

and no movement 

in limited, 

above 

detectable 

the crown 

Pa 

from 

movement, 2-3 cm 

root inoculations. 

in 

All isolates were tested for ice nucleation activity with only 

3 isolates testing positive. These 3 were from 374 4 year old stem 

tissues of 2 trees from different orchards. 

PERSISTANT, NON-LYTIC BACTERIOPHAGE IN STRAINS OF 

XANTHOMONAS CAMPESTRIS PATHOVARS PRUNI AND VESICATORIA 

D.F. Ritchie. Department of Plant Pathology, N.C. State 

Univ., Raleigh, NC 27695. 378 

PATHOGENICITY AND BIOTYPE DETERMINATION OF AGROBACTERIUM ISO- 

More Moresthant2 than 25 strains stratins of each eachp e pathovar thov were ugar w reasydoring assayed y for 

peraistant, non-lytic phage by centrifugally removingSteUnvriyMssipitae 

LATES Graves. FROM Dept. MUSCADINE. of Plant K. Pathology L. Thies, and D. Weed E. Griffin, Science, and Mississippi C. H. 

S396 

bacteria from 18-24 h broth cultures and spotting 1 Aul of 

supernatant on the lawn of each bacterial strain in 0.6% 

top agar. Phage were detected in cultures of all strains, 

Detection Detectin Tinesoame in some strains depended dpnded on the ndicat?6 on th strain 

to srion 

State University, Mississippi State, MS 39762. 

Although Agrobacteria are classified on the basis of pathogeni- 

city, three distinct 

ciy the'itntgop 

groups (Biovars 

Boas1 1 2 .ad3 and 3) are r recognized 

eo 

the basis of biochemical tests. Crown gall on grapes (Vitis 

used. Titers among strains ranged from 10 to 10 vinifera) is considered to be due to Agrobacterium tuefaciens 

pfu/sl. Attempts to "cure" a strain using acridine orange biovar 3. A high incidence of Agrobacteria were isolated from 

were unsuccessful. Phage infectivity was destroyed by 10 vascular fluids, galls, and roots of muscadine (Vitis rotundisin 

exposure to > 1% chloroform but most phage strains folia) throughout Mississippi. Selective sedia and diagnostic 

were not inactivited by 10 sin exposure to 90C. Phage tests were used to determine biovars, while lateral bud culnucleic 

acid was not sensitive to RNase A but was tures, detached suscadine leaf assays, and whole plants were 

sensitive to sung bean nuclease. This suggests these use todtrieptoe'iyo slts l he ivr 

phags cntai saMA.were isolated. Biovar 3 was the predominant biovar isolated 

from vascular fluids. Gall induction by both biovar 1 and 3 was 

375 demonstrated. Additional isolates are being assayed to determine 

the relationship between biovar and crown gall development 

VARIATION IN AGGRESSIVENESS OF XANTHOMONAS CAMPESTRIS PV. in muscadine. 

CITRUMELO ASSOCIATED WITH CITRUS BACTERIAL SPOT (CBS) IN 

FLORIDA. J. H. Graham and T. R. Gottwald, University of 

Florida, Lake Alfred 33850 and USDA-ARS, Orlando, FL 32803. 379 

Wound-inoculated detached leaves of Swingle citrumelo and TRANSMISSION AND REISoLATION OF A BACTERIUM ISOLATED FROM 

Duncan grapefruit were used to characterize strains of Xc pv. GREENING-INFEcTED CITRUS IN SOUTH AFRICA. R-J Chippindall and 

citrumelo from citrus nurseries and to distinguish these-- V H Whitlock, Department of Microbiology, University of the 

strains from Xc pv. citri causing Asiatic citrus canker. CBS Witwatersrand, P 0 Wits, 2050, South Africa. 

strains varied in aggressiveness based on the extent and'" 

persistence of watersoaking and the development of necrosis. A culture of a bacterium repeatedly isolated from greening- 

Aggressiveness on detached leaves was correlated with that on infected citrus in South Africa was shown to induce characterwound-inoculated 

leaves in the greenhouse and field. In vitro istic greening symptoms when mechanically inoculated into citrus 

inoculations clearly distinguished the flat-spreading T-sions and the organism reisolated from test plants, thereby fulfilling 

of CBS from the erumpent, callus-like reaction produced by Xc Koch's postulates. The bacterium was also isolated from symptopv. 

citri. In 4 nursery outbreaks, strain aggressiveness wa-s matic cucumber connected via dodder to naturally infected citrus. 

related to severity of leaf symptoms and spatial distribution Based on these findinqs, this bacterium was thought to be the 

1182 PHYTOPATHOLOGY

causative agent of greening, however, insect vector transmission area, while the susceptible check had more than 70%. DSI may 

of the isolate was negative, and inoculated plants did not ex- help breeders predict partial resistance of rice genotypes that 

hibit the presence of the gentistic acid marker of greening dis- do not encounter virulent races of P. oryzae during tests. 

ease. Also, antisera raised against the isolate failed to consistently 

detect greening in citrus known to be infected with 

the disease. Results suggest that this organism, identified as 384 

a Clavibacter spp. on the basis of morphology and fatty acid 

profiles, is not the causative agent of citrus greening per PROTECTION OF YIELD BY COMBINING RESISTANCE AND TOLERANCE TO 

but a probable component of the "greening syndrome". SEPTORIA TRITICI BLOTCH OF WHEAT. Z. Eyal, Department of Botany, 

Tel Aviv University, Tel Aviv 69978, Israel. 

380 

Selection for high 1000-kernel weight (TKW>4Sg) was performed 

in early generations of crosses between the moderate-resistant 

MORPHOLOGICAL COMPARISONS OF MALES OF ONE ISOLATE EACH OF HET- 

ERODERA GLYCINES, H. CRUCIFERAE AND ONE OF THEIR HYBRIDS. L. 

I. Miller, Dept. of Plant Path., Phys., & Weed Sci, VPI&SU,- 


Comparisons were made of male characters of 21 specimens each 

of one isolate of Heterodera glycines(M) cultured on 'Lee' 

soybean, one isolate of H. cruciferae(C) cultured on 'Market 

Prize' cabbage and one of their hybrids(MC) cultured on soybean. 

Dimensions in Pm were as follows-length(LTH) :M 

1275-1600 (mean 1428, s.d. ± 106.5), C 930-1270 (1095 ± 100.7), 

MC 1050-1510 (1256 ± 111.6); stylet knobs to dorsal gland orifice 

(DGO) :M 3.0-4.9 (3.7 ± 0.6), C 3.9-4.9 (4.3 ± 0.3), MC 

2.3-3.5 (2.7 ± 0.3); breadth of stylet knobs(KBS) :M 4.0-5.2 

(4.6 ± 0.4), C 3.7-5.0 (4.4 ± 0.4), MC 4.4-5.7 (5.0 + 0.3). 

cultivar Musala"S" (CIv4YT, CM16780) and the susceptible, high- 

TKW cultivar Lakhish and with the tolerant cultivar Miriam, 

under sever epidemics induced by a mixture of Septoria tritici 

isolates. "Frontal" resistance from Musala"S" was detected in 

selected, tolerant F5 lines using isolate ISR398, and the 

"background" tolerance by using ISR8036 to which Musala"S" is 

susceptible. Some Musala"S"/Lakhish lines expressed the 

resistance of Musala"S" to ISR398 and retained the high TKW of 

Lakhish under ISR8036 epidemic. Some Musala"S"/Miriam lines 

expressed resistance to ISR398 and TKW as high or higher than 

that of Miriam when subjected to ISR8036. Lines were selected 

which combine "frontal" resistance to ISR398 and high kernel 

weight retention to ISR8036, together with earliness and highyieldind 

potential. 

Characters LTH and DGO were significantly different (P=0.05) 

for M, C and MC. Character KBS for M and C was not significantly 

different; however dimensions of the MC hybrid differed 

significantly from the M and C parents. The MC hybrid reproduced 

on soybean, cabbage and 'US75' sugarbeet. 

385 

COMPONENTS OF RESISTANCE OF TOBACCO TO PHYTOPHTHORA PARASITICA 

VAR. NICOTIANAE. Keith Jones and H. D. Shew, Department of 

Plant Pathology, North Carolina State University, Raleigh, NC. 

Four cultivars of tobacco with field resistance ranging from 

381 highly resistant to susceptible were used. Root growth was 

quantified in a controlled environment and in the field using 

RESISTANCE TO WHEAT LEAF RUST OF LAND CULTIVARS AND THEIR root observation chambers. Data were collected using computer 

DERIVATIVES. Beatriz A. Perez and A. P. Roelfs, Department of based digitizing equipment. The highly resistant cultivar 

Plant Pathology, University of Minnesota and Cereal Rust produced roots more slowly than the other cultivars, reducing 

Laboratory, USDA/ARS, St. Paul, MN 55108. the number of pathogen propagules encountered. Inoculum 

Many wheats (Triticum aestivum) have been derived from Americano efficiency was observed by inoculating zoospores onto 

25c, Americano 26n, and/or Americano 44d. Durable resistance to undisturbed roots of plants in controlled and field conditions, 

leaf rust in several modern wheats has been related directly to and was least on roots of the highly resistant cultivar. Lesion 

resistance from Americano 44d. Seedlings and adult plants of expansion on roots also was followed visually. Maximum lesion 

these wheats, selected cultivars, near-isogenic lines used for expansion was measured by plating root samples onto selective 

race identification, and Thatcher were tested with isolates of medium, and with an ELISA system. The ELISA system was used to 

Puccinia recondita f. sp. tritici. Some cultures differentiated quantify the amount of fungal tissue in root lesions on the four 

Americano 25c from Americano 26n and Americano 44d. Seedlings of cultivars. These results show that root production and inoculum 

Americano 25c were resistant to avirulent cultures on TcLrl6. efficiency are important components of field resistance. 

Seedlings and adult plants of Buck Manantial, an Americano 25c 

derivative, were immune to all cultures tested. Seedling and 

adult plants of Americano 26n and Americano 44d were 386 

susceptible. The resistance of Americano 44d was moderately 

severe in adult plants. Seedlings of Marcos Juarez Inta, an GENETIC CONTROL OF IMMUNITY TO TURNIP MOSAIC VIRUS IN WINTER 

Americano 44d derivative, had a high infection type but was low OILSEED RAPE (Brassica napus ssp. oleifera) AND THE EFFECT OF 

to moderately severe in adult plant stage. FOREIGN ISOLATES OF THE VIRUS. J. A. Walsh, AFRC Institute of 

Horticultural Research, Wellesbourne, Warwick, CV35 9EF, UK. 

Immunity to a UK isolate of turnip mosaic virus (TuMV) was 

382 studied in eight lines of oilseed rape. Six of these lines 

were homozygous for the immunity factor and two were from 

Invitrscreening forresistance to grayleaf spot inmaize. VMBes_,JM heterozygous parents. Segregation ratios in the F 2 generations 

Perkins, AS Wang, ME Coker,JB Mlcic,DS Cheng. Sungene of reciprocal crosses between homozygous immune lines and 

Technologies Corp., 2050 Concourse Dr., San Jose, CA 95131 homozygous susceptible cultivars showed that immunity was 

controlled by a single dominant nuclear gene. The immunity was 

Isolates of Cercospora zeae-maydis were collected from the field and confirmed by the inability to detect virus particles in 

cultured in liquid media. Cell-free filtrates were prepared that proved 50% mechanically inoculated plants by back inoculations, ELISA, and 

toxic to immature embryos and 90% toxic to callus cultures of maize. Over ISEM tests. Plants were immune to repeated inoculations and 

250 plants were regenerated from the surviving callus and grown to aphid transmissions. The immunity was effective against two 

maturity in the greenhouse. Progeny of these plants showed a6O% increase German isolates of TuMV. However, a Greek isolate partially 

in resistance to the filtrate. Field resistance to gray leaf spot is being tested. overcame the immunity causing local infection only and Canadian 

383 

and Danish isolates overcame the immunity completely causing 

systemic mosaic-type symptoms. 

PREDICTION OF PARTIAL RESISTANCE TO RICE BLAST USING 

MULTILOCATION TRIALS. S.W. Ahn, O.V. Seshu, and J.M. eonman. 387 

International Rice Research Institute, P.O. Box 933, Manila, COMPARATIVE STUDIES OF RESISTANCE IN PEACH GENOTYPES TO 

Philippines. MONILINIA FRUCTICOLA. J.E. Adaskaveg, A.J. Feliciano, snd J.M. 

Assessment of partial resistance to blast is often impossible Ogawa. Dept. of Plant Pathology, University of California, Davis. 95616 

for some cultivars because virulent races of Pyricularia oryzae 

are not present at the screening site. Thirty-nine rice culti- Blossoms and fruit of peaches were evaluated for resistance to _M. 

vars were evaluated for partial resistance in upland miniplots. fructicola. Blossoms were spray inoculated with a spore suspension (2 

Results were analyzed to determine the relationship between the X 104 conidia/ml) and incubated with free-moisture for 48 hr in the 

partial resistance assessment and disease severity index (OSI) . laboratory (20 C) or 48-72 hr in the field (16-20 C). In both tests, 

The OSI is the average score of compatible reactions for a percentage of anthers infected was 10-00% less in resistant (Bolinha, 

cultivar in the International Rice Blast Nursery (IRBN) trials. Kakamas) than in susceptible genotypes (Slamn, Loadel, Tufts, Flavor- 

IRBN is a ,sultilocation trial conducted at more than 20 sites in crest) after 48 hr. Incubation > 72 hr resulted in no differences 

different countries yearly. OSI was positively correlated with between genotypes. Non-wounded fruits were spray inoculated with a 

diseased leaf area (r=0.77) and log diseased leaf area (r=0.B0) . spore suspension and incubated as above for 2-16 hr at 20 C, air-dried, 

Seventeen cultivars with DSI less than 5.5 had 10% diseased leaf and evaluated after 72 hr. Percentage of infected, inoculated fruits 

Vol. 79, No. 10, 1989 1 183

increased from 0% (Bolinha) and 60% (Corona) after 2 hr of wetness to resistance against two compatible isolates. NIs CL03TTP, 

12.5-25% (Bolinha) and 100% (Corona) after8 hr of wetness. Histological C104LAC, C1O1PKT, and C105TTP-4 showed 29-61% fewer lesions than 

studies of fruit, in similar stages of development, demonstrated C039. NIs C104PKT and C102PKT differed little from C039. Thus, 

morphological differences in the epidermal tissue between resistant certain NIs have residual resistance compared to the recurrent 

(Bolinha, Kakamas) and S-genotypes (Corona, UCL)18-8-11). Generally, parent. Efforts are underway to pyramid resistance genes from 

R-genotypes had a thicker cuticle, compact epidermal cells, and fewer the NIs showing residual resistance. 

trichomes that originated from the first cell layer of the epidermis. 

388 392 

EVIDENCE FOR CYTOPLASMIC OR MATERNAL DETERMINANTS IN THE 

VARIATION IN TOLERANCE TO BENOMYL AMONG COLLETOTRICHUM REACTION OF WHEAT CULTIVARS TO TAN SPOT. Shabeer, A., W. W. 

GLOEOSPORIOIDES ISOLATES FROM MANGO. R.T. McMillan, Jr., Bockus, and B. L. Norman, Dept. of Plant Pathology, Kansas State 

Michael M. Moss, L.R. Bowling,and L. Stempel, University of Univ., Manhattan, KS 66506. 

Florida, IFAS, Trop. Res. & Educ. Center, Homestead, Florida 

USA, 33031. 

Two tan spot-resistant winter wheat cultivars, Red 

and 

Chief 

Auburn 

(RCH) 

(ABRN), were crossed with susceptible TAMI05. 

In 1987 and 1988 a mango grove located in Dade County Florida Parent and F1 seed of reciprocal crosses was produced in the 

USA lost over 50% of the crop to mango anthracnose caused by C. 

g1oeosporioides. 

greenhouse. 

The 

Plants 

grove 

were 

was 

grown 

sprayed 

in a randomized 

with 1 1/2 

block 

ibs of 

design with 

oeosporo ideskly Te gover asd sperayedto4 

grehue-lnswr-rw 

withk1 1/2er lbsfr 

i admzdbokdsg 

set 9 replications, 10 plants/entry/replication, 

benomyl 

inoculated 

weekly in flower 

with a 

and every 3 to 4 weeks after fruit set conidial suspension (1 3 ,000/ml) of Drechslera tritici-repentis, 

by the owner in 1987 and by a professional grove management and placed in a mist chamber. The top 

company 

3 leaves 

in 1988, 

of each 

with 

plant 

no noticeable control of anthracnose in were rated on a 0-5 scale. Average leaf ratings using 

both 

ABRN 

years. 

as 

In the summer of 1988, 100 infected fruits were the resistant parent were: ABRN - 0.79, 

harvested 

ABRN X 

randomly 

TAM105 (F 1 ) = 

from the grove from which 84 single spore 1.80, TAM105 X ABRN (F1 ) 2.18, and TAM105 - 

colonies 

2.50. Av 

were isolated. The isolates were screened at 0, 1, 10, ratings using RCH were: RCH - 0.91, RCH X TAM105 (F 1 ) = 

and 100 ppm 

2.10, 

of benomyl. Out of 84 single spore colonies, nearly TAM105 X RCH (F 1 ) = 2.35, and TAM105 = 2.75. 

40% 

Ratings 

were tolerant 

of F 

to 10-100 ppm of benomyl, while 1 

60% were plants when the resistant parent was used 

sensitive, 

as a female 

showing little or no radial growth. These results significantly (E = 0.05) less than when it was used as ale 

may explain the lack of anthracnose control by benomyl in the indicating reciprocal effects. 

grove. 

389 393 

INFECTION OF PEPPER MESOPHYLL PROTOPLASTS WITH PEPPER MILD INDEPENDENT SELECTION OF RESISTANCE TO USTILAGO SCITAMINEA AND 

MOTTLE VIRUS USING ELECTROPORATION. J.L. Jacobs and C.T. SUGARCANE MOSAIC VIRUS (SCMV) IN SUGARCANE. M. P. Grisham, B. 

Stephens. Michigan State University, East Lansing, MI 48824. L. Legendre, and J. W. Dunckelman, USDA, ARS, Sugarcane Research 

Unit, Houma, Louisiana 70361. 

Mesophyll protoplasts of pepper (Capsicum annuum) were inoculated 

with pepper mild mottle virus (PMMV) by electroporation. Sugarcane 

The protoplasts, 

smut caused 

derived 

by Ustilago 

from 

scitaminea 

cotyledons 

and 

of 

sugarcane 

seedlings grown mosaic (SCM) caused by SCMV are important 

in 

diseases 

vitro, were 

of Louisiana 

obtained by treatment with an enzyme solution sugarcane. One-hundred 

containing 

sugarcane 

1.0% 

clones 

(w/v) 

from 

Cellulysin, 

each of four 

0.5% (w/v) Driselase, 0.1% biparental crosses of parents 

(w/v) Macerase, 

with different 

CPW 

combinations 

salts and 0.4 

of 

M sorbitol at pH 6.0. reactions 

Infection 

to U. scitaminea 

was 

and 

achieved 

SCMV 

with 

were evaluated 

several direct 

for 

current pulses resistance t6the two pathogens 

of 10 

in 

msec 

separate 

at a 

field 

field 

tests. 

strength 

No 

of 0.36 to 0.57 kV/cm and association was found between 

a virus concentration 

resistance or susceptibility 

of 100 pg/ml in 

to U. 

a 0.7 M mannitol solu- scitaminea and to SCMV 

tion. 

within 

The 

or 

proportion 

among the progeny 

of infected 

of the 

protoplasts 

four 

was quantified crosses. The 

by 

breeder 

staining 

can 

with 

therefore 

viral coat 

concurrently 

protein-specific 

select for 

antibodies conju- resistance to U. scitaminea and SCMV, or 

gated 

independently 

to fluorescein 

select 

isothiocyanate. The percentage of viable for resistance 

protoplasts 

to one without 

after 

increasing 

electroporation 

the probability 

was determined 

of 

by Evan's selecting for 

blue 

susceptibility 

exclusion. 

to 

Variables 

the other. 

that 

Among 

influence 

the four 

the optimum uptake crosses, the number of clones 

of 

that 

whole 

developed 

virus 

smut 

particles 

was greater 

(PMMV) 

in 

into protoplasts of pepper one cross in which the female 

are 

parent 

being investigated. 

was susceptible to U. 

scitaminea, but the number of clones with SCM did not diTf'er. 

390 394 

HERITABILITY OF RESISTANCE TO WINTER STRESS FACTORS IN DACTYLIS The Effect of Ethylene and Silver Thiosulfate on the Disease 

GLOMERATA. Anne Marte Tronsmo, Norwegian Plant Protection Expression of Colletotrichum lagenarium on Cucumber Leaves. 

Institute, P.O.Box 70, N-1432 AS-NLH, Norway. C. Biles, F. Abeles, and C. Wilson. USDA-ARS, Appalachian Fruit 

Research Station, 45 Wiltshire Road, Kearneysville, WV. 

In Norwegian climate winter survival is a critical stage in the 

cultivation of perennial grasses. Winter injury is caused by CUcumber seedlings were sprayed with 1mM silver thiosulfate 

snow molds and several abiotic factors. Improved winterhardiness (STS) or gassed with 100 ppm ethylene. After 24 hr, the first 

is an important objective in nour grass breeding, and we try to true leaves were inoculated with Colletotrichum lagenarium. 

obtain this by artificial testing. The progress depends on the Ethylene and water-treated control leaves exhibited high levels 

variation and the heritability of the traits. Earlier investi- of lesion development, whereas, STS-treated plants had 

gations have shown that there exists a great variation in snow significantly fewer lesions. Tests on culture media showed that 

mold resistance and freezing resistance. The heritability was STS was not fungitoxic to C. lagenarium. STS is known to be an 

studied in half sib families from polycross fields of Dactylis inhibitor of ethylene action. These data suggest that ethylene 

gIomrsta.. The broad sense heritability (h 2 ) for the polycross may play a role in lesion development and tissue senescence in 

progeny were 0.60 for freezing resistance, 0.49 for resistance cucumbers. 

to Typhula ishikariensis, and 0.42 for resistance to Fusarium 

nivale, which indicates good possibility for progress by selection 

for these traits. 395 

391 

31AND 

RESIDUAL BLAST RESISTANCE OF NEAR-ISOGENIC RICE LINES. 

J.M. Bonman, T.I. Vergel de Bins, and D.J. Nackill, International 

Rice Research Institute, P.O. Box 933, Manila, 

Philippines. 

THE REGULATION OF THE PEA DISEASE RESISTANCE RESPONSE GENE, 

DRRG-49, VIA BOTH AP-l DNA ATTACHMENT SITES AND TOPOISONERASES 

THEIR DNA CONSENSUS SITES. C. C. Chiang, A. Pettinger 

and L. A. Hadwiger, Department of Plant Pathology, Washington 

State University, Pullman, WA 99164. 

Topoisomerase II consensus site clusters, which specify 

A set of near-isogenic rice lines (NI) was developed through 

backcrossing and recurrent selection for resistance to specific 

isolates of Pyricularia oryzae. The recurrent parent, C039, is 

widely susceptible to tropical races of the pathogen. Nls were 

tested for complete resistance against a range of isolates, and 

7 groups were identified based on reaction patterns. C039 and 

Nls with different resistance genes were tested for partial 

scaffold attachment, exist on regions 3' and 5' to the DRRG- 

49 structural gene providing the potential for a chromosomal 

loop. Also, AP-l sites are present that recognize specific 

transcriptional proteins. In Fusarium solani challenged pea 

endocarp, there is a sharp increase in topoisomerase I 

activity and a release of chitosan heptamer. DRRC gene 

regulation in terms of topography and torsional stresses of 

chromosomal loops and by transcription factors, topoisomerases 

1184 PHYTOPATHOLOGY

and chitosan heptamer will be discussed. We propose the found to be exclusively the purportedly rare Al mating type. 

regulation of the DRRG-49 can result from major alterations More than 80 native fynbos plants were recorded as new Al 

in chromatin as well as from the more specific hosts. Isolates from cultivated forests and agricultural crops 

transcription factor-interaction involving DNA elements in the were predominately the A2 mating type and occurred on hosts 

gene's promoter regions. similar to those previously recorded elsewhere. The occurrence 

of the Al mating type on cultivated crops was limited to 

proteas, grapevines and pines in regions adjacent to native 

396 fynbos vegetation. 

REGULATION OF CAPSULAR POLYSACCHARIDE SYNTHESIS IN ERWINIA SP. 

BY rcsA. K. F. Poetter, F. R. Bernhard, and D. L. Coplin. 400 

Dept. of Plant Pathology, The Ohio State University, Columbus, 

OH 43210 ISOZYME COMPARISONS OF EIGHT PYTHIUM SPECIES. W. Chen, R. W. 

Schneider, and J. W. Hoy. Dept. of Plant Pathol. and Crop 

The rcsA gene product activates capsular polysaccharide Physiol., La. Ag. Exp. Station, La. State Univ. Ag. Center, 

biosynthesis in enteric bacteria. The nucleotide sequence of Baton Rouge, LA 70803. 

the rcsA gene from E. stewartii was determined and its 

predicted amino acid sequence had substantial homology with Starch gel electrophoresis of 13 enzymes was used to compare 

the homologs of the E. coll (64%) and K. aerogenes (58%) rcsA eight Pythium species (total 204 isolates) collected from North 

genes. The 3' end of the proteins were highly conserved (79- America, Hawaii, Japan, Korea and Australia. Banding patterns 

91%). A cosmid clone from an E. amylovora library complemented were determined for each isolate and similarity coefficients 

E. stewartii and E. coli rcsA mutants and stimulated were calculated for each pair of isolates. The matrix of 

expression of cps::lac fusions and EPS production in these similarity coefficients was then subjected to principle 

species. An rcsA::Tn5 mutation from E. stewartii was crossed component analysis. Isolates of morphological species 

into the E. amylovora chromosome and the resulting mutant was generally formed clusters. Clusters of P. ashanidermatum and 

avirulent and nonmucoid. Preliminary nucleotide sequence P. deliense, and P. arrhenomanes and P. graminicola could not 

comparisons of the rcsA genes in E. stewartii, E. amylovora, be distinguished.- P. irregulare and P. s pinosume and P. 

and E. herbicola suggest strong conservation of rcsA in the myriotylum and P. ultimum clustered independently, but 

genus Erwinia. overlapped. Isozyme variation within species was sometimes 

397 

associated with differences in geographic origin or morphology. 

Host differences did not affect variation. The significance of 

the findings in regard to Pythium taxonomy will be discussed. 

QUANTITATIVE GENETIC ANALYSIS OF NORTHERN LEAF BLIGHT AND 

LEAF FRECKLES AND WILT RESISTANCE IN A CORN SYNTHETIC. 

Hidayat Rahman, M. 

Science 

L. Carson 

Department, 

and 

Box 2109, 

Z.W. 

Brookings, 

Wicks, 

SD 

III, 

57007. 

SDSU, Plant 401 

NON-RANDOM DISTRIBUTION OF VIRULENCE AND PHENOTYPIC DIVERSITY 

Two hundred North Carolina Design I progenies from SDPPS maize IN TWO POPULATIONS OF PUCCINIA RECONDITA IN CANADA. J.A. 

synthetic were evaluated during the summer of 1988 in two Kolmer, Agriculture Canada, Winnipeg MB R3T 2M9 Canada. 

separate experiments for northern leaf blight (NLB) and leaf 

freckles and wilt (LFW) resistance at Brookings, SD. For LFW, 

plants were inoculated at 4 and 6 leaf stages with a mixture 

of four isolates of C___. miohiganese subsp. nebraskense. In the 

NLB experiment, ground corn leaf tissues infested with 

Exserohilum turcicum (Race I) were placed in the plant whorls 

at the six and eight leaf stages. Disease progress was 

recorded at weekly and biweekly intervals for LFW and NLB 

respectively. Variance component analysis of the Design I 

progenies was conducted to estimate type and magnitude of gene 

action involved in the inheritance of resistance to these two 

diseases. Heritability estimates and genetic correlations 

between reactions to these two diseases will be discussed. 

The eastern and prairie populations of Puccinia recondita in 

Canada were examined for non-random distributions of virulence 

and levels of phenotypic diversity. In the prairie region 

where resistant cultivars have been grown and polymorphisms 

for unneeded virulences are low, virulences to Lrl and Lr2a 

appeared to be positively associated from 1960-1974, but was 

negatively associated from 1975-1980. Non-random 

distributions of other virulences in the prairie region were 

also observed. In the eastern region where susceptible 

cultivars are grown and unneeded virulences are common, 

positive associations were found among virulences to Lr2c, 

LrB, Lr3ka, Lrtt, and LrL8. Based on the infection types of 

the UN differentials, the eastern population had a higher 

level of diversity as measured by the Shannon index than the 

prairie population in the three years examined. The nonrandom 

distributions of virulence observed in both populations 

are characteristic of asexual cereal rust populations. 

HETEROKARYOSIS AND VEGETATIVE COMPATIBILITY IN THE THREE 

VARIETIES OF LEPTOGRAPHIUM WAGENERI. P. J. Zambino and T. 

C. Harrington. Department of Botany and Plant Pathology, University 402 

of New Hampshire, Durham, NH 03824. 

Nitrate non-utilizing mutants were selected from strains of the INTROGRESSION OF DISEASE RESISTANCE FROM WILD RICES TO ORYZA 

inditrtenon-u t n pathoged mrest 

fromographm stgeraions chorate- SATIVA. R. Nelson, A. Amante, N. Oliva, R. Dalmacio, L. Sitch 

indigenous forest pathogen Leptograp/hium wageneri on chlorate- and H. Leung. Depts. of Plant Pathology and Plant Breeding, 

containing media. Pairings were made between mutants of complement- International Rice Research Institute, Los Bafios, Philippines 

ing phenotype on a minimal (nitrate) medium (MM). In some pairings, 

dense hyphal growth (complementation) occurred along the area of Resistance to blast and bacterial blight was evaluated in 3 

contact between the mutants. Subcultures from complementing areas tetraploid wild rices - 0. longiglumis, 0. minuta, and 0. 

grew both faster and denser than auxotrophic mycelia on MM. Rarely, ridleyi. Each species was resistant to a blast fungus isolate 

hyphal tip (HT) transfers from complementing subcultures Were and to six races of the bacterial blight pathogen. By backprototrophic 

on MM, and these prototrophs had isozyme markers of crossing and embryo rescue, BC1, BC2, and BC3 plants with 

both of the original mutant strains. All examined hyphal tips were different: numbers of 0. minuta chromosomes were obtained from 

multinucleate. Complementation occurred in pairings among mutants 0. sanyoa X 0. minuta crosses. Five BC1 and one BC2 plants 

from 19 tested strains of var. wageneri, indicating one vegetative showed complete resistance to bacterial blight. Since 0. 

compatibility (VC) group. There were three geographically-separated longiglumis and 0. ridleyi were sexually incompatible with 0. 

VC groups among 20 tested strains of var. ponderosum. The greatest sativa, attempts were made to introduce wild species DNA byvariability 

was found in var. pseudotsugae, which had 10 VC groups transformation via the pollen tube pathway. Repetitive DNA 

among 28 strains. No complementation was found between varieties, sequences specific to the wild species were isolated as probes 

to follow the introgression of alien DNA. Dispersed wild 

species-specific sequences might eventually be used to isolate 

399 closely-linked disease resistance genes in interspecific 

BIOGEOGRAPHY OF PHYTOPHTHORA CINNAN4ONI MATING TYPES IN SOUTH recombinants. 

AFRICA. S.L o reasn Dept. of Plant Pathology, 

Oklahoma State Univ., Stillwater, OK 74078-9947. 

Mating types were determined for more than 3000 P. cinnamomi 

isolates from agricultural and natural ecosystems in South INHERITANCE OF VIRULENCE IN PHYTOPHTHORA MEGASPERMA F.SP. 

Africa. Certain biogeographic patterns emerged from analysis GLYCINEA. Ravindra G. Bhat, A. F. Schmitthenner and B. A. 

of the distribution and host associations of the Al and A2 McBlain*, Dept. of Plant Pathology and *Dept. of Agronomy, Ohio 

mating types. In indigenous forests, the A2 mating type was State Univ., Wooster, OH 44691. 

most prevalent, but both mating types freguently occurred 

together at the same sites. All isolates from native fynbos The genetics of host-pathogen interaction in soybeanvegetation 

and rivers draining fynbos mountain catchments were Phyt0Dhthora meqasperma f.sp. qilycinea (Pti) system has been 

403 

Vol. 79, No. 10, 1989 1185

hampered by lack of suitable technique to clarify the complete sequences obtained for 17S and 25S RNAs from AG 4 show 

pathogen's genotype. A simple and efficient method was high overall similarity with other fungi, and decreased similarity when 

developed to obtain inbred single oospore cultures of PEmc. compared to plants, animals and more primitive eukaryotes. We have 

Field isolates of Pin races 1 and 4 were selfed to study the applied the recently developed polymerase chain reaction (PCR) to 

segregation of virulence genes. Host differentials for a e a 

hypocotyl inoculation test included two universal suscepts and enzymatically amplify and sequence phylogenetically informative DNA 

cultivars with single resistance genes Rps, Rpsl-b, Rpsl-k or segmentsfromeach AG withinR solani. Preliminary data basedon 

Rps6. Progeny data indicated that race 1 is conditioned by a partial sequences fromb25S RNAshow as much asr4% nucleotide 

single dominant gene and avirulence is recessive. Race 4 divergence between different AG's. A preliminary phylogenetic analysis 

virulence seems to be conditioned by two genes, one of which based on partial PCR sequences from 6 rDNA segments (including genic 

appears to be the same gene as for race 1. The dominant gene as well as non-genic regions) will be presented. 

for race 1 and another gene which is recessive appear to be 

required for race 4 virulence. 

404 

GLYCEOLLIN ELICITATION BY AVIRULENT SINGLE OOSPORE 

PROGENY OF PHYTOPHTHORA MEGASPERMA F.SP. GLYCINEA. A. 

F. Olah and R. G. Bhat, Dept of Plant Pathology, Ohio 

State Univ., Wooster, OH 44691. 

Virulent and avirulent single oospore progeny of 

Phytophthora meiasperma f.sp. glycinea were hypocotylinoculated 

into normally suceptible or resistant 

soybeans. After 48 hrs, growth of the fungus was 

measured using ELISA and glyceollin elicited was 

determined by HPLC. All avirulent isolates produced no 

disease but only some of these elicited glyceollin in 

amounts that would account for the lack of disease. 

Familial patterns for glyceollin elicited will be 

presented and comparison made to interactions of 

soybean with P. cactorum and Aspergillus sp. 

405 

EVALUATING TOBACCO GENOTYPES FOR RESISTANCE TO PHYTOPHTHORA 

PARASITICA TL, Miller, VAR. and NICOTIANA M.T. Ni~lsen. USING DETACHED Department LEAVES. of Agronomy, E.C. TeTford, 

University of Kentucky, Lexington, Ky. 40546. 

t of 

408 

HERITABILITY 408 

OF HYGROMYCIN RESISTALNCE IN TRALNSFORMHED STRAINS OF 

FUSAPRUM MONILIFORME. John F Leslie* and Martin B. Dickman**, 

*Department of Plant Pathology, Kansas State University, 

A nondestructive technique to screen tobacco germplasm for 

resistance to EPy~ophthora parasitica Dast. var. nicotiana 

(Breda de Haan) waisdev-vToped to facilitate selection in a 

breeding program. Leaves (7-9 cm long) were excised, sterilized 

for 30 sec. in 0.05% NaOCl3, and inoculated with four 

on 

1-cm-diameter 

oatmeal 

mycelial plugs of 2-d-old P. 

agar. 

p grown 

Leaves were incubated undeP rThlF---midi ty 

in a growth chamber at 27 C with constant light. Number of 

infections and percent leaf area infected were recorded 

daily for six days after inoculation. Genotype rankings 

for infection number and percent infection were similar to 

Manhattan, KS 66506; and **Department of Plant Pathology, 

University of Nebraska, Lincoln, NE 68543. 

Fusarium moniliforme strains transformed with the hygromycin B 

phosphotransferase gene conferring resistance to 100 mg/L 

hygromycin B were crossed with strain FKMA-59 which is 

auxotrophic for both nicotinic acid (nic) and pyridoxine (pdx). 

Both nic and pdx segregated in Mendelian ratios following 

meiosis, but hygromycin B resistance segregated in a 2:1 ratio 

of sensitive to resistant. Some of the progeny were resistant 

to 250 mg/L hygromycin B, even though the parental transformants 

field resistance rankings of the same genotypes. The new 

technique was not effective for detecting race specific 

resistance derived from Nicotiana longiflora. 

were all sensitive to this level of the drug. Hygromycin- 

resistant progeny from one cross were backcrossed to a wild-type 

strain. Progeny of these second generation crosses also showed 

the 2:1 segregation of sensitive to resistant, indicating that 

resistant progeny were not more 

original transformants. 

genetically stable than the 

406 

VEGETATIVE COMPATIBILITY GROUPS IN ASPERGILLUS FLAVUS. 

P. Bayman and P. J. Cotty, USDA, ARS, Southern Regional 409 

Research Center, P.O. Box 19687, New Orleans, LA 70179 409 

ANALYZING THE GENETIC STRUCTURE OF SEPTORIA TRITICI 

Aspergillus flavus causes aflatoxin contamination of cottonseed, POPULATIONS USING RESTRICTION FRAGMENT LENGTH 

corn and other crops. This fungus occurs in both soil and liv- POLYMORPHISMS. B. A. McDonald and J. P. Martinez. Dept. Plant 

ing plant tissues. populations of A. flavus are morphologically Pathology and Microbiology, Texas A&M University, College Station, TX 

and physiologically diverse. iowever, little is known about 77843-2132. 

genetic relatedness of A. flavus strains from different crops or 

localities. We isolated more than a hundred strains of A. RFLPs are under development as tools for studying the population genetics of 

flavus from cottonseed and from soils of various crops in south- the wheat pathogen Septboria tritici. Probes were developed by cloning 0.5western 

Arizona. 'To estimate genetic diversity and distribution 2.4 kb fragments from a total DNA digest of S. tritici. Random Sau3A 

"in A. flavus populations, we selected nitrate non-utilizing fragments cloned into the vector pGEM4 were used to probe total DNA 

mutants of these strains and assigned them to vegetative compat- extracted from six S. tritici isolates digested with six different restriction 

ibility groups (W!Gs) via complementation tests. Most V12Gs were enzymes. A high level of genetic variation was observed among the six 

represented by more than one isolate. Several VCGs included isolates tested using 23 individual probes. RFLPs were detected for 91 of the 

both soil and cottonseed isolates, or isolates from fields 130 probe-enzyme combinations tested. Two probes detected apparent 

planted to different crops. A. flavus populations from Georgia deletions. Four probes hybridized to repetitive DNA; two of these probes 

corn were compared to those from Arizona cotton. These data may be useful for DNA fingerprinting. An rDNA probe from yeast did not 

provide a preliminary measure of the population structure of A. detect useful polymorphisms. These RFLP probes will be useful for studying 

flavus and may result in strategies for control of aflatoxin.- genetic variation, genetic relatedness and gene flow in S. tritici populations. 

407 410 

SEQUENCE ANALYSIS OF RIBOSoMAL RNA GENES FROM TOXICITY AND TOXIN PRODUCTION OF Fusarium ISOLATES FROM 

RHIZOCTONIA SOLANI. D.GOonzalez and R.Vilgalys. Department NORTHEASTERN CHINA. Weiping Xie and Chester d. Mirocha, 

of Botany, Duke University, Durham NC 27706. Department of Plant Pathology, University of Minnesota, St. 

Paul, MN 55108, USA; Hangqing Li and Xue Wang, Department of 

Restriction analyses have revealed little variation between the ribosomal BilgHingagUivrty HrbnP..Cn. 

RNA genes of different anastomosis groups (AG's) in R.solani, One hundred and two i sol ates of Fusari um spp. were i sol ated from 

suggesting that these fungi are closely related. Analysis of nearly soil, corn and wheat samples col lected from northeastern China. 

1186 PHYTOPATHOLOGY

Rice cultures of the isolates were checked for toxicity in rat for control of these diseases on wounded pears and apples. 

feeding tests. Twenty-five isolates acutely toxic to rats were Three types of wounds were made: cut, nail, and bruise (with 

analyzed for toxin production by thin layer (TLC) and gas broken skin. The fruits were dipped in a conidial suspension 

chromatography (GC). The mycotoxins found were T-2 toxin, HT-2 (1 x 1O conidia/ml) of the pathogen with various 

toxin, scirpentriol, monoacetoxyscirpenol, diacetoxyscirpenol, concentrations of pyrrolnitrin ranging from 6-200 ug/mi. 

neosolaniol, acuminatin, 8-acetoxyneosolaniol, 8-acetoxy T-2 Following treatment, one-half of the fruit was stored at 24 C 

tetraol, deoxynivalenol, 15-acetyl deoxynivalenol and for 6 days and the other half at 2 C for 30 days, after which 

zearalenone. The identities of the toxins were confirmed by gas the diameter of the wound originating rots was measured. 

chromatography-mass spectrometry (GC-MS). All the isolates Complete control of both diseases was achieved under both 

analyzed were negative for wortmannin and fusarochromanone. storage conditions. However, higher concentrations of 

pyrrolnitrin were required for control at 24 C. The wound type 

had a profound effect on the control; cut wounds were the 

easiest and bruise wounds most difficult to control. 

411 

SURVIVAL OF ERWINIA CAROTOVORA ON TOMATO LEAVES. J. A. Bartz 

and D. E. Concelmo. 

Gainesville, 32607. 

Plant Pathology Dept., Univ. of Florida, 

415 

Erwinia carotovora survived at least 20 days in wounds on 

leaves of greenhouse-grown tomatoes, but seldom more than 6 

days on nonwounded tissues. About 10% of 106 cfu deposited 

on a pin-puncture wound were recovered after the leaf surface 

had dried; only 0.4% or less were recoved from a similar 

deposition on nonwounded leaves. Wounds infested with 103 

cfu did not have detectable populations (> 10 cfu) immediately 

after the suspension dried. However, populations up to 7 X 

102 cfu were found after 6 days. Survival for 20 days was 

Detection corn debria of from Aspergillus Iowa corn flavus fields. in soil, J. F. corn Shearer, debris N. and K. non- Baker, 

L.E. Sweets and L. N. TiffanyJ.Iowa State University, Ames, IA 

50011. 

Samples of soil and of stalk and cob debris were collected from 

40 corn fields in eight Iowa counties following the 1988 har- 

vest. Non-corn debris samples were collected from fencelines, 

waterways or ditches adjacent to 11 of the fields. Soil samp 

were tested for Aspergillus flavus by sprinkling approximately 

associated with the larger initial population, but such 0.5 gm of soil onto M3S1OB agar plates. After a three day incupopulations 

did not appear to increase. Recovery of the E. 

carotovora was enhanced if leaves were exposed to dew before 

being sampled. Asymptomatic tomato leaves can support 

epiphytic populations of E. carotovora that may eventually 

contaminate or inoculate fruit. 

bation period at 370 C, plates were visually examined for A. 

flavus colonies. A. flavus was detected in all 40 soil samples. 

Pieces of cob and stalk pith approximately 1 cm in diameter were 

pulled from freshly broken samples and plated on M3S10B agar 

plates. Plates were incubated at 370 C for 3 days and visually 

examined for presence of A. flavus colonies. Seventy percent of 

412 

the cob pieces and 42 percent of the stalk pieces were positive 

for A. flavus. When random pieces of non-corn debris were 

tested on M3S10B agar, 57 percent were positive for A. flavus. 

GLYCOSIDE FORMATION OF THE MYCOTOXIN ZEARALENONE IN LIQUID 

FERMENTATION BY Rhizopus sp. J. Plasencia and C. J. Mirocha. 


Paul, MN 55108. 

When a Rhizopus sp. is incubated in presence of zearalenone, 

two major conversion products are found in the culture medium. 

Normal phase thin layer chromatography of the two products in 

chloroform-methanol (4:1) give Rf values of I (0.32) and II 

(0.26) indicating their high polarity as compared with 

zearalenone. Treatment of the products with 8-glucosidase 

yielded zearalenone as the aglycon and a carbohydrate identified 

as D-glucose by mass spectrometry. Highest activity of the 

enzymes involved in the bioconversion is found within the 

mycelium. Both products are found in larger amounts 

to intracellularly the isolation than of cell-free outside the extracts mycelium. which This contained observation the lead most 

active enzyme fraction. 

The bioavailability of zearalenone in its conjugate form to 

laboraorratswill be s ed. initscondiameter 

laboratory rats will be studied. 

416 

HYDROSTATIC PRESSURE AND WOUND DIAMETER INFLUENCE 

ENTRY OF FUNGAL SPORES INTO PEAR WOUNDS DURING 

IMMERSION DUMPING. David Sugar and R.A. Spotts, 

oregon state University, Southern Oregon 

Experiment Station, Medford, OR 97502. 

Wounds in pear fruit are important infection 

courts for fungi causing postharvest decay of 

pear, and dump tanks harbor spores which may 

contact wound tissue during immersion dumping. 

Wound immersion diameter depth and affect hydrostatic penetration pressure of spores due toof 

Penicillium expansum and Phialophora malorum into 

wounds. Pears with wounds 0.4, 0.5, 1, 2 and 6 mm 

x 2 mm depth were immersed 2 min at 

depths of 0, 10, 20 40, 60, 80 and 100 cm in 

413 

water containing 10 spores of either fungus. At 

wound diameters < 2 mm, incidence of infection 

increased with depth. Infection of wounds 6 mm in 

BIOCONTROL OF POSTHARVEST ROTS OF PEACH AND APPLE WITH THE diameter was independent of immersion depth. 

YEASTS HANSENIASPORA UVARUM AND DEBARYOMYCES HANSENII. 

R. J. McLaughlin, M. E. Wisniewski, C. L. Wilson, and 

E. Chalutz*, USDA-ARS, 45 Wiltshire Rd., Kearneysville, WV 

25430, and *VolcaI% i Center, Bet Dagan, Israel 50250. 

Peach and apple fruit were artificially wounded, treated with 

417 

COMPARISON OF CEREAL SEEDLING BIOASSAYS FOR Fusarium TOXINS. U. 

Bosch, D. R. Johnson, C. J. Mirocha and J. A. Percich, 

lO6-109 cfu/ml suspensitns of eight yeast strains (H. Department of Plant Pathology, University of Minnesota, St. 

uvarum, 0. hansenii, or Zygosaccharomyces rouxii), an-d Paul, MN 55108. 

challnged-with 10'-105 spores/ml of two postharvest fungal 

pathoqens for each fruit. Strain 138 of H. uvarum was most 

effective in reducing Rhizopus rot in- peach. Marginal 

reduction of brown rot of peach and Penicillium rot of apple 

was conferred by several strains, including D. hansenii strain 

Mycotoxin bioassays using seedlings of barley (Hordeum vuloare 

' Larker' ), rice (0rvza sati va ' Lemont' ), wheat (Tri t icum 

aestivum 'McNair 701') or wild rice (Zizania palustris 'Meter') 

were evaluated for sensitivity to the Fusarium toxins 

US-7. Strain 138 and 0. hansenii strain-s US-7 and 101 

significantly reduced Botrytis rot in apple. Biocontrol 

ability of yeast strains was affected by culture age, cell 

concentration of the yeast and pathogen, timing of challenge 

inoculation, and presence of CaC1 2 (0, 1, and 2%) in the 

yeast cell suspensions. 

deoxynivalenol (DON) and T-2. Ten germinating seeds of each 

cereal were grown in DON or T-2 solutions of 20, 10, 2, 1, 0.8, 

0.6, 0.4, 0.2 or 0.1 vg/ml. Coleoptile lengths were measured 

after 2 days (barley, wheat and wild rice) or 3 days (rice). The 

no-effect level for DON and T-2, respectively, were 0.2 and 0.2 

pg/ml for wild rice, 2.0 and 4.0 pg/ml for wheat, 6.0 and 4.0 

pg/ml for barley and 6.0 and 1.0 pg/ml for rice. All bioassays 

provided simple visual indicators for the two toxins, but the 

414 

sensitivity of the wild rice assay permitted detection of ng 

amounts. 

CCNTROL OF STORAGE ROTS OF PCNE FRUITS WITH PYRROLNITRIN 

ISOLATED FROM ANTAGONISTIC PSEUDOMONAS CEPACIA. W. Janisiewicz 

and L. Yourman, USDA, ARS, AFRS, Kearneysville, WV, and 3. 418 

Roitman and N. Mahoney, USDA, ARS, WRRm, Albany, CA. 

TOXICITY TO OUCKLINGS OF FUSARIUM NYGAMAI ISOLATED FROM MILLET 

Pyrrolnitrin, isolated from Pseudomonas cepacia which is known SEED FROM NIGERIA. N. B. Onyike, P. E. Nelson, Dept. of Plant 

to control grey-mold (Botrytis cinerea) and blue-mold Pathology, Penn State Univ., University Park, PA 16802, and 

(Penicillium expansum) on pome fruits after harvest, was used w. F.O0. Marasas, South African Med. Res. Coun., Tygerberg 7505. 

Vol. 79, No. 10, 1989 1187

Pearl millet, Pennisetum typhoides, was collected in Nigeria Maize white line mosaic virus (MWLMV) was transmitted from 

from seed stored in homes, sold in markets, or left unharvested MWLMV-infected roots to roots of Seneca Chief sweetcorn 

in the field. All Fusarium cultures obtained from 100 seed of seedlings in a modified slant-board hydroponic system. 

each of 7 samples cultured on a pentachloronitrobenzene selec- Transmission was accomplished by direct placement of root 

tive medium were identified. The most prevalent species inoculum onto seedling roots. ELISA was used to determine root 

recovered was F. nygamai which made up 42.4% of the total, infection and it consistently detected MWLMV in roots of 

These cultures consisted of strains that produced short or long inoculated seedlings treated with Tilt, Benlate, Diazinon, 

chains of microconidia and 45 cultures representing both strains Ridomil, gentamycin, chloramphenicol, vancomycin, streptomycin, 

were selected for toxicity tests. Cultures were grown on auto- or lincomycin-spectinomycin. Similar frequencies 

claved 

of 

yellow corn kernels in 2 liter flasks & incubated at 25C transmission (90-100%) occurred in roots inoculated with 

for 3 wks. The medium was dried at 55C for 24 hr and ground inoculum placed 1-2 cm distance from test seedlings, inoculum 

into a fine meal. The moldy meal was mixed 50% by weight with contained in polycarbonate bag filters (pbf) of 0.2-8.0 gm pore 

commercial chicken mash, and fed to ducklings ad lib. Of the sizes, 0.1-0.3 gm of inoculum, and inoculum heated at 30-60 C. 

cultures tested, 94% were toxic, causing the death of all 4 MWLMV transmission was reduced to 30% when inoculum was 

ducklings in 2.5 days with an average feed intake of 26 g. contained by a pbf of 0.05 gm pore size. 

419 

423 

A RAPID QUANTITATIVE IMMUNOASSAY FOR ERGOTAMINE. R.A. 

Shelby and V. C. Kelley, Departments of Plant Pathology 

anT'Rotany and Microbiology, Auburn University, AL 

36849. 

BEAN MILD MOSAIC VIRUS AS A CONTAMINANT IN GREENHOUSE VIRUS 

STUDIES. P. Sepulveda and A.W. Saettler. ARS, USDA, Dept. 

of Botany and Plant Pathology, Michigan State University, 

East Lansing, MI 48824. 

Anti-ergotamine antibodies were produced by immunizing 

rabbits with bovine serum albumin-ergotamine conjugate. The 

resulting partially purified polyclonal antiserum was used to 

develop a quantitative competitive inhibition (CI) ELISA 

capable of detecting ergotamine at the nanogram level. In 

cross-reactivity tests with 22 related alkaloids there was 

measurable reactivity only with ergotamine, ergocristine, and 

ergostine. Using the assay it was possible to measure 

ergotamine in spiked samples of wheat, rye and millet. Ergot 

(Claviceys purpurea) sclerotia were detectable in wheat flour 

at a level of 0o.1 w/w. Quantitative measurements of 

ergotamine in ergot sclerotia of wheat and fescue as well as 

endophyte (Acremonium coenophialum) infected fescue seed were 

in the range reported for ot'i-e detection methods. 

During host range studies of several strains of bean yellow 

mosaic virus (BYMV), we observed virus symptoms in several 

plant species reported to be resistant to BYMV. We also observ- 

ed mild mosaic symptoms on a number of the noninoculated 

control bean plants. Mild mosaic symptoms developed in plants 

of the Domino bean cultivar ten days after inoculation with 

sap from the control plants exhibiting symptoms. Large numbers 

of spherical virus particles, 28 nm diameter, were observed 

with TEM in leaf dip preparations negativelly stained with 

2% ammonium molybdate. Identity of the spherical particles 

as bean mild mosaic virus (BMMV) was determined using agar 

gel double diffusion and immunosorbent electron microscopy; 

antisera was provided by Dr. F. Morales, CIAT. Further studies 

revealed the presence of B21V as a contaminant in several 

of our strains of BYMV. 

420 424 

A MODEL THAT PREDICTS ALLOWABLE STORAGE TIME OF DRY EDIBLE BEANS A NEW VIRAL DISEASE OF CYAMOPSIS TETRAGONOLOBA 

BASED 

(L) 

ON 

TAUB. 

FACTORS 

M.E.C. 

INFLUENCING INVASION BY STORAGE FUNGI. J. D. R and H Ben-Moshe. Department of Microbiology, University 

Pokorny and R. A. Meronuck, Department of Plant Pathology, 

University 

of 

of 

the 

Minnesota, 

Witwatersrand, 

St. Paul, 

Johannesburg, 

MN 55108. 

P 0 Wits, 2050. 

Cyamopsis tetragonoloba (L) Taub. (guar) was found 

A model has 

to 

been developed 

exhibit 

that predicts allowable storage time symtoms of reduced leaf size and inflorescence number 

for 

(pods 

dry edible beans (Phaseolus vulgaris) stored at 80 and 85% exhibited 50% sterility) and often the stems 

relative 

elongated 

humidities 

and 

and at 18, 24 and 30 0 C. The beans used to remained green after the plant had set seed. Investigations 

develop the model were collected from warehouse, cooperative and revealed a flexuous rod (750 x 15nm) which 

farm 

is 

locations 

transmissible 

in low, middle and high altitude regions of to several Phaseolus vulgaris cultivars, 

Rwanda, Africa. 

Vigna unguicul 

The percent of surface disinfected seeds which and Lycopersicum esculentum L. by mechanical 

yielded 

inoculation. 

Aspergillus glaucus in culture (r 2 =.6383), length of Symptoms included systemic red veins and chlorosis. 

previous st rage (r7=.6459), and the relative humidity of future Nonpersistent transmission with the aphid, Myzus 

storage 

persicae 

(r =.3393) were selected as significant predictor (Sulzer) was also successful in some cases. 

variables 

ELISA tests, 

for determining 

and 

allowable storage time on the basis of immunoblots of polypeptide bands separated by PAGE gave positive 

stepwise regression analysis. Allowable storage time was results with several potyvirus 

determined 

(BCMV, BYMV, 

from 

SMV 

when the 

and PVY) 

beans became a substandard grade. The antisera. These symptoms have been named "green sterile" 

model is 

and 

designed to predict storability for storage lengths up "little leaf" and are unlike any other viral diseases 

to 

reported 

6 months and shows potential as a management tool to assist in guar. These symptoms appear to be associated 

tropical 

with a potyvirus 

and developing countries in the safe storage of dry but confirmation of this virus being the sole causal agent 

edible beans. is required. 

421 

EFFECTIVENESS OF FUNGICIDES FOR CONTROL OF MYCOTOXIGENIC 42 

FUNGI AND MYCOTOXINS IN PEANUTS. K. L. Bowen and P. A. A GENE FOR RESISTANCE TO WHEAT STREAK MOSAIC VIRUS ON CHROMOSOME 

Backman, Dept. of Plant Pathology, Auburn University, AL. 6 OF MAIZE. M. 0. McMullenI and R. Louie 2 , USDA-ARS, Depts. of 

Agronomy 1 and Plant Pathology 2 , OARDC, The Ohio State 

The fungicides, terbutrazole and flutolanil, used for control University, Wooster, Ohio 44691 

of southern stem rot, (Sclerotium rolfsii), and limb and pod 

rot (Rhizoctonia solani) in peanuts, have also been found to Wheat streak mosaic virus (WSMV) induces generalized mosaic 

reduce fungal damage affecting seed quality. Peanut pods symptoms in selected maize inbreds. During 1088, WSMV was 

harvested from plots treated with foliar applications of these detected in many lines in our maize nursery. WSMV symptoms were 

fungicides were evaluated for levels of infestation by associated with the expression of the polymitotic (po) marker in 

Aspergillus app., other seed-borne fungi, and aflatoxins, a B73 genetic background. The polymitotic locus is on the short 

Flutolanil-treated peanuts showed no consistent differences in arm of maize chromosome 6. An isolate of WSMV from these 

Aspergillus app, incidence or aflatoxin levels measured by naturally-infected plants was used to mechanically inoculate 

ELISA when compared to chlorothalonil or nontreated controls. maize plants segregating po/po or po/+. After inoculation, all 

However, terbutrazofe-treated peanuts had 56.4% less kernel po/po plants (10) exhibited generalized mosaic symptoms, all 

infection by Aspergillus spp. than peanuts receiving standard p0/+ plants exhibited either symtomless (14) or delayed, limited 

foliar sprays for leafspot control with chlorothalonil, symptom (5) responses. All 873 plants (+/+) were symptomless. 

Measured aflatoxin concentrations in terbutrazole treated These results indicate that there is a gene on chromosome 6 

peanuts were 25% lower than in chlorothalonil treated peanuts. affecting resistance to WSMV. RFLP analysis places this gene 

either on the short arm of chromosome 6 or on the long arm 

proximal to the RFLP marker locus UMC-59. 

422 

TRANSMISSION STUDIES OF MAIZE WHITE LINE MOSAIC VIRUS. R. 426 

Louie, J. j. Abt, and J. K. Knoke, USDA/ARS and Ohio Stat-e 

Univ., Wooster, OH 44691. A VIRUS DISEASE COMPLEX OF SWEET POTATO FROM PUERTO RICO. 

1188 PHYTOPATHOLOGY

M. Laakso and J. W. Moyer, Dept. of Plant Pathology, North CLOSTEROVIRUSES ASSOCIATED WITH GRAPE LEAFROLL 

Carolina State University, Box 7616, Raleigh, NC 27695-7616. DISEASE. J. S. Hu, D. Boscia, and D. Gonsalves, Dept. of Plant 

Pathology, Cornell University, NYSAES, Geneva, NY, 14456, USA. 

We report the etiology of a disease of sweet potato which 

occurs in Puerto Rico. This disease is the result of a Stable hybridoma cell lines secreting monoclonal antibodies (mAB) tothe 

synergistic interaction between sweet potato feathery mottle NY-1 isolate of closteroviruses associated with grape leafroll disease 

virus (SPFMV) and a nonmechanically transmitted virus-like (GLRaV) were produced. The mAB reacted with the NY-l isolate and 

agent. The predominant symptoms of the disease complex consist several serologically related isolates, but not with isolates in other 

of severe stunting and general chlorosis of sweet potato. This serotypes. The reactions were the same in 5 different kinds of ELISA, 

is in contrast to the typical symptoms associated with SPFMV. ISEM, dot-immunoblotting, and Western blotting assays. The serological 

Symptoms in sweet potato and I. setosa caused by SPFMV alone reactivities of the mAB were found to be as good as those of rabbit 

were consistent with those caused by other isolates of SPFMV. polyclonal antibodies for the detection of the virus in grape leaf tissue in 

The virus-like agent causes small diffuse chlorotic spots and double antibody sandwich direct ELISA. With the double gold labelling 

ringspots in sweet potato and a distinct mosaic in I. setosa. electron microscopy technique, we were able to detect mixed infections of 

Antiserum which reacts with a 29 kd protein has been prepared grapevines with different serotypes of GLRaV. A very sensitive Western 

from partially purified preparations and a cDNA cloned which blotting assay was used to estimate the molecular weight of virus coat 

are both specific for plants infected with the nonmechanically protein of the GLRaV from partially concentrated samples using the mAB. 

transmitted agent. 

431 

THE ULTRASTRUCTURAL ASPECTS OF INFECTION OF ZEA MAYS 

CV. SILVER QUEEN BY SORGHUM YELLOW BANDING VIRUS 

(SYBV). C.E. McClelen and R.W. Toler, Texas A&M University, College 

Station, TX 77843 

Rapidly expanding leaves from Zea mays cv. 'Silver Queen' inoculated 

30 days prior with SYBV and healthy controls, were ultrastructurally 

viewed and compared to determine cytological effects and cellular 

location of the virus. Specific infection by SYBV was confirmed by 

Ouchterlony tests. SYBV was found throughout the infected leaves in 

all cell types. The highest concentrations were observed in the 

epidermal and parenchyma cells. The virus was packed into large 

vesicles which had expanded into the vacuolar space and cytoplasmic 

areas adjacent to the chloroplasts and nuclei. A large reduction in 

starch accumulation was noted in the chloroplasts of infected leaves. 

Mitochondria appeared swollen and contained reduced and irregular 

cristae. SYBV was associated with and seen in the endoplasmic 

reticulum. No viral particles were detected in the nuclei. 

A NEW SOURCE OF WHEAT STREAK MOSAIC VIRUS RESISTANCE. 

N. A. Tuleen and R. W. Toler, Department of Soil and Crop Sciences and 

48Department of Plant Pathology and Microbiology, respectively, Tex 

IDENTIFICATION AND INCIDENCE OF CUCURBIT VIRUSES IN SOUTH A&M University, College Station 77843. 

LOUISIANA. F. J. Fernandes, R. A. Valverde, and L. L. Black, Eight accessions of Agropyron intermedium from Iran and Russia were 

Department of Plant Pathology and Crop Physiology, Louisiana crossed to Triticum aestivum cv Chinese Spring. The 42 chromosome F 1 

Agricultural Experiment Station, Louisiana State University hybrids displayed a high level of resistance to wheat streak mosaic virus 

Agricultural Center, Baton Rouge, LA 70803. (WSMV). These F 1 plants were pollinated by Chinese Spring and plants 

with 61-63 chromosomes saved and then backcrossed to Chinese Spring 

A survey for viral diseases was conducted during 1988 in the for two or three generations. Resistant progeny with the lowest 

cucurbit producing areas of south Louisiana. Samples from chromosome number were selected for additional backcrossing. Resistant 

plants showing virus-like symptoms were collected from different monosomic chromosome additions, ie. plants with 21" of wheat 

locations and tested for the presence of five cucurbit viruses chromosomes and a univalent chromosome from Agropyron were obtained 

using ELISA and host reaction. Identification of the viruses from crosses of three different accessions; two from Iran and one from 

was confirmed by dsRNA analysis and electron microscopy. Out of Russia. Disomic additions were selected in the self progeny. Two of the 

178 samples analyzed, 104 (58%) were found to be infected with additions were morphologically similar and may involve the same 

papaya ringspot virus (PRSV), 69 (39%) with watermelon mosaic Agropyron chromosome, while the third had a different head type. In 

virus (WMV), 68 (38%) with zucchini yellow mosaic virus (ZYMV) crosses involving the remaining accessions, plants with lower 

vr 60 (34%) (3M%), with 68 cucumber (38%) wit mosaic zuc virus yellw (CMV), maic and vr 13 (7%) (7%)w with chromosome WSMV. numbers were associated with a lower level of resistance to 

tobacco ringspot virus (TRSV). Mixed infections were common. 

Crops tested included squash, watermelon, cantaloupe, and 

cucumber. 

432 

433 

RELATIVE IMPORTANCE OF TRANSIENT VERSUS RESIDENT 

429 APHIDS IN THE SPREAD OF MAIZE DWARF MOSAIC VIRUS IN 

49 SORGHUM. J. D. Alexander and R. W. Toler, Dept. of Plant Path. and 

SEVERE MAIZE CHLOROTIC DWARF DISEASE CAUSED BY DOUBLE INFECTION 

WITH MILD VIRUS ISOLATES. R. E. GingerY' and L. R. Nault 

McoilTETMCleeSaT.783 

2 , USDA- 

- " " 'sorghum 

ARS and Department of Plant Pathology 

Spread of maize dwarf mosaic virus, strain A (MDMV-A), in 

by aphids was investigated. Two types of sorghum, one 

1 , and Department of Entomology 

susceptible to infection by MDMV-A via aphid vectoring (Pioneer 8199) 

2 , The Ohio State University-Ohio Agricultural Research and 

Development Center, Wooster, OH 44691. 

Two maize chlorotic dwarf virus (MCDV) isolates that caused only 

mild symptoms in maize were isolated from johnsongrass and independently 

maintained in maize by serial transfer with leafhopper 

vectors. Isolate #1 differed from isolate #2 by producing milder 

vein-clearing, less stunting, and delayed anthesis. Double infect 

ion with isolates #1 and #2 in the greenhouse produced severe 

stunting and leaf-tearing symptoms similar to those attributed 

to MCDV in the field. Both isolates reacted with anti-MCDV serum 

in ELISA tests. The molecular weights of two of the three capsid 

proteins of isolate #1 were different from those of the correand 

one relatively resistant to infection by MDMV-A (Tx2786) were 

planted in pure stands as well as mixed stands of approximately 25:75 

and 60:40 %, respectively. Half of the plots were treated with systemic 

insecticide (Aldicarb). Disease incidence was recorded 10 weeks after 

emergence. Regression analyses showed the relationship between plant 

mixture proportions and the infection levels to be highly linear, 

indicating no dilution of the rate of virus spread among the susceptible 

plants. This suggests that the mode of any secondary spread typically 

spanned, and possibly included, several plants at a time since intervening 

resistant plants did not affect the virus spread. The insecticide 

treatment resulted in a 3%/ average increase in disease. These results are 

consistent with virus spread mainly due to the series probing activities 

of transient aphids whose activity may be aggravated by insecticides, 

rather than by aphids colonizing infected plants and then spreading the 

sponding proteins of isolate #2. Because of these differences, virus. 

plants could be scored for infection with isolate #1 by SDS-polyacrylamide 

gel electrophoresis of isolated virions. 

430 

430 

434 

ENDOGENOUS ANTIvlRAL PROTEINS: IMPLICATIONS FOR SELF-RECOGNITION. 

Meyer Chessin and Allan Zipf, Division of Biological Sciences, 

USE OF MONOCLoNAL ANTIBODIES IN THE STUDY OF University of Montana, Missoula, Montana 59812. 

Vol. 79, No. 10, 1989 1189

Many higher plants contain endogenous proteinaceous inhibitors after inoculation all seedlings from susceptible P. x hortorum 

of virus infection which are typically ineffective when assayed cultivars were dead or severely blighted. Seedlings of P. 

on the source host. An inhibitor from Datura stramonium has capitatum, P. frutetorum, P. fulgidum, P. fruticosum, P. 

been tested on a wide host range, and its specificity is inquinans and P. zonale were not significantly different from 

manifested at the intrageneric level using TMV as test virus. P. x hortorum in reaction to X. c. pv. pelargonii. However, 

The mechanism of specificity of the pokeweed inhibitor may Pelargonium reniforme seedlings were significantly more 

involve ribosome function since it reduces cell-free protein resistant than P. x hortorum, and P. cordifolium seedlings 

synthesis using wheat ribosomes but not with pokeweed ribosomes. were significantly more resistant tha-n P. reniforme. This is 

Recent work with the Datura protein suggests that it acts as a the first reported screening of P. cordifolium for resistance 

competitive inhibitor of virus establishment. Can differences to bacterial blight. This specie may be useful for breeding 

in cellular receptors for virus therefore account for some of resistance into horticulturally important cultivars. 

the specificity, as well? 

435 SPREAD OF BACTERIAL SPOT DURING THINNING 

CHARACTERIZATION TOMATOES 

OF DIRECT-SEEDED 

OF EXTRACELLULAR PECTIC ENZYMES PRODUCED 

AND 

Pohronezny, 

MANAGEMENT WITH 

Michael 

BACTERICIDE 

A. Moss, Wilbur 

HAND WASHES. 

Dankers, 

Ken 

BY STREPTOMYCES SPECIES. F.R. Spooner Jr. 

and James 

and 

Schenk, 

R. University 

Hammerschmidt. 

of Florida, IFAS, 

Department 

Tropical Research 

of Botany 

and 

and 

Education 

Plant Pathology, Center (TREC), Homestead, 

Michigan 

FL 33031. 

State University, East Lansing, MI 48824. 

The spread of bacterial spot during thinning of direct-seeded 

Isolates of Streptomyces pathogenic on potato (Solanum tomatoes was studied in two experiments at TREC. In late 

tuberosum L.) and nonpathogenic isolates of Streptomyces spring 1988 (warm and humid), bacterial spot incidence was less 

were observed to produce extracellular pectinases when grown when plants were thinned in the afternoon after foliage was dry 

in medium containing 1% polygalacturonic acid. Enzyme activity (44%) vs. those thinned in the morning when laden with dew 

was detected for all isolates by thiobarbituric acid, reducing (87%). In non-thinned rows, disease incidence was as low as 

sugar and viscometric assays. Pectolytic activity was optimal 5%. Ethanol (70%) and 10% povidone-iodine, applied as hand 

at pH 8.4 and was stimulated by millimolar concentration washes between exposure to diseased foliage and thinning of 

of calcium. Pectolytic activity was minimal or nonexistent hills, reduced disease incidence 65% and 81%, respectively. 

at pH 4.8 for most isolates. This is indicative of pectate Ethanol or povidone-iodine hand washes reduced populations of 

lyase activity with minimal polygalacturonase. The production the pathogen (106 cfu/ml) by 97% or more in most cases. In 

of extracellular pectinases did not correlate with pathogenici- late fall 1988 (cool and dry), disease incidence as reduced 

ty on potato for Streptomyces species. from 55% to zero by simply waiting until plants were dry 

436 

439 

before thinning. 

BACTERIAL STUNT DISEASES OF BLUE GRASS AND BERMUDA IN CALIFOR- 

NIA AND 

440 

THEIR CONTROL BY HARRY FERTILIZER AND PLANT PROTECTANT. COMPARISON 

M.J.Thirumalachar, 

OF RESISTANT 

Jeersannidhi 

AND 

Anderson 

SUSCEPTIBLE 

Institute,POB-506, 

LETTUCE CULT- 

Locust 

IVARS 

st.Walnut 

TO 

Creek, 

CORKY 

CA 

ROOT 

94596 

IN 

and 

FLORIDA. 

Marvin D.Whitehead, 

L. E. Datnoff 

817 

and 

Clifton Rd.,N.E.,Atlanta, GA 30307. 

R. T. Nagata, University of 

Research 

Florida, 

and 

Everglades 

Education 

Xylem 

Center, 

limited 

Belle 

bacterium 

Glade, 

Clavibacter 

33430. 

cynodontis causing Bermuda 

(=Burkholderella)xyli 

grass stunt disease 

subsF. 

is spaFsely distributed 

Corky 

in Contra 

root 

Costa 

of lettuce 

,but a similar 

(Lactuca 

stunt 

sativa 

disease L. )is 

of Poa 

a disease 

caused 

pratense 

by a gram 

L.disfiguring 

negative bacterium. 

lawns with linear 

Little 

rows of 

informa- 

dead fuzzy 

stolons 

tion 

and brown 

is available 

patches is 

on 

widespread.The 

yield losses 

identity 

due to 

of 

this 

the 

disease 

causal organism 

in 

which 

commercial 

is close to the 

lettuce 

Bermuda 

production, 

stunt organism is 

especially 

organic 

on 

being determined.Lawn 

soils. Susceptible 

mowing machines 

('Ithaca' 

and 

and 

sprinklers 

'Shawnee') 

spread the 

infection. and 

Harry 

resistant 

Fertilizer ('Raleigh' 

and Plant and 

Protectant 'Southbay') 

(HF-PP) develocrisphead 

lettuce 

ped at Jeersannidhi 

cultivars 

Anderson 

were planted 

Institute is 

in 

a phytobactericide 

a randomized 

and complete 

fungicide 

block 

containing 

design 

organic 

in fields 

and inorganic 

with various 

nitrogen 

cropping 

sources as 

plant food.Spraying 

histories. 

HF-PP 

Root 

at 

disease 

40 cc per 

severity 

gallon of 

was 

water 

significantly 

after lower 

lawn 

(P

443 

The stability of antigenic determinants recognized by 

three monoclonal antibodies (mAbs) specific to strains of 

Xanthomonas campestris pv. oryzae (Xco) was examined. 

Philippine and Texas strains were serially passed through 

rice plants over a period of 20 wks. Samples from the 

passages were evaluated with mAbs using ELISA. The 

reactivity of the mAbs to the Philippine strains did not 

change over the 20 wk period. The weakly virulent Texas 

strains could not be recovered for the full period. 

However, their reactivity to the mAbs did not change. 

Thus, the antigenic determinants recognized by the mAbs 

are considered stable for Xco. A dot blot using the mAbs 

was developed for testing field samples of Xco. 

SELECTIVE CHEMICALS FOR ISOLATION OF XANTHOMONAS CAMPESTRIS PV. 

ORYZAE (ISHIYAMA) DYE. Wuqiao Yuan, Department of Plant 

Pathology & Physiology, Clemson University, Clemson, SC 29634 

Cephradine (40 ug/ml), cephaloridine (1 ug/ml), crystal violet 

(4 ug/ml), methyl violet (3 ug/ml) and nalidixic acid (4 ug/ml) 

all permitted growth of Xanthomonas campestris pv. oryzae in 

liquid media and were 5 tested separately by replica plating _sugarcane, with 

XANTHOMONAS OFFICINARUM SP. NOV., A SECOND ORGANISM INVOLVED 

IN SUGARCANE GUMMING DISEASE. M. Qhobela and L. E. Claflin. 

Dept. of Plant Pathology, Kansas State Univ., Manhattan 66506 

A critical examination of 22 strains of Xanthomonas campes- 

Aritipa] ea min tio of2 tans of Xtmon as oe 

trisg v. vasculorum, revealed two the distinct inCitant groups of gumming of organisms. disease The of 

5 chinese and two IRRI (PSC-79 & PXO-86) strains of X. c. oryzae 

and 34 bacteria from rice seeds. All chemicals permitted growth 

of all X. c. oryzae strains. Crystal violet inhibited all 7 

Gram+, 7-of-17 non-yellow, Gram- and 1 of 6 yellow, oxidative, 

Gram- bacteria while methyl violet inhibited 4 of the same Gram+ 

and 4 of the same non-yellow, Gram- bacteria. Nalidixic acid (1 

ug/ml) inhibited all 3 yellow, fermentative, Gram bacteria, 

Cephradine inhibited 7 non-yellow, Gram- and 5 Gram+ bacteria 

whileephr adine inhibited 4ofthesame non-yellow, and5Gramscribed Gramwhile 

and 3 of cephaloridine the same Gram+ inhibited bacteria. 4 of Five the yellow, same non-yellow, oxidative, Grambacteria 

were not suppressed by the chemicals. Cycloheximide 

(>100 ug/ml) inhibited X. c. oryzae. Combinations of chemicals 

should be tested for isolation of X. c. oryzae. 

first group (16 strains) consisted of X. campestris pv. 

vasculorum sensu stricto; whereas the other consisted of a 

yet u es s pees t he two org a 

yet undescribed Xanthomonas species. The two organisms 

differed from each other in serological reactivity, host 

range, DNA homology values, DNA restriction endonuclease 

patterns, and by membrane protein profiles on sodium dodecyl 

sulfate poly-acrylamide gel slabs. Strains of the unde- 

Xanthomonas were pathogenic on sugarcane and tiger- 

grass Strains but of not this pathogenic undescribed on maize, Xanthomonas sorghum, clearly or pearl formed millet. a 

distinct group and the name Xanthomonas officinarum sp. nov. 

is proposed for this organism. 

448 

444 XANTHOMONAS CAMPESTRIS PV. ZINNIAE STRAINS RAPIDLY PRODUCE 

CONTROL OF WATERMELON FRUIT BLOTCH BY SEED IIEAT-TREATMENT. G. C. LARGE QUANTITIES OF EXTRACELLULAR PROTEASE. X. P. Sun and 

Wall, Agricultural Experiment Station, College of Agriculture and D.F. Ritchie. Dept. of Plant Pathology, N.C. State Univ., 

Life Sciences, University of Guam, Mangilao, GU, 96923. Raleigh, NC 27695. 

Pseudomonas pseudoalcaligenes subsp. citrulli is the causal agent of The substrate azocasein and milk agar overlaid on sucrose 

fruit blotch, foliar lesions, and seedling blight on watermelon. peptone agar plates were used to detect and quantify 

Seed infested with this pathogen develops into infected seedlings. protease. Larger quantities of extra-cellular protease 

Symptoms are first apparent as watersoaked lesions on the were detected more rapidly in strains of X. campestris pv. 

cotyledons. Most infected seedlings die, as the infection spreads zinniae than were detected in strains of pvS. oruni, 

into the stem. Splashing water carries the iuoculuis to other nearby campestris, vesicatoria, pelarqonii, or oryzae. Protease 

plants. Surviving plants, and those infected later, harbor the was produced in a defined medium lacking casein or other 

bacteria in foliar lesions. Under prolonged rainy weather and warm proteins. A crude enzyme preparation of at least 10-fold 

temperatures, the pathogen attacks the fruit. Infected fruit have concentration was obtained by precipitation of bacteriala 

reduced shelf life. Other fruit in contact with these may also 

become infected. Heat treatment of infested seed at 50' C for 20 cell-free aupernatant with 60% ammonium sulfate. 

min effectively controlled disease development in seedlings in a 

completely randomized experiment with 4 replications, repeated 3 

times. Seed were heat-treated by placing them in gauze sacks and 

immersing in a waterbath held at constant temperature, then growing 449 

in pots in the greenhouse. INDOLEACETIC ACID PRODUCTION BY EPIPHYTIC BACTERIA 

ASSOCIATED WITH PEAR FRUIT RUSSETTING. E. Clark and S. E. Lindow, 

Department of Plant Pathology, University of California, Berkeley, CA 94720. 

445 

Russetting of Bartlett pear fruit at harvest is significantly increased by application 

of bacterial producers of indole-3-acetic acid (IAA) to pear flowers at 50% 

STRAINS OF XANTHOMONAS CAMPESTRIS ISOLATED FROM AMBARELLA bloom. IAA producers and non-producers were isolated from pear fruit and leaf 

(SPONDIAS CYTHEREA SONN.) IN THE FRENCH WEST INDIES BELONG TO surfaces; IAA producers consisted primarily of fluorescent Pseudomonas app. and 

PV. MANGIFERAEIDICAE. 0. Pruvost, IRFA/CIRAD, Laboratoire de yellow-pigmented enteric spp. A cloned DNA fragment encoding the two 

Phytopathologie, B.P. 180, 97455 SAINT PIERRE Cedex, ile de la enzymes in the IAA biosynthetic pathway of the gall-forming pathogen 

Pat•ologie 

Reunion; and J. Luisetti, INRA, Station de Pahlgcpresence 

vegetal e, Route de Saint Clement, Beaucouze, 49000 ANGERS, 

France. (0. Pruvost) 

Pseudomonas savastanoi was radiolabelled and used as a probe to test for the 

of related sequences in IAA-producing epiphytes. Cross-hybridization 

to DNA isolated from several IAA-producing strains was detected by Southern 

blot analysis at low stringency but not at high stringency; hybridization was not 

Based on pathogen ici ty on Mango (Mangi fera i ndica L. ) and 

Ca shew (Anacardium occidental e L. ), api gmented Xatooa 

campestris strains isolated from Ambarella (Soondias cvytherea 

observed with DNA from the non-producers tested. This result indicates that at 

least part of the IAA biosynthetic pathway of some epiphytic IAA producers may 

resemble that of characterized pathogenic species. 

Sonn.) in the French West Indies were identified as pv. 

mangiferaeindicae,. Mango isolates of that pathovar, however, 

are avirulent on Ambarella and on Mombin (Spondias mombin). 

Tuthese Ambarella strains are probably new pathogenic 

450 

COMPUTER-AIDED) ANALYSIS OF RADIOLABELED PROTFEIN PROFILES OF 

Thumso atooa apsrsp.mnieaidce BACTERIAL ISOLATES FROM CORN ROOTS. C.Hendrick, D.Haefele, 

fom-fXnhmnscmeti p agfraidce " 

J.Marlow. Microbial Genetics Division, Pioneer Hi-Bred 

The relationship of the Ambarella strains to Mango under International, Inc., Johnston, IA 50131. 

determined. 

natual inthe ondiion renh Wet Idie nees t bewe 

446 

are evaluating methods for fingerprinting bacteria selected 

as potential biocontrol agents for seedling diseases of corn. 

To assess the variability in total radiolabeled protein 

profiles between closely related bacteria of diverse geographic 

origin, we analyzed 58 isolates of Pseudomonas fluorescens and 

12 isol~.tes of Serratia plymu/thica from corn roots. Proteins 

STABILITY OF ANTIGENIC DETERMINANTS OF XANTHOMONAS 

CANPESTRIS PV. ORYZAE DETECTED BY MONOCLoNAL ANTIBODIES. 

P. 0. Roberts, A. M. Al~varez, and A. A. Benedict. 

University of Hawaii. Honolulu, HI 96822. 

from each isolate were labeledTby incorporation of 

3 5 

S-methionine, separated by SDS-PAGE, and visualized by autoradiography 

or by scanning dried gels with a 2-dimensional 

beta-scanner. Cluster analysis of scan data showed that the P. 

Vol. 79, No. 10, 1989 1191

fluorescens isolates fell into at least 7 groups and the S. rot. Meloidogyne hapla was the most 

plymuthica 

common 

isolates 

plant-parasitic 

fell into 4 groups. There was no nematode found in roots or soil. Others included Pratylenchus, 

correlation between the geographic source of an isolate and its Criconemella, and Tylenchorhynchus spp. 

electrophoretic group. The method is rapid and reproducible 

and is useful for subgrouping closely related bacterial 

isolates. 

455 

451 STROMATIC HYPHAL INOCULUM FOR KABATIELLA ZEAE. C. A. Martinson. 

Department of Plant Pathology, Iowa State 

CELL 

University, 

WALL-MACERATING-ENZYME 

Ames, 

ACTIVITY IN MYCENA CITRICOLOR. Iowa 50011 

J.P. Tewari, and D.V. Rao, Department 

Un iver s ity of 

o f Plant 

A lbert Science, a , Edm onton , A lbert a , Canad Kabatiella 

a , T6G 2 P5 zese, 

. the causal 

K b t 

agent 

e l 

of 

e e 

eyespot 

h a 

of 

s l 

maize, 

a e t o 

was 

grown in reciprocal shake culture on potato 

y s o 

dextrose 

f m i e 

broth 

a 

a- 

Mvcena mended with 0.1% 

of coffee citricolor, yeast 

produces the extract 

oxalic causal (PDB) 

acid agent of the American leaf spot black stromatic hyphae formed. 

for 

in About 

5 

culture 250 

to 7 

ml 

days, 

and in stromatic 

until 

infected culture was seeded into P0B in a 10 liter fermentor; yields hyphal of 

tissue. As a result of this, the pH of coffee cell wall 122 an seeged wto equiv o ster harvestmedium 

was lowered to 4.4, a condition which is known to favor 122 and 138g (dry wt. equiv.) of stromatic hyphae were harsmacerating-enzyme 

activity. However, the enzyme 

esterase 

pectin 

was 

methyl 

absent and significant levels of 

ed after 3 and 

ed, ground, 

4 days 

coated 

respectively. 

onto #37 white quartz 

Stromatic 

sand, 

,id hyphae 

air drisd 

were 

over 

- 

polygalacturonase (PG) and cellulase (Cx) were not producedf night and could be stored dry for one year. About 

Furthermore, 4x10 lesions 

the fungus hydralized 

Furtermrethe 

ungs 

cellulose 

hdroized 

(CI) 

celuloe 

at a slo w 

(C) a a 

developed/g 

lowfollowing 

of dried stromatic hyphae 

whorl 

on a 

inoculation. 

susceptible variety 

With routine resistance screening, 

rate. The lesion tissue also showed low PG and Cx activities. aouto1img strloatic hyha ws used ergdyand; abut1 

This suggested that tissue deterioration during lesion about I mg stromatic hyphae was used per g dry sand; about 

development was not primarily caused by macerating enzymes. sand was directed into the plant whorl with a pipet modified 

Extensive deposition of calcium oxalate crystals was into an hour-glass type applicator. Sand inoculum sporulate 

associated with the cell wall material. These results the whorl and continued to fall back into the whorl and resposuggested 

a primary and direct role of oxalic acid in tissue rulated several times. Inoculation were made any time of day. 

deterioration through the formation of calcium oxalate. 

456 

452 PATHOGENICITY OF ROTYLENCHULUS RENIFORMIS ON COTTON. Julio C. 

SMALL, CONJUGATABLE PLASMID IN COPPER-RESISTANT STRAINS OF Borbon, William E. Batson, Jr., and Gary W. Lawrence, Dept. of 

XANTHOMONAS CAMPESTRIS PV.VESICATORIA. V. Dittaponqpitch, Plant Pathology and Weed Science, Mississippi State University, 

D.F. Ritchie, and R.G. Upchurch. Department of Plant Miss. State, MS 39762. 

Pathology, N.C. State Univ., Raleigh, NC 27695. The effect of the reniform nematode (Rotylenchulus reniformis 

Thirty-two strains of Xanthomonas campestris pv. Linford & Oliveira) on the growth of cotton seedlings (Gossypvesicatoria 

isolated from pepper and tomato were tested ium hirsutum L.) "Stoneville 112" was determined in a greenfor 

sensitivity to 200 Aig/ml copper sulfate in sucrose house study. Soil was infested with 0, 1,000, 5,000, 10,000, 

peptone agar. Sixty percent were copper resistant. 15,000, and 20,000 young adults/500 g of soil. Plant height was 

Plasmid profiles indicated the presence of at least two measured weekly for 10 weeks. At harvest, fresh and dry weights 

plasmids in all strains. All copper-resistant strains of shoots and roots were recorded. Plant height was significontained 

an approximately 3 kbp plasmid. This plasmid cantly reduced by an initial population density of 10,000 nemawas 

transferred via conjugation to copper-sensitive todes after seven weeks. Initial nematode population densities 

strains. Transconjugates contained the 3 kbp plasmid and of 1,000 or higher suppressed shoot growth. Initial nematode 

were copper resistant. Preliminary anaylsis indicated the population densities of 15,000 and 20,000 were required to replasmid 

was digested by restriction enzymes PstI, Sau3A, duce root growth. 

AluI, and TaqI, but not by EcoRI, BamHI, HindIII, or XhoI. 

457 

453 EFFECTS OF CYST NEMATODE RACE 5 ON SUSCEPTIBLE SOYBEANS AT FOUR 

EXISTENCE OF CHITINASE ACTIVITY IN MATURE CORN KERNELS (ZEA PLANTING DATES. R. P. Pacumbaba and W. Tadesse. Dept. of 

MAYS). J. N. Neucere and T. E. Cleveland, USDA, ARS, Southern Plant and Soil Science, Alabama A&M University, Normal 35762 

Regional Research Center, P.O. Box 19687, New Orleans, LA 

70179 Effects of soybean cyst nematode race 5 (SCN) on susceptible 

Essex and Lee were investigated in 1, 15, 30 April and 14 May 

Reducing infection by Aspergilli species that produce afla- planting dates for 3 seasons. The means of the two soybean 

toxins in corn is an area of intense interest. One speculation cultivars for the three-year period at different planting dates 

is that chitinase is a resistance factor involved in inhibiting were significantly different for the size and number of 

fungal growth. Mature kernels of yellow corn and white corn nodules, soil temperatures, plant heights, number of cysts, SCN 

were assayed for chitinase activity. Results showed activity rate and % SCN field infestation, and yield. For 14 May 

primarily in germ tissues with disparity between the two planting date, significantly larger size of nodules, increased 

varieties. The highest activity per quantity of protein was number of nodules and cysts, higher SCN rate and % SCN field 

observed in the germ of white corn. Immunochemical assays infestation were noted as well as significantly lower yield and 

showed different profiles; two precipitin bands present in the shorter plant height. Planting susceptible soybean cultivars 

endosperson of white corn were not detected in either the germ in cyst nematode race 5 infested soil earlier than the regular 

of white corn or whole kernels of yellow corn. Strategies for planting dates in Northern Alabama (14 May-14 June) increases 

purification and characterization of active components will be plant height and yield but decreases the number of cysts, SCN 

discussed, rate and % SCN field infestation. These indicated that the 

soybean cultivars tested escaped SCN infestation in the field. 

454 

OCCURRENCE OF PEANUT POD ROT IN OKLAHOMA: 1983-85. A. a. 

Filonow and C. C. Russell. Department of Plant Pathology, 

458 

ITRCIEEFCSO OASUBNML N UTVRO NI 

Okaom taeUnvrst, tllaer K.707.DENCE 

Peanut fields in Bryan, Caddo, Hughes, and Okfuskee counties 

were surveyed for pod rot from early September to mid October. 

OF PHOMOPSIS SEED DECAY IN SOYBEAN IN MISSISSIPPI. K. W. 

Roy, N. H. Buehring, W. F. Jones, and K. S. McLean. Mississippi 

State University, Drawer PG, Mississippi State, MS 39762. 

Thirty plants per acre from 2-8 acres per field were sampled. In 

1983, 41.7% of 36 fields had pod rot as diagnosed by symptoms In a 6-yr (1980-1985) field study the soybean cultivars Tracy 

and isolations of Pythiu~m myriotylum or Rhizoctonia solani from and Centennial were sown in soil treated with 0, 31.5, 75.0, or 

diseased pods. In 1984 and 1985, 73.1% of 26 fields and 43.5% of 150 kg/ha muriate of potash (K). At growth stages R2 and R4 fo- 

46 fields, respectively had pod rot. Mean disease incidence for liage was treated with benomyl (0.56 kg a.i./ha) or left unall 

fields in 1983, 1984, and 1985 was 6.1%, 21.3%, and 5.8%, treated. Mature seeds were assayed for Phomopsis 10ngicolla. 

respectively. In 1904 no linear correlation(r=-0.09) was found Phomopsis seed decay b e i t n was w e n u l l c reduced a p c i u e m 

by e s K o in p o 1982, s f 1983, o m n d and i v 1985. 

d u a f i l d sS i g n i f i c a n t K b y c u aewend 

l1 t i po v a r i rot iu incdne 

n t e r a c t i o n s o Over c c u 3pyears r r e d i n P. 1 9 8 myods 2 tyrum a n d 

oin R.vsolanied 1983. In 1982, 31.5, 15.0, 

an pd 

and 

otiniene.Ovr 

150 kg K 

eas 

reduced 

. 

seed 

yrotlm 

decayi 

were 

f . oln 

isolated 

Centennial by 52, from symptomatic pods 

73, and 86%, respectively, 

in whereas 42-60% or only 30-35% 

75 kg 

of the K reduced (30%) seed decay in Tracy. In 1983, 75 and 150 kg K 

fields, respectively. Sclerotium rolfsii was found in rotted reduced seed decay in Centennial by 46 and 71%, respectively, 

pods in 52-79% of the fields, suggesting a possible role in pod whereas K had no effect on Tracy. In 1985, 150 kg K reduced 

1192 PHYTOPATHOLOGY

seed decay by 10%. Phomopsis seed decay was reduced by benomyl seedlings from P. pumila 'Mando' were poor hosts, while ten others supported 

alone in 1984 (27%) and 1985 (8%) but was increased in 1981 substantial population increases. The phenotype for host suitability appears to be 

(64%) and 1983 (65%). In 1980, benomyl increased seed decay by segregating in the seedling populations from these two sources. Other selections 

68% i n the absence of K but had no effect i n its presence. may be identified as unsuitable hosts for C. xenoplax in the near future. 

459 463 

EFFECTS OF SOIL INOCULUM DENSITIES OF FUSARIUM MONILIFORME ON PHYTOPYTHORA SHUCK AND KERNEL ROT OF PECAN FRUIT C. C. 

GRAIN SORGHUM GROWTH AND ROOT DEVELOPMENT. D. K. Tuopay, L. E. Reilly , F. F. Hendrix, Jr. and M. W. Hoýchkiss 

Trevathan, and G. W. Lawrence. Dept. Plant Pathology and Weed IUSDA-ARS, P.O. Box 87, Byron, GA 31008, Dept. of Plant 

Science, P. 0. Drawer PG, Mississippi State, MS 39762. Pathology, University of Georgia, Athens, GA 30602. 

The relationship of inoculum concentration of Fusarium monili- Phytophthora shuck and kernel rot of pecan Carya illinoensis 

forme and growth of grain sorghum (Sorghum bicolor) variety (Wang.) K. Koch. is a new disease of pecan fruit. The causal 

DeKalb 59 was determined under greenhouse conditions. Micro- agent is Phytophthora cactorum (Leb & Cohn) Schroet. The 

conidial inoculum was incorporated in sterile soil mix at disease was first observed on maturing fruits in central GA in 

concentrations of 0, 2.0, 17.0, 1.7 x 102, 1.7 x 103 and 1.7 x early September 1988. Yield and quality of the kernels was 

104 conidia per gram. Growth of leaves and shoots was measured reduced by >50% in some central GA orchards and to a lesser 

weekly from the base of the plant to the tip of the uppermost extent in south GA. Symptoms developed within 4-6 da, usually 

leaf for eight weeks. At the end of this period, plants were starting at the attachment end of the fruit with a distinct 

lifted from soil and shoot fresh and dry weights and root fresh margin between the necrotic and healthy tissue. The shucks 

and dry weights were recorded. F. moniliforme was recovered turned brown, dried over a 2-3 wk period and stuck tightly to 

from 69% of stem and root sections of plants growing in soil the shell. The kernel had a dark brown seed coat with gray 

infested with the fungus. Plant height, shoot fresh and dry rotted endosperm. Koch's postulates were satisfied by 

weights and root fresh and dry weights were significantly laboratory and field innoculations of nut clusters. Symptoms 

reduced at inoculum levels of 1.7 x 102 conidia per gram of appeared in 4 da for laboratory and 6-8 da for field 

soil and greater. innoculations. 

460 464 

SEEDLING BLIGHT OF GRAIN SORGHUM CAUSED BY GIBBERELLA THAPSINA. 

Douglas J. Jardine, Department of Plant Pathology, Kansas State 


T.A. Frisina, R. D. Milholland, and R. A. Fong. Sporangial ano 

oospore production by Phytophthora fragariae in roots of 

strawberry plants. North Carolina State University, Box 7616 

A new species of Gibberella, G. thapsina was consistently 

isolated from mature sorghum (Sorghum bicolor) plants. 

Laboratory inoculations of three-day-old sorghum seedlings (cv. 

'Pioneer 8515') were made by dipping the roots into a spore 

suspension (10 cfu/ml) and incubating for three days at 27 C. 

Seedling roots dipped in sterile distilled water served as the 

control. Three Kansas isolates, FKMF 1321, FKMF 1337 and FKMF 

1427, induced symptoms typical of seedling blight on grain 

sorghum. Symptoms consisted primarily of the formation of 

reddish-brown lesions at the base of root laterals, and after 

3 days, death of the infected lateral. On some plants, the 

entire root system was necrotic. No symptoms developed on the 

roots of the control plants and G. thapsina was reisolated from 

a rvirulent 

inoculated roots but not from the controls. 

Raleigh, North Carolina 27695-7616. 

Sporangial production by P. fragariae was greatest when roots 

of the susceptible strawberry cultivar Tennessee Beauty were 

incubated for 8 days at 15 C following inoculation with a 

highly virulent isolate (NC-I) of race Pf-2. Duration of root 

incubation in unsterile soil leachate (2-6 days) for the total 

8 day incubation period was not a significant factor in 

sporangial production. Sporangial production decreased after 

8 days incubation. In contrast, oospore production increased 

between days 6 and 12. Temperature optima for maximum 

sporangial production by race Pf-2 were 15"20 C, whereas, 

temperature optima for sporangial production of a weakly 

isolate of race Pf-7 (ATCC 18638) were 20-24 C. 

Race Pf-7 produced less sporangia/root than Pf-2 at all 

temperatures tested. 

461 

IN VITRO EFFICACY OF FUNGICIDES AGAINST CERCOSPORELLA RUBI. 

Barbara J. Smith, USDA-ARS, Small Fruit Research Station, Biological and cultural factors associated with citrus replant 

P. 0. Box 287, Poplarville, MS 39470. problems in Texas. M.Skaria and C.Farrald, Texas A&I 

University Citrus Center, P.O. Box 1150, Weslaco, TX 78596. 

Rosette of blackberry, caused by Cercosporella rubi, is severe 

in the southeastern U.S. and is not adequately controlled by Three-year-old 'Marrs early orange' (Citrus sinensis) trees 

currently registered fungicides. Twenty-two fungicides were on sour orange (C. aurantium) rootstock show strikingly 

screened in vitro for efficacy against C. rubi at 3 to 5 rates heterogeneous growth characteristics. Trunk diameter at an 

ranging from 0.05 to 500 .ug a.i./ml incorporated into potato inch above the budunion and plant height range between 0.9 to 

dextrose agar prior to pouring into petri plates. Mycelial 7.0 cm and 60 to 180 cm, respectively. Thirty percent of the 

plugs of C. rubi were transferred to each plate, and colony total trees are stunted. Two fungi,Phytophthora parasitica and 

diameter was measured after 7 days. The fungicides were placed Ganoderma lucidum, and a nematode, Tylenchulus semipenetrans, 

into 5 efficacy groups based on the lowest level of each have been found associated with the severely stunted trees. 

fungicide causing a significant growth reduction of C. rubi Star ruby grapefruit (C. paradisi), which occupied this 10 

compared to its growth on unamended PDA: 1) 0.05 pg/rsl: acre block, had a high incidence of P. parasitica infection. 

flusilazole, propiconazole, captafol, benomyl, ferbam, These trees were freeze-killed in 1983. The land was prepared 

diniconazole, bitertanol, myclobutanil; 2) 0.5 pg/mi: DCNA, for replanting in 1984 by cutting the trees at the soill 

fenarimol, maneb, iprodione, mancozeb; 3) 5 .zg/ml: dodine surface, leaving the stump and the root system in the soil. 

triadimefon, triforine, metalaxyl, folpet ; 4 ) 50 Aig/al : Association of cultural as well as biological factors 

anilazine, cupric hydroxide, captan; 5) 500 ug/ml: sulfur, including the above mentioned organisms as causal agents of 

citrus replant problems will be discussed. 

462 466 

PRUNUS SELECTIONS UNSUITABLE AS HOSTS FOR CRICONEMELLA 

XENOPLAX. S. W. Westcott, III and E. I. Zehr, Department of Plant Pathology and HARRY FERTILIZER AND PLANT PROTECTANT IN TOMATO CROPPING. 

Physiology, Clemson University, Clemson, SC 29634. Marvin D.Whitehead and N.J .Thirumalschar .817 Clifton Road, N.E. 

Atlanta, GA 30307, and Jeersannidhi Anderson Institute, POB.506 

Selections of Prunus are being screened for suitability as hosts to Criconemetla Locust St .Walnut Creek, CA 94596. 

xenoplax and ultimately for use as rootstocks. Seeds were collected from trees Harry Fertlizer and Plant Protectant (H-F-PP) is a systemic, 

maintained in the Prunus Germplasm Collection at Clemson University. Seedlings non-phytotoxic bactericide and fungicide containing organic 

were grown in steamed sand infested with C. xenoplax (100-300/plant) for periods and inorganic nitrogen fertilizers. In experimental plots of 

of 9-15 wk in a greenhouse. Seedlings that supported little or no reproduction by the BURPEE SUPER-STIEAK tomato variety in Atlanta ,pre-fertilized 

nematode were tested in a similar manner one or more additional times. Those that with farm yard manure and commercial booster fertilizers, 

consistently were poor hosts for the nematode were propagated by rooting cuttings foliar sprays on 30th and 40th .days after planting a~ere given 

and were tested again for host suitability to C. xenoplax. From a collection of 18 

seeds from P. besseyi seven seedlings were identified as poor hosts for C. xenoplax 

as follows : In treated plots, Hf-PP at concentration of four 

tablespoonfuls in a gallon of wat.,r, and in control plots 

(no detectable reproduction by the nematode). Eleven seedlings from P. besseyi 

supported about a ten-fold increase in nematodes in 10 weeks, that was similar to 

Dithane Z-78 one tablespoon per gallon were used.The leaf bli- 

ghts by Alternaria solani and Cladosporium fulvum were complethe 

reproduction seen on P. persica 'Nemaguard' seedlings. Similarly, four tely controlled by HF-PP, and moderate in Dithane Z-78 plots. 

Vol. 79, No. 10, 1989 1193

HF-PP plots gave 30% more yield, and the fruits were uniformly 

2 to 2.5 lbs in size, while in controls it was 1.75 lbs mostly. 

Disease control, plant growth stimulation and foliar nutrition 

by HF-PP was responsible for this improved cropping. 

467 

rot/blackleg, were observed, however Verticillium wilt and to 

a lesser extent Rhizoctonia canker seem to be the major 

disease problems. With Verticillium wilt there is an 

apparent association with rotation practices and use of 

susceptible cultivars. These two diseases will be the focus 

of continuing studies. 

EVALUATION OF SOIL SOLARIZATION FOR CONTROL OF VERTICILLIUM 

WILT IN CHERRY TOMATO. J. A. Liebman, D. P. Morgan, L. 

Epstein, and M. J. Jimenez. University of California, 

Berkeley, 94720. 

Solarization limited by the controls need to many apply soil-borne plastic mulches plant diseases, to soil during but is the 

sumier. If the field is planted there is too much sbade; if 

tbmmer.Ifthe field is 

471 

ROOT AND CROWN DECLINE OF GLOBE ARTICHOKES WITH AN 

UNKNOWN ETIOLOGY. J. C. Correll and S. F. Colbert. Department of Plant 

Pathology, University of California, Berkeley, CA 94720 

Artichokes (Cynara scolymus) have been grown extensively in the Monterey 

County region of California for approximately 80 years; it is not unusual to find 

flo tegwrlostes there seaons pruc t;io. 

the field 

fields 

is fallow 

which 

the 

have 

grower loses 

been 

the 

cropped 

season's 

exclusively 

production. 

to artichokes 

Recently, a 

for 

root and 

10-15 

crown 

years. 

We investigated the use of solarization to control Verticillium observed in 

decline 

numerous 

problem 

fields. 

of 

Symptoms 

unknown 

include 

etiology 

a dark 

has 

brown 

been 

wilt of drip-irrigated 

to 

cherry 

black root 

tomato, a crop which normally is discoloration in the cortex as well as the stele. Larger 

staked, 

roots 

so 

(2-4 

the 

cm) 

soil 

have 

surface 

been 

is not shaded. Compared to non- observed with severe cortical discoloration 

solarized 

and necrosis 

controls, 

of the stele. 

solarization 

Advanced 

significantly reduced soil root decline symptoms include a complete collapse 

inoculum 

of the stele 

density 

where 

of 

the 

the 

tissue 

pathogen and led to significantly develops soft rot type symptoms. This 

increased 

soft rot appearance 

yields 

can 

the 

progress 

following 

into the 

season. The effect was most above ground crown. Work has been initiated 

dramatic 

to examine 

when 

the 

mulches 

cause and 

were 

possible 

applied at time of transplanting control of this problem. Several fungi, including Fusarium 

(17 April) 

solani, 

rather 

Cylindrocarpon 

than later in the season (25 June or sp., and Rhizoctonia solani have been frequently isolated from necrotic root tissue. 

30 July). Solarizing cropped ground was slightly more Also the stunt nematode, Merlinius sp. has been isolated from soils with the 

effective than solarizing fallow ground, and did not unduly decline syndrome. Grubs of the cribrate weevil (Otiorhynchus cribricollis) also 

interfere with normal agronomic practices, have been observed to cause substantial feeding injury to roots. A working 

hypothesis is that the root decline problem is the result of a disease complex. 

468 472 

GROWTH OF PINK ROOT RESISTANT AND SUSCEPTIBLE ONION CULTIVARS VIRAL DISEASES ASSOCIATED 

IN 

WITH 

ORGANIC 

CUCURBITS 

SOIL INFESTED 

CROPS IN 

WITH 

HAWAII. 

PYRENOCHAETA 

J. J. 

TERRESTRIS. P. M. Cho, D. E. Ullman, T. L. German, and D. Custer. P. 

Coleman, 

0. Box 269, 

L. A. Ellerbrock, and J. W. Lorbeer, Departments of University of Hawaii, Kula, HI 96790. 

Vegetable Crops and Plant Pathology, Cornell University, 

Ithaca, NY 14853. 

Farm surveys were conducted on three Hawaiian islands including 

Maui, Molokai and Oahu to determine the major mosaic viruses 

Onion cultivars Kodiak and Paragon, reported to be resistant infecting cucurbit crops. High disease incidences (greater than 

and susceptible to pink root, respectively, were grown in 30%) were associated with all zucchini plantings sampled. 

organic soil, not previously cropped to onion, in the Disease incidences in cucumber and watermelon plantings were 

greenhouse in pots and outdoors in flats. Blended mycelium of lower at less than 10%. Zucchini yellow mosaic virus (ZYMV) was 

P. terrestris was mixed with soil in half the treatments; the the only virus found on Molokai farms, the predominant virus on 

control soil was unamended. Green leaf weight and bulb weight Maui crops, and also found on Oahu. Watermelon mosaic virus 1 

at harvest were not decreased in the presence of P. terrestris (WMVl) was the predominant virus on Oahu; and accounted for 3%, 

for either cultivar. Plants in amended soil had more pink 29% and 49% of infected plants on Maui. Cucumber mosaic virus 

roots at harvest than plants in unamended soil, but overall (CMV) and watermelon mosaic virus 2 (WMV2) were not detected on 

pink root levels were low. P. terrestris was isolated only any of the surveyed farms. Forty-two different aphid species 

from plants grown in the amended soil. Pink color of onion have been identified on Maui. Three known vectors predominate 

roots from control soil appeared to be due to naturally including Aphis gossypii, A. craccivora, and Myzus persicae. 

occurring fungi with pink mycelium which were isolated from JMS stylet oil delayed the onset of disease and disease 

such roots, but were not identified. incidence. 

469 

INCIDENCE AND DISTRIBUTION OF ESCAROLE NECROSIS IN CICHORIUM POLYMERASE EXPRESSION MODULATION BY RIBOSOMAL 

ENDIVIA IN FLORIDA. R. N. Raid, R. T. Nagata, U. of Florida, FRAMESHIFTING IN RED CLOVER NECROTIC MOSAIC VIRUS. Z. 

Everglades Research and Education Center, Belle Glade, 33430 Xiong, S. A. Lommel, Department of Plant Pathology, North Carolina 

and L. L. McDaniel, ATCC, Rockville, MD 20852. State University, Raleigh, NC., 27695. 

Commercial fields Commrcia of f Cichorium Cihorum filds endivia ediva L. L.weresureyedfor were surveyed for Nucleotide (RCNMV) genomic sequence RNA-1 analysis revealed of the that red the clover polymerase necrotic gene mosaic apper virus 

escarole necrosis, caused by an uncharacterized virus (ENV), (RCNMV)egenomic RNA-1 revealedthatithe polymersetgee from Nov 1988 to ear 

Mar 1989. Six geographical locations in the to be regulated by a frameshift 

Everglades 

mechanism. 

Agricultural 

To definitively establish 

Area were sampled. Plants were visually the capability of ribosomal 

observed 

frameshifting 

for symptom 

at 

development 

the identified 

and indirect-ELISA 

sequence, 

was used to reporter gene fusions where constructed and 

verify diagnoses. 

assayed 

Escarole necrosis was detected -- in 5 of 6 and quantity of fusion protein product. A portion of the 

for 

galactosidase 

the presence 

locations and in 11 of 12 fields sampled. Disease incidence gene was fused downstream (in a translational ranged context) from to 0 to the 

894 plants per one hectare block (0 to 1.26%). frameshift region. Bacterial colonies transformed with the construct 

Incidence and spatial distribution within the field appear to turned blue in the presence of x-gal. However, 

be related the blue 

to color the was 

proximity and orientation of neighboring much less intense when compared to a construct 

sugarcane 

where 

fields or canals, 

the 

suggesting 

frameshift 

the possibility that the region was eliminated and the f-galactosidase 

virus 

gene 

is insect-vectored. 

was fused inframe 

Sequential observations suggest that before the frameshift region. The differential blue reactions 

there 

suggest 

is little or no secondary infection. Results indicate that ribosomal frameshifting acts as a down regulatory 

that 

mechanism 

although 

for 

escarole necrosis is widespread in south Florida, polymerase expression. The detailed expression of these constructs in 

it is of little importance on C. endivia at the present time. vitro and in vivo will be reported. 

40SEQUENCE ANALYSIS OF THE ZUCCHINI YELLOWS MOSAIC VIRUS COAT 

IMPORTANCE OF SOIL AND TUBER-BORNE DISEASES OF POTATO IN PROTEIN GENE. Guowei Fang and Rebecca Grumet, Horticulture 

SOUTH DAKOTA. .-. .... Ga eneg SDSU, Plant Science Department, Michigan State University, East Lansing, MI 41824. 

Department, Box 2109, Brookings, SD 57DD7.'''• 

Zucchini yellows mosaic virus (ZYMV) is a highly aggressive 

Observations in commercial potato production fields as well member of the potyvirus group that infects cucurbit crops 

as direct contacts with growers were made during 1985 - 1988 (cucumbers, squashes, melons). We seek to genetically-engineer 

to determine the importance of soil and tuber-borne disease resistance to this disease. To this end we have cloned and 

problems in northeastern South Dakota. In randomly selected sequenced the ZYMV coat protein gene. A ca. 1.5 Kb clone from 

fielda, visual observations were made to determine presence the 3' end of the ZYMV genome was cloned into a Bluescript 

or absence of diseases, and where necessary, laboratory vector and digested to form a set of nested deletions for use 

diagnosis was used as confirmation, Estimates of disease in Sanger dideoxy sequencing reactions. Predicted amino acid 

severity were made where possible. Numerous diseases, sequence shows many features in common with other potyviral 

including common scab, Fusarium tuber decay, and soft coat proteins. These include the conserved motifs MVWCIENGTSP, 


473

LARYAFD, QMKAA, FGLDG and TERH, in the central portion and 3' ization with PSTV-specific probes. The data showed that the 

end of the gene. The sequence data, putative 5' and 3' ends of PSTV cDNA sequences are integrated into potato and tobacco genthe 

gene, and comparisons to other potyviral coat protein omic DNA. Expression of the chimeric PSTV "genes" will be 

sequences will be presented. examined by Northern blot hybridization. Regenerated tomato 

shoots currently growing on selective media will be analyzed as 

described above. 

475 

OCCURRENCE OF A SMALL RNA IN MAIZE CHLOROTIC DWARF VIRUS 479 

(MCDV)-LIKE PARTICLES. X. Ge, D. T. Gordon and R. E. Gingery cDNA CLONING OF THE WHEAT STREAK MOSAIC VIRUS GENOME. K. R. 

(USDA), Dept of Plant Pathology, and M. D. McMullen (USDA), Zajula and S. A. Lommel, Department of Plant Pathology, 

Dept of Agronomy, Ohio State Univ., Wooster, OH 44691. Raleigh, NC. 

A slower sedimenting alon virus-like wih i rae-znalcenrifued MDV particle irins (SSVLP) was repratons detected 

along with MCDV virions in rate-zonal centrifuged preparations 

from MCDV-infected maize. SSVLP morphology was identical to 

that of MCDV (isometric, ca. 30 nm dia.) except that cores of 

SSVLPs were penetrated with uranyl-acetate. The buoyant 

densities 

respectively. 

of SSVLP 

SSVLP 

and 

was 

MCDV 

positive 

were 1.295 

in ISEM and and 1.442 immuno- g(CSS04 )/ml, 

Wheat streak mosaic virus (WSMV), a putative member of the 

potyvirus group, is composed of a single-stranded RNA genome 

of approximately 8.5 kb and a capsid protein of 42kDa. A 2.1 

kb 3' terminal cDNA clone was synthesized by oligo--dT priming. 

This clone has been sequenced and the capsid protein domain 

oligonucleotide identified. Primer primer extension homologous cloning to was the performed 5' terminus using of the an 

precipitated with MCDV-specific antiserum. SDS-PAGE revealed 

three MCDV virions. capsid proteins ssRNA of of about corresponding 1 kb was purified mol. wts. from for SSVLPs SSVLP and 

10-kb ssRNA from MCDV virions. The 10-kb RNA hybridized with 

ranoml priNA 

randomly primed 

probessynts 

cDNA probes synthesized 

iz fromThe 10-kb 

from the 

RNA 

l-kb RNA,dermn. 

oligo clote per homologous 

existing to the existing clone. A clone virus was specific identified. 3.2 kb Together clone not these homologous clones 

represent over 75% of the genome. Nucleic acid sequence, in 

vitro translation products, and a partial genome map are being 

whereas the 1-kb RNA hybridized only weakly with cDNA from the 

10-kb RNA. We hypothesize that MCDV capsid proteins encapsidate 

a RNA related to MCDV-genomic RNA to form the SSVLP. 

determined. 

480 

476 

The Effects of Chemical Fixatives on Wheat Soil-borne Mosaic 

Viruses, Dr. R. L. Grayson and Mary Sue Mayes, Dept. of Plant 

Path., Phys. and Weed Sci. , VPI&SU, Blacksburg, VA 24061-0331. 

EFFECT OF GENOMIC POSITION ON THE EXPRESSION OF THE TOBACCO 

MOSAIC VIRUS MOVEMENT PROTEIN. J. N. Culver, K. M. Lehto, and 

W. 0. Dawson. Dept. of Plant Pathology, University of 

California, Riverside, CA 92521. 

Tobacco mosaic virus (TMV) mutants with various deletions in 

the coat protein (CP) gene were utilized to study the effect of 

genomic position on the expression of the viral 30 K movement 

protein. Vi ral protei ns puri f ied "f rom i nocul ated Ileaves were 

Viral suspensions of wheat soil-borne mosaic virus (WSBMV) were 

examined to determine the effects of fixation on particle morphology. 

Serum sensitive electron microscopy (SSEM) in combination 

with primary antibodies and secondary gold label antibodies 

were the techniques utilized. Gluteraldehyde and osmium tetroxide 

(OS0 4 ) were both shown to have a deterimental effect on WSBMV 

morphology. Results indicate that these chemicals alter the condition 

Gluteraldehyde of the protein had the coat most and severe cause effect a loss on in WSBMV. viral Disassocia- integrity. 

analyzed using a Western blot technique to quantify the ratio 

of viral 126 K to 30 K proteins for each mutant. The ratio of 

126 K/3D K allowed a comparison of viral infection to 30 K 

levels for each sa mle. a com utantson o vingrparta infe tion twere s 

levels for each sample. TMV mutants having partial deletions 

in the CP gene showed a 3 to 4 fold increase in 30 K levels 

when compared to wild type TMV, while mutants having the entire 

CP gene deleted showed a 10 fold increase in 30 K levels. A 

mutant having a full-length but non-translatable CP gene produced 

30 K levels equal to that of wild type TMV. This study 

demonstrates that positioning of the 30 K gene nearer the 3' 

terminus of TMV yields higher levels of protein. 

had i 

tion of the viral coat was observed after one hour. OSO 4 had an 

effect on the viruses after exposure of 8+ hours. When viruses 

exposed to more than one fixative, the viral distortion was 

much more severe. WSBMV exposed to gluteraldehyde and OS0 4 for 

time periods in excess of three hours showed the most severe 

effects. These results may indicate why little success has been 

achieved in observing WSBMV in thin sections. 

481 

477 

INFECTIVITY OF MUTATED cDNAs OF THE POTATO SPINDLE TUBER VIROID 

(PSTV). DO.K. Lakshman and S.M. Tavantzis, Dept. of Botany & 

Plant Pathology, Univ. of Maine, Orono, ME 04469. 

SEQUENCE ANALYSIS OF DEFECTIVE INTERFERING RNAS OF 

TOMATO BUSHY STUNT VIRUS. D A Knorr, T J Morris. 

Plant Pathology Dept., University of California, 

Berkeley, 94720. 

Defective interfering (DI) RNA B1O, derived through 

ten serial passages of tomato bushy stunt virus in 

Nicotiana benthamiana, was analyzed by cDNA cloning 

Five mutanta were constructed from a 358-base coDA clone 

(pAV401),of PSTV and subcloned at the Bamn asite of "Riboprobe" 

plasmids pSP64 and pSP65. Mutant LT1 baa an A addition after 

baae 53. Mutants APM4 and APM6 have abort deletions (baaes In 

97 and 286-288, reapectively) at the two Aval aitea of LTI. In 

addition, APM6 baa an inaert of 24 baaea next to the deletions. 

Mutant ST4 carriea a two-base deletion (339 and 340) at the 

sty ite of LTIl whereas E9 is a four-base deletion (146-149) 

at the Eag site of LTI. Seedlings of the tomato cv. Rutgers 

were inoculated with BlamHi-digested plasmids carrying monomers 

of the above PSTV cOMA mutants. Seedlings inoculated with 

pLTI, and p5T4 exhibited symptoms and contained PSTV RMA. 

Experiments are underway to determine the infectivity of dimeric 

or trimeric cOMA and the respective (+) RNA transcripts of 

the above mutants. 

and sequencing. DI B1O is a population of TBSV 

deletion mutants, -600 nt in length, that is effic- 

iently replicated and encapsidated, and also protects 

the host from lethal necrosis associated with helper 

virus infection. Six independent cDNA clones of DI 

BIO each contain the 5' viral leader seguence followed 

by an internal block of m250 nt from the polymerase 

domain. 3' termini include stop codons for the viral 

P19 and/or P21 ORFs, and a deletion of 179 nt of non- 

translated seguence. Size differences between the BI0 

cDNAs is due primarily to variation of the junction 

site between the polymlerase and 3' domains. 

482 

478 

A TRANSGENIC PLANT APPROACH TO STUDYING VIROID PATMOGENESIS. 

S.M. Tavantzis, D.K. Lakshman, L.C. Burian, and B.P. Bandy. 

Dept. of Botany & Plant Path. Univ. of Maine, Orono, ME 04469. 

CONTROL OF CROWN ROT OF BANANA IN THE WINDWARD 

ISLANDS BY A THIABENDAZOLE-COATED PAD. A. Johanson, 

F.J. Proctor, J.R. Cox, and M.J. Jeger, Overseas 

Development Natural Resources Institute, Central 

Avenue, Chatham Maritime, Kent ME4 4TB, U.K. 

I h idadIlnsbnnsaectfo h 

snthem a indwpackd Islnd th bfel.ana absr benutfo h 

Site-specific, short deletions or insertions were introduced 

int o aPST th seuene cDA conean suseqenty vn-the 

int o a th STVcDA seuene coneandsubequntl veified 

by sequencing. Full-length or portions of the mutated 

PSTV cDNA were subcloned, in a plus or minus orientation and 

cellulose pad coated with the fungicide 

thiabendazole (TBZ) is applied to the cut surface of 

banana hand to absorb latex and to control crown 

rot. Crown rot, the most commercially important 

post-harvest disease of bananas, is caused by 

usaimeVeral fungimoiifreincludinganCOlletotrp ichumidro musae' 

under the control of the CaNV 35S promoter, in the plant expression 

vector pBIl2l and mobilized to the Agrobacterium tumefaciens 

strain LBA4404. Leaf discs of potato, tobacco, and 

• ' 

tomato were inoculated with Agrobacterium cultures carrying the 

PSTV sequences. Regenerated potato and tobacco shoots that 

rooted on selective media were analyzed by Southern blot hybrid~- 

Fusaryium moniformen cons' a pl--irTlroesiue-idcae 

1limi[d penetration of TBZ into the tissues from the 

pad. The mean level of TBZ in the surface 2mm was 

B.9 mg/kg; in the next 5mm, 0.7 mg /kq. Below this, 

levels were undetectable by HPLC. Studies showed 

that some insensitivity to TBZ is present in field 

populations of the causal fungi. 

Vol. 79, No. 10, 1989 1195

483 

RELATIVE RESISTANCE OF WHEAT CLASSES AND CULTIVARS TO 

INVASION BY STORAGE FUNGI. D. Mauer, USDA-ARS, U.S. Grain 

Marketing Research Laboratory, Manhattan, KS 66502. 

The variability in moisture content (MC) of a lot 

seeds 

of grain 

may predispose 

or 

fungi. There is little 

the 

uniformity 

entire lot 

in 

to 

the 

the 

MC 

spoilage 

among single 

by storage 

seeds 

from the same lot of soybeans. 

The moisture content of individual seeds collected from 

Wheat samples representing the major classes and cultivars 

were obtained from throughout the U.S. The 256 samples were 

inoculated with spores of Aspergillus glaucus, stored 3 weeks 

at 25 C and 83% relative humidity, and evaluated for visible 

mold, internal mold invasion, and equilibrium moisture 

content. The three red wheat classes, hard red winter, soft 

red winter, and hard red spring were similar in their average 

mold susceptibility. Durums were slightly more susceptible 

than the red wheats, and their equilibrium moisture contents 

were lower. Western white wheats, including club wheats, 

were consistently among the most susceptible, but eastern 

whites averaged among the most resistant. Resistance was 

hard affected red by spring both cultivar, cultivar and Stoa, growing was very location. resistant, However, one 

regardless of where grown. 

maturing 

drying single 

soybean 

seeds 

plants 

at 103 

in 

C 

the 

for 72 

field 

hours 

was 

in 

determined 

copper cups. 

by oven- 

The averages MC and variation between the lowest and highest 

moisture content value were found for three cultivars of soybean 

at harvest. Elgin 87 15.24% (11.15%-25.42%) range 14.30%, Hardin 

13.20% (10.00%-19.10%) range 9.15%, and Corsoy 79 12.20% 

(11.30%-14.32%) range 3.00%. 

The greater the MC at harvest the greater the variability 

the 

in 

MC of soybean seeds. The effect of cultivar on moisture 

content variability was found to be significant. 

488 

DIACERIOXYSCIRPENOL, NIVALENOL, FUSARIN C AND ZEAPAR tI FORMA- 

TION POTATO, BY GEOGRAPHIC 

GRAIN, AND 

ISOIATES 

PASTURE 

OF FUSARIUM CROCEWELLENSE 

HERBAGE. FROM 

Ronald F. Vesonder, 

Northern Regional Research Center, Agricultural Research 

484 Service, USDA, 1815 N. University St., Peoria, IL 61604. 

RED ROT OF MUSKMELON: A POSTHARVEST DECAY. B. D. Bruton, Eighteen Fusarium crookwellense 

USDA/ ARS, Lane, 

isolates 

OK 74555; 

from the 

S. C. 

continents 

Redlin, 

USDA/ 

Contract 

ARS, A , B Scientist, 

Beltsville M MD 

of 

20705; 

Australia, 

J. K. 

Europe, 

Collins, 

and 

ability 

North 

Okla. 

America 

StateExrcsfocrnemntdwheahFaiuiolews 

to 

were 

elaborate S ompared 

mycotoxins 

for their 

Univ., Lane, 

on 

OK; 

corn 

P. Perkins-Veazie, 

at 25 C 

USDA/ARS, 

for 2 

Lane, 

weeks. 

OK. Extracts analyzed from by thin corn layer fermented chromatography with each 

(TIh) Fusarium 

and gas 

isolate 

chromatogwas 

A decay exhibiting red discoloration in muskmelon (Cucumis melo raphy/mass spectroscopy (GC/MS) for mycotoxins. Toxins detec- 

L.) fruits has been observed in postharvest storage studies and ted were zearalenone (13 isolates), fusarin C (11 isolates), 

on occasion been involved in load rejection of melons grown in nivalenol (4 isolates), and diacetoxyscirpnol (2 isolates 

Southeastern Oklahoma. Epicoccum nigrum was consistently iso- Zearalenone and fusarin C were found to be expressed by isolated 

from decay areas exhibiting the red discoloration. lates from each continent, while nivalenol was detected in the 

Koch's postulates were fulfilled in controlled inoculation Fusarium isolates originating from Australia and one isolate 

studies of muskmelon fruit. This is the first report of E. from the United States. These studies suggest this species is 

nigrum as a pathogen of muskmelon. Inoculations of cucumber, a producer of zearalenone and other mycotoxins which could 

tomato, yellow apple, and pear proved the fungus to be path- cause problems with reproduction and well-being of livestock. 

ogenic to these fruit; whereas, eggplant displayed no decay. 

Fungal sporulation occurred only on tomato. Glucose, fructose, 

and sucrose, representing the three primary sugars found in 489 

muskmelon fruit, were incorporated into separate culture 

media. Mycelial extension was greatest on potato glucose agar RAPID-CYCLING BRASSICAS FOR HANDS-ON TEACHING OF PLANT 

at 20°C and very limited at 1,5, and 30*C. A proposed common PATHOLOGY. P. H. Williams, Dept. of Plant Pathology, 1630 Linden 

name for the disease is red rot. Drive, University of Wisconsin, Madison, WI 53706. 

485 Rapid-cycling stocks of Brassica rapa and five other 

species 

Brassica 

have been developed together with inexpensive selfcontained 

growing systems that are suitable 

RESISTANCE OF 

for 

CITRUS FRUIT 

exploratory 

EXOCARP TO PENCILLIUM DIGITATUM. 

J. W. 

learning 

Eckert, M. 

in 

Ratnayake, 

the classroom. 

and 

The 

J. 

materials 

Sievert. Dept. 

are suitable 

of Plant 

for 

investigations 

Pathology, 

of growth 

University 

and development 

of California, 

(plants 

Riverside, 

flower in 

CA 

14 

92521. days and cycle in 35 days); reproduction (flowering, pollination, 

Wounds in the exocarp developed fertilization, 

resistance to embryogeny, 

infection cell 

by and 

P. molecular 

(mendelian, biology); genetics 

digitatwi cytoplasmic, 

within 24 hrs molecular, 

at 250C. quantitative, 

The degree population, 

of resistance breeding, 

correlated 

selection 

with 

and 

the 

evolution); 

amount of lignin, physiology 

determined (hormones, 

spectrophoto- CA, 

auxin, cytokinins, 

metrically 

photosynthesis, 

as lignin thioglycolate, 

respiration, nutrition, 

associated 

water 

with the walls relations, 

of cells 

tropisms surrounding and photoresponses); 

the wound site. 

and 

Lignin ecology 

formation (chemicals 

was and symbionts, 

increased at pests, 

high pathogens 

relative and 

humidity. microbes). 

The thioglycollate A number of 

deri- explorations 

vative 

in host-pathogen 

was extracted 

relations 

and digested 

using Albugo, 

with CuO. 

Plasmo- 

A TLC of the 

digest diophora, 

revealed Leptosphaeria, 

three Fusarium, 

UV-absorbing Xanthomonas, 

spots with TuMV 

Rf and CaMV 

values have been 

corresponding 

developed. Instructional 

to p-hydroxybenzaldehyde, 

materials and genetic 

vanillin, 

stocks 

and are available through 

syringaldehyde, 

the Crucifer Genetics 

characteristic Cooperative 

components 

at the 

of lignin. above address or at 608/262-8638. 

486 VARABIITYIN HEEQULIBIUMMOITUE CNTET O THEE 490 

VARABIIT INTH 

VIRUS STATUS OF SEVERAL UNITED STATES SMALL FRUIT 

EQILBRIM MISURE COTEN O THEE 

CULTIVARS 

GERMPLASM 

OF SOYBEANS. 

COLLECTIONS. 

F. A. Lazzari and 

Joseph 

R. A. Meronuck, 

D. Postman, 

National 

USDA 

Clonal Germplasm 

Department 

Repository, 

of Plant 

33447 

Pathology, 

Peoria 

University of Minnesota, St. Road, Corvallis, Oregon 97333. 

Paul, MN 55108. 

Equilibrium The Moisture U.S.D.A. 

Content (EMC) maintains 

data are necessary collections 

to (Frg ar of 

ia) strawberry 

design , drying and storage regimes 

currant 

for hygroscopic 

and gooseberry 

products such 

(Ribes) 

b~ad15'kT'ry and raspberry (Rubus) , 

asganadses blueberry 

oba 

and 

storageagri fungi adseswhen stored oyenwith sesexcess edsaesbetdt aemoisture. ujce o piaeb Germplasm cranberry R~po--mr (Vaccinium) , - at-the Corvallis. National Cultivated Cl onal1 

The average EMC as determined by oven drying single seeds varieties 

using copper 

and wild 

cups showed preserved as clones or relatives 

that after as seed. of 

120 Clonal 

these 

days accessions 

crops 

of storage 

are 

at the are tested 

same relative 

for latent 

humidity 

virus 

(RH) and 

infection 

temperature, 

by 

different 

ELISA, 

cultivars inoculation 

of soybeans 

of sensitive 

reach different 

indicator 

moisture contents• 

plants, and 

visual inspection 

Three lots of 

of 

Hardin, 

clones. 

Corsoy 79, 

Plants 

and Elgin 

found 

87 soybeans 

to be 

stored virus infected 

at 75% RH 

are 

resulted 

subjected 

in an 

to 

EMC 

heat-therapy 

of 13.50%, 14.20%, 

and in 

and 14.61% vitro meristem 

resecivly amecutiar 

culture. 

Te kptat80%RHrechd 

Resulting plants 

n MC 

are 

respectively The s1,ame c6u60varespetvl;adwe kept at80RHrahdnEM retested 

accessions 

for viruses, 

when 

and 

tests 

replace 

are negative 

infected 

of1.0% 168% an 166% epciey n hnkp t identity 

and 

has 

plant 

85% RH they reached an EMC of 17.58%, 

been 

17.95%, 

verified. 

and 19.01%, 

The percent 

negative of 

clonal 

virus 

accessions available 

respectively. 

to researchers 

This study reveals 

' 

that 

is 

different 

as follows: 

cultivars 

69% 

of 

Ofo400 

soybeans 

.2rag[_ia 

reach clones, 

160 Ribes 

49% 

clones, 

of 

63% 

different 

of431-RubiB 

levels of 

clones, 

moisture 

and 

content 

78% 

under the same conditions, of 3WVaccinium clones. ""- 

487 491 

MOISTURE VARIABILITY IN SEEDS OF THREE SOYBEAN CULTIVARS BEFORE INCIDENCE OF TOMATO RINGSPOT VIRUS IN GRAPE IN VIRGINIA. D.C 

HARVEST. Flavio A. Lazzari and R. A. Meronuck, Department of Bays and S.A. Tolin, Department of Plant Pathology, Physiology 

Plant Pathology, University of Minnesota, St. Paul, MN 55108. and Weed Science, VPI&SU, Blacksburg, VA 24061. 

1196 PHYTOPATHOLOGY

In an initial survey of ten commercial vineyards in Virginia PAV were measured by enzyme-linked immunosorbent assay (ELISA) 

to determine the incidence of tomato ringspot virus (TmRSV), the and by purification. Oats were inoculated with viruliferous 

cultivars Chardonnay, Cabernet Sauvignon, Cabernet Franc, Sau- Rhopalosiphum padi 7, 15, or 21 days after planting. Symptoms 

vignon Blanc, White Riesling, Vidal 256 and Merlot, and the of BYDV-RPV-IL infection were mild in all lines. Root and shoot 

rootstocks S04 and Kober 5BB, were sampled. Weed species dande- samples were collected from 8 to 33 days after inoculation. The 

lion, broad-leaf and narrow-leaf plantain, and white clover were time of peak virus concentration, as measured by ELISA, varied 

also sampled. ELISA and indicator plants were used to detect with the age of plants at inoculation. No consistent pattern 

virus and confirm its identity. Only one commercial cultivar, of virus titer was found in tolerant and susceptible sister oat 

Vidal 256, at two locations, was positive for the TmRSV. Eight pairs. BYDV-RPV-IL concentrations were higher in susceptible 

vines each of S04 and Kober 5BB rootstocks were positive at a plants at some sampling times and in some tissues, whereas at 

single location, and isolates of virus from these plants are other times or in other tissues the titer was higher in the 

being studied further. Infected dandelions were found at the two tolerant sister oats. Yields of purified BYDV-RPV-IL were 

locations which had the infected Vidal, but the virus was similar from tolerant and susceptible sister oat lines. 

slightly different from that in grape. No other weed species 

were infected, suggesting that wild hosts do not play a role in 

the epidemiology of TmRSV in grape in Virginia. 496 

VIRIONS OF A MISSISSIPPI ISOLATE OF SUBTERRANEAN CLOVER RED 

LEAF (SOYBEAN [W4RF)-LIKE IIYIEOV OBSERVED IN PHLOCI Of 

492 INFECTED SUBTERRANEAN CLOVER. M. R. McLaughlin, USDA, Alt, 

DETECTION OF VIRUSES IN FLORIST GERANIUM USING A SIMPLIFIED Crop Science Research Laboratory, Forage Research Unit, P. 0. 

METHOD OF dsRNA ANALYSIS. S. T. Adkins and S. T. Nameth, Dept. B 5367, Mississippi State, MS 39762-5367. 

of Plant Pathology, The Ohio State University, Ohio Agricultural Excised petioles from symptomatic leaves of Trifolium 

Research and Development Center, Columbus, OH 43210. subterraneun L. cv. Geraldton, experimentally infected with 

A simplified method for analysis of viral-associated dsRNA the 'Meteora' isolate of subterranean clover red leaf (soybean 

detected the presence of a number of viruses in infected geran- dwarf)-like luteovirus (Phytopathology 78:1584), were fixed in 

ium plants. Greenhouse-grown florist geraniums inoculated with Karnovsky's fixative, treated with RNase to remove riboscmes, 

known viruses such as CMV, TMV, TRSV, PFBV and commercially- postfixed in osmium, stained in uranyl acetate, and embedded 

produced geraniums showing symptoms of virus infection were in Spurr' resin. Thin sections wre stained in uranyl 

extracted and analyzed. One to 7 gr. of geranium tissue was acetate and lead citrate and examined by electron microscopy. 

sampled. Samples were phenol extracted and subjected to cellu- Electron-dense diamtercreebsereddn spherical virus thucyoplatednuclusvacules 

particles about 22nm in 

lose column chromotography. The purified dsRNA was analyzed diameter were observed in the cytoplasm, nucleus, vacuoles, 

with a mini electrophoresis unit (125 V for 1 hr.) on 5% poly- and plasmodesmata of some phloem cells. Vesicles containirg 

acrylamide vertical gels. The complete extraction and analysis electron-dense fibrils, but no virus particles, were observed 

process took 4 hrs. DsRNA profiles and molecular weights of in the cytoplasm of some infected cells. Consistent with 

known viruses were compared with dsRNA profiles from plants luteovirus cytcpathology, only phloem cells contained virions. 

infected with unknown viruses. The precision and the accuracy 

of the assay was dependent on the size of the tissue sampled and 

the type of virus present. This method provides a rapid and 497 

reliable means of detecting viruses in geraniums. 

INFECTION OF SWEET CORN AND WHEAT WITH MAIZE 

WHITE LINE MOSAIC VIRUS BY ARTIFICIAL INOCULATION. 

493 L. Zhang and T. A. Zitter, Department of Plant Pathology, Cornell 

EFFECTS OF PMV-SAD ON ST. AUGUSTINEGRASS. Q. B. Heidel University, Ithaca, NY 14853 

and R. W. Toler, Dept. of Plant Pathology and Microbiology, Texas 

A&M University, College Station, TX 77843. Maize white line mosaic virus (MWLMV) was transmitted by wounding 

healthy corn or wheat seed and adding purified virus to the wound site. 

Texas Common St. Augustinegrass, Stenotaphrum secundatum (Walt.) The MWLMV-infected corn or wheat seedlings exhibited typical 

Kuntze, was mechanically inoculated with individual isolates of N and 

W of panicum mosaic virus-St. Augustine decline strain (PMV-SAD). MWLM symptoms. Of the major cereal crops (corn, rice, barley, 

Infected and uninfected St. Augustinegrass was transplanted in late sorghum, oat, wheat, etc.) tested with this technique, only corn and 

May to one meter square field plots established on a modified sand root wheat were found to be infected. Results were confirmed using ELISA 

zone. Plants were observed through the growing season and into and cDNA techniques. No significant difference in susceptibility to 

November to determine effects on growth and development due to MWLMV was found among 11 sweet corn varieties with three different 

PMV-SAD. Percent coverage was reduced significantly (a=0.05) in 

virus infected grass. Stolon number, internode length, leaf length and genes (sh2, su, se) encoding sugary phenotypes. This technique can 

width, root length and dry and fresh weights of leaves, stolons and easily provide a continuous supply of diseased material. 

roots were not significantly affected over a six month period. 

494 498 

DETECTION OF TOMATO SPOTTED WILT VIRUS IN IMPATIENS USING 

DESMODIUM YELLOW MOTTLE VIRUS AND CUCUMBER MOSAIC VIRUS BIOTINYLATED MOUSE MONOCLONAL ANTIBODIES. H. T. Hsu and R. H. 

INFECTING WILD GROUNDNUIT (Apios americana). R. A. Valverde, R. 

Provvidenti, and C. A. Clark, Dept. of Plant Pathology and Crop 

Lawson, USDA-ARS, Beltsville, Maryland 207J5A 

Physiology, Louisiana Agricultural Experiment Station, Louisiana 

State University, Agricultural Center, Baton Rouge, LA 70803 

Vi rus-speci fic bioti nyl ated monocl onal antibodies were utilIi zed 

in ELISA for detection of tomato spotted wilt virus (TSWV) in 

and Cornell University, Geneva, NY 14456. the Impatiens cultivar, Mojave. Rooted cuttings from naturally 

infected plants were grown in a greenhouse and leaf samples 

Wild groundnut (Apios americana), a native legume of eastern removed from the apex, mid-portion and base of the stem were 

North America is being investigated for potential domestication 

in Louisiana. Two virus isolates: desmodium yellow mottle virus 

tested for TSWV. Leaves from the apex tested positively for 

TSWV while those lower on the same stem were negative. Cuttings 

(DYMV) and cucumber mosaic virus (CMV) were obtained from 

different accessions in experimental plots in Baton Rouge. 

Symptoms in wild groundnut induced by both viruses were 

were made from TSWV-positive and TSWV-negative portions of the 

same plants and grown for about 3 months. Plants from all 

cuttings tested positively for TSWV. Plants from cuttings of 

indistinguishable and consisted of a mild yellow mottle. Both infected Impatiens grown in high temperature (27 C day; 24 C 

viruses were identified by host reaction, serology electron night) or low temperature (21 C day; 18 C night), with 16 and 8 

microscopy and dsRNA analysis. Cucumber mosaic virus was hr light and dark periods, all tested positively for TSWV.. At 

isolated from 12 out of 20 plants showing virus-like symptoms. 

Desmodium yellow mottle virus was found in 3 out of 20 plants 

tested. 

the lower temperatures, leaves were symptomless while necrosis 

and some leaf distortion was present at the higher temperatures. 

495 499 

H. N. Fouly and C. J. D'Arcy. Titers of barley yellow dwarf 

MAIZE DWARF MOSAIC VIRUS-VENEZuELAN STRAIN INFECTION IN Sorghum 

bicolor AND Zea maya BUMOLE SHEATH CELLS. F. Mayobre and N. L. 

virus-RPV-IL in tolerant and susceptible sister oat lines.Maol. nsiuoVezandenvtgciesCntfas 

Dept. 

4 709.'' 

of Plant Pathology, Univ. of Illinois, Urbana, IL 61801- C.M.B.C., Apdo. 21827, Caracas 1020 A, Venezuela. 

The purpose of this work was to deterssine MD)MV-V infection ef- 

The titers of barley yellow dwarf virus-RPV-IL (BYDV-RPV-IL) in fects on the ultrastructure of bundle sheath cells (BSC) and 

two pairs of sister oat lines tolerant and susceptible to BYDV- compare them to those of mesophyll cells (MC) of Sorghum hi color 

Vol. 79, No. 10, 1989 1197

(L) var. Wray and Zea mays (L) var. Ohio 28. Seedlings were ino aromas, and related compounds, including various allyl, 

culated with MDMV-V. After 20 days, leaf samples were processe-d sulfide, thiocyanate and isothiocyanate compounds, were tested 

for electron microscopy study. Bundle, rod, laminate and pin- for stimulatory activity. Most active was dodecyl isothiowheel 

virus inclusions were observed within BSC and MC cytoplasm cyanate (C12-NCS), followed by decyl-NCS and nonyl-NCS. 

but not associated with organelles. Chloroplasts and mitochon- Octadecyl-NCS was slightly active. With dodecyl-NCS at 

dria degenerating membranes were observed in both cell types. concentrations of 1 to 100 pl/L, germination ranged between 65 

Increased starch accumulation was observed in BSC chloroplasts and 85% at 21 days, 18 C. With decyl-NCS, germination ranged 

but not in MCchl. Cytoplasmic, nuclear, and tonoplast membranes between 70 and 30% at concentrations of 1 to 50 pl/L. With 

were disrupted and, ingrowth and outgrowth were seen. Virus ef- nonyl-NCS, germination ranged from 55 to 15% at concentrations 

fect was more evident in MC than in BSC. The results indicate of 1 to 25 pl/L. These isothiocyanate derivatives are much 

that BSC can provide the necessary energy for virus multiplica- slower acting than our previously reported hexane extract of 

tion, functioning not only as a passive transporter of the virus thistle roots. However, they are the first compounds of known 

particles but as active agent in the infection process, structure found to stimulate teliospores of P. punctiformis. 

500 504 

MOLECULAR CLONING OF A TOMATO HMG CoA REDUCTASE GENE AND ITS 

DEFENSE-RELATED EXPRESSION. H. S. Park, C. J. Denbow, and C. L. 

Cramer, Department of Plant Pathology, Physiology and Weed 

Science, Virginia Polytechnic Institute and State University, 


3 

-Hydroxy-3-methylglutaryl CoA reductase (HMGR) mediates a key 

rate-limiting step in isoprenoid biosynthesis leading to produc- 

MUTANTS OF CERCOSPORA KIKUCHII ALTERED IN CERCOSPORIN SYNTHESIS 

AND PATHOGENICITY. Robert G. Upchurch, D. Carey Walker, and 

Margaret E. Daub. Department of Plant Pathology, N.C. State 

University, Raleigh, NC 27695-7616. 

Five mutants altered in the synthesis of the photosensitizing 

phytotoxin, cercosporin, were isolated following UV mutagenesis 

of conidia of the soybean fungal pathogen Civrcospora kikuchii. 

tion of phytoalexins, rubber, electron transport components, 

sterols, carotenoids, gibberellins, and abscisic acid in plants. 

We have isolated a full-length tomato genomic clone encoding 

HMGR based on cross-hybridization with yeast HMG1 (Basson, et 

al. 1986, PNAS 83:5563). Partial sequence analysis reveals 

regions exceeding 65% nucleic acid and 70% derived amino acid 

identity with yeast HMGR. Southern anal ses of tomato DNA 

probed with this clone reveal multiple HAGR genes. In Northern 

blots, HMGR sequences hybridize to mRNA of 2.4 to 2.7 kb. HMGR 

mRNA levels are greatly increased in early-log phase cells 

treated with Verticillium albo-atrum or Fusarium oxysporum 

cell-wall components suggesting defense-related induction, 

The toxin mutants were found to be of two types: type i 

synthesizes no toxin on all laboratory media tested and type 2 

has reduced toxin synthesis on rich (PDA) medium only. Parental 

isolate PR synthesized cercosporin on all media tested. 

Cercosporin synthesized by the mutants ranged from 0 to 36% of 

the parental isolate. Soybean cultivars Lee and Centennial were 

inoculated by three methods in the glasshouse: mycelial plug 

inoculation and by atomizing conidia and mycelial fragments onto 

leaf, pod and stem surfaces. In addition, detached pods were 

inoculated by conidial and mycelial suspensions. Mutants lacking 

the ability to synthesize toxin on all laboratory media tested 

did not incite leaf, pod or stem lesions. 

501 

STRAIN-SELECTIVE RESPONSE OF LEAF DISCS OF CHRYSANTHEMUM MORIFOLIUM TO SILICON ENHANCES RESISTANCE OF 

AGROBACTERIUM 

BARLEY TO POWDERY 

TUMEFACIENS 

MILDEW 

IS AFFECTED BY HORMONE PRECONDITIONING. A.L. (Erysiphe graminis f. sp. hordei). D. Jiang, 

Bush 

R. 

and 

J. Zeyen 

S.G. Pueppke, 

and V. 

Department of Plant Pathology, University of Missouri, Russo, Department of Plant Pathology, University of Minnesota, 

Columbia, MO 65211. St. Paul, MN 55108. 

Mildew susceptible barley cvs. (Atlas and Proctor) were grown in 

The tumorogenic response by leaf discs of two cultivars (Gem and Puritan) low mineral content peat, uniformly fertilized and watered with 

of Chrysanthemum morifolium to two strains (B6 and Chry 5) of Agrobacterium distilled water. Silicon supplied plants (Si+) were given 10 g 

tumefaciens reflects the response of whole plant stem inoculations. Hormones of CaSIO 3 or Na 2 SiO 3 per kg of peat; whereas, silicon deficient 

had a preconditioning effect on the discs, enhancing tumor production, plants (Si-) were untreated. Si+ plants grew more quickly and 

but not changing the strain X cultivar response. Leaf discs were cultured had significantly higher dry weights and Si contents than Sion 

water agar, or MS medium plus or minus hormones for 3 days before plants. When inoculated, Si+ plant leaves had far fewer and 

submersion for 5 minutes in A. tumefaciens (10 8 cells/ml). The leaf discs smaller mildew colonies than did Si- leaves, and mildew 

were cultured on water agar for 3-5 days, then moved to MS medium minus sporulation was greatly reduced. Microscopic examination showed 

hormones. Tumors were visible within 1 week of co-cultivation. Tumor mildew germling penetration efficiency (# of germlings producing 

number was greatest when the discs were pretreated with hormones. Gem, haustoria .- # of encounter sites) was greatly reduced on Si+ 

for example, produced tumors in response to Chry 5 but not B6 in stem leaves, and was associated with unpenetrated host cell papillae. 

inoculations. Leaf discs on water agar produced an average of 1 tumor/disc The decrease of fungal germling penetration efficiency was 

in response to either strain. On MS + 0.1 mg 2,4-D/1 the values were 2 positively correlated with total Si concentrations in Si+ 

and 14 tumors/disc in response to B6 and Chry 5, respectively, leaves. Si may be a structural component of epidermal cell 

walls, making fungal penetration difficult. 

503 

STIMULATION OF GERMINATION OF TELIOSPORES OF PUCCINIA50 

PUNCTIFORMIS BY SEVERAL ALKYL ISOTHIOCYANATE DERIVATIVES.50 

R. C. French, USDA-ARS, Frederick, MD 21701 DEGRADATION OF CELLS AND TISSUES OF SELECTED DICOTS AND GRAMIN- 

EOUS MONOCOTS BY PECTATE LYASE AND XYLANASE. C. A. Rodrigues 

Volatiles from onion and garlic tissues stimulated germination and E. J. Braun, Dept. of Plant Pathology, Iowa State Univ., 

of teliospores of P. punctiformis. Known components of these Ames, IA 50011. 


505

The primary cell walls of grasses contain far less pectic C.J. Lamb2 and R.A. DixonI. 'Plant Biology Division, The Nobl1 

material and far more xylan than the walls of dicots. Our goal Foundation, P.O. Box 2180, Ardmore, Oklahoma 73402. 

2 

plant 

is to determine which enzymes are necessary for the degradation Biology Laboratory, Salk Institute, P.O. Box 85800, San Diego, 

of grass tissues. Endopectate lyase (PL) and endoxylanase iso- California 92138. 

lated from a corn stalk rot strain of Erwinia chrysanthemi Chalcone synthase (CHS) catalyzes a key step in isoflavonoid 

(SRI20A) were tested, both alone and together, for cell-killing phytoalexin biosynthesis. The cis-acting regulatory sequences 

and tissue macerating ability. Levels of PL which killed 50- of CHS were examined using chimeric genes consisting of the 

60% of root cap cells isolated from beans and cucumber killed 5'-flanking regions of bean CHS fused with the coding sequence 

only 10-15% of the cells isolated from oats and corn. Addition of a "promoter-less" Q-glucuronidase (GUS) gene. Transgenic 

of xylanase to PL preparations had no effect on bean cells but tobacco plants containing these constructs were assayed for 

had an additive effect on oat cell death and a synergistic GUS activity after various inducing treatments. UV 

effect on corn cell death. In tissue maceration studies, the 

number of leaf cells released by PL treatment was over 230% 

irradiation or application of HgCl 2 caused a 4-fold increase 

in GUS activity within 6 or 50 h, respectively. Infiltration 

greater than the control for tobacco, 170% for cowpea, 16% for of a nonpathogenic isolate of Pseudomonas syringae into 

oats and 16% for corn. Maceration experiments using xylanase transgenic tobacco leaves caused a several-fold increase in 

are currently in progress. the GUS activity surrounding the hypersensitive lesions. 

These studies will help define the factors which coordinate 

508 

defense gene expression. 

EXTRACELLULAR PROTEASES OF XANTHOMONAS CAMPESTRIS 

PV. MALVACEARUM. R. K. Gholson, C. Rodgers, and M. Pierce. Dept. 

of Biochemistry, Oklahoma State University, Oklahoma Agricultural 

512 

Experiment Station, Stillwater, OK 74078. BIOTINYLATION OF TOBACCO SUSPENSION-CULTURED CELLS AND 

EFFECTS ON PLANT-BACTERIA INTERACTIONS. G.H. Harmon and C.J. 

Stain 3-1 of X. campestris pv. malvacearum (Xcm) produces at least three Baker. USDA-ARS. Microbiol. and Plant Pathology Lab., 

extracellular proteases, which showed different patterns of peptide bond 

cleavage. The protease-deficient mutant PM2 produced barely detectable levels 

Beltsville, MD 20705 

of protease in culture under optimal inducing conditions, amounting to about Two biotinylating reagents, sulfosuccinimidobiotin and 

6% of that produced by the parental strain per bacterium at the same stage of sulfosuccinimidyl 6- (biotinamido)hexanoate were used to 

culture. Purification of polypeptides from culture supernatants and assays for label surface plasma membrane proteins of N. tabacum 

protease activity showed the presence in PM2 cultures of the major neutral suspension cells. Biotinylation of cells at pH 6.0 for one 

protease P1 of Xcm. Whether the other two proteases are also present at hour prior to inoculation with Pseudomonas syringae pv 

reduced levels is being investigated. In pathogenicity tests in susceptible Acala 

44, PM2 grew less well than stain 3-1, and the growth yields, although only 

differing by a factorof2 or 3 at an inoculum concentration of 5 X 10 

syringae did not affect the H+ uptake/K+ efflux response 

observed in controls. Protoplasts isolated from biotinylated 

6 cfu/ml, cells were viable and not subject to easy rupture. However, 

differed by more than a factor of 10 when inoculation was with 108 cfu/ml. biotin labeling of protoplasts after cell wall removal 

We have demonstrated homology between the Xcm genome and a genomic resulted in an increased number of nonviable and/or fragile 

clone of the protease structural gene from X. campestris pv. campestris given cells. Protoplast cell surface plasma membrane proteins 

to us by M. Daniels. We will report on cloning of this homologous DNA. labeled with either biotin reagent could be detected on 

western blots. These results show that biotinylation of 

intact cells may be a useful tool for studying plasma 

509 

membrane involvement in plant-bacterial interactions. 

ACTIVITY, ISOZYME PATTERNS AND CELLULAR LOCALIZATION OF 

PEROXIDASE AS RELATED TO SYSTEMIC RESISTANCE OF TOBACCO TO 

BLUE MOLD INDUCED BY PERONOSPORA TABACINA AND TOBACCO MOSAIC 

VIRUS. X. S. Ye, S. Q. Pan and J. Kuc, Department of Plant 513 

Pathology, University of Kentucky, Lexington, KY 40546-0091 ORGANIZATION OF PECTINS IN POTATO TUBER CELL WALLS AND THEIR 

HYDROLYSIS BY PECTATE LYASE FROM PSEUDOMONAS VIRIDIFLAVA. 

Stem injection with P. tabacina or leaf inoculation with TMV K. Sasaki, G. Nagahashi & C.-H. Liao, USDA/ARS, Eastern 

in tobacco cultivar Ky 14 induced systemic resistance to blue 

mold and TMV. The treatments also elicited a systemic increase 

Regional Research Center, Philadelphia, PA 19118 

in peroxidase activity which was positively correlated with To understand the mechanism of tissue maceration by pectate 

induced resistance. Upon challenge with P. tabacina, lyase from P. viridiflava (SF 312), we first studied the pectin 

peroxidase activity was increased earlier and more rapidly in organization in potato tuber cell walls as a natural substrate. 

the induced plants than in controls. The greatest increases in Chemical and ultrastructural analyses revealed that nearly half 

peroxidase activity were found in intercellular fluids and 

cell wall fractions. Isozyme patterns of peroxidase were 

of pectic polymers were held in the region nearer the inner 

surface of the cell wall by Ca 2 +bridges and the rest in the 

detected on isoelectric focusing gels and showed an increase middle lamella by covalent linkages. Next, potato tuber cell 

of some anionic peroxidases as a function of induced walls were treated with purified pectate lyase to study how this 

resistance. Some salt-soluble cell wall proteins also were enzyme attacks these different groups of pectic polymers. After 

higher in the induced plants as compared to control plants. 3 h at 30 °C, 70% of the galacturonic acid was solubilized from 

cell walls and after 24 h, 85%. The results indicate that this 

510 

enzyme can hydrolyze most pectic polymers including the middle 

lamella of the potato tuber cell walls. Further studies of the 

degradation of cell wall structure by pectate lyase will also 

CONSTITUTIVE CONJUGATES OF DAIDZEIN AND GENISTEIN NAY PLAY be presented. 

MULTIPLE ROLES IN EARLY RACE SPECIFIC ANTIBIOTIC RESISTANCE IN 

SOYBEAN. I. L. Graham, Dept. of Plant Pathology, The Ohio 

State University, Columbus, OH 43210. 

514 

In earlier work we demonstrated that constitutive pools of 

conjugates of the glyceollin precursor, daidzein, are utilized 

in a race specific manner for glyceollin synthesis in 

Phytophthora megasperma f. sp. glycinea (PNG) infected soybean 

cotyledon tissues. Both the rate of hydrolysis of the conju- 

CORRELATION BETWEEN IN VITRO SYNTHESIS 1 OF PHENYLACETIC 2 ACID AND 

VIRULENCE IN RHIZOCTONIA SOLANI. S.N. Tavantzis, B.L.Perkins, 

2 1 

R.J. Bushway, and B.P. Bandy. Depts. of IBotany and Plant 

Path. and 2 Food Science, Univ. of Namne, Orono, NE 04469. 

gates and the subsequent utilization of daidzein for glyceollin 

are race specific events. Although the rate of conjugate A study conducted by our group and data reported by other remhydrolysis 

is not affected by light, the rate of glyceollin earch teams have shown that hypovirulent isolates of R. solani 

biosynthesis from free daidzein is. In the light, glyceollin (AG 3) promote an increased rate of growth in potato. Earlier 

synthesis is well underway within 24 hr with only moderate work has suggested that in vitro cultures of R. solani produce 

accumulation of free daidzein. In the dark, however, free phenylacetic acid (PAA) which caused disease symptoms on potato 

daidzein (and closely related genistein) accumulate to high attributed to this pathogen (Frank & Francis, Can. J. Bot. 54, 

levels within 24 hr followed only later (24-48 hr) hy glyceollin 2536-2540). We carried out a study to determine the amount of 

accumulation. We hypothesize that under those conditions where PAA and derivatives produced by AG 3 isolates representing a 

glyceollin elicitation is inefficient, the isoflavones them- wide spectrum of virulence. FAA and derivatives were partitionselves 

may play an additional role as direct antibiotics, ad in an organic solvent and quantitated by HPLC. The amount of 

PAA present in the culture filtrates was proportional to the 

511 virulence of the corresponding R. solani isolate, and ranged 

from 45 to 595 ug of FAA per gram dry weight of mycelium. There 

FUNCTIONAL ANALYSIS OF CHALCONE SYNTHASE PROMOTER SEQUENCES was no correlation between the presence or amount of FAA deriva- 

FROM BEAN IN TRANsGENIC TOBACCO. B.A. StermerI, J. Schmid 2 , tives (rn-ON, p-ON, and n-OH) and virulence. 

Vol. 79, No. 10, 1989 1199

515 Withdrawn 519 

516 

DIRECT DETECTION OF 0-1,3-GLUCANASE ISOZYMES ON POLYACRYLAMIDE 

ELECTROPHORESIS AND ISOELECTROFOCUSING GELS. S. Q. Pan, 

X. S. Ye and J. Kuc, Department of Plant Pathology, University 

of Kentucky, Lexington, KY 40546. 

A procedure is described to assay isozymes of 0-1,3-glucanase 

directly on polyacrylamide electrophoresis (PAGE) and isoelectrofocusing 

(IEF) gels by using 2 ,3,5-triphenyltetrazolium 

chloride. The reagent reacts with reducing sugars released by 

0-1,3-glucanases from the substrate laminarin. Acidic and 

neutral isozymes of 0-i,3-glucanase were detected and quantified 

on 17.5% native PAGE gels run with an anodic discontinuous 

buffer system. A significant linear relationship (a = < 

0.01, R = 0.991) was observed between amounts of 0-i,3-glucanase 

loaded and intensities of bands stained with the reagent 

on native PAGE gels. A fuller isozyme pattern was obtained on 

7.5% IEF gels with a pH range of 3.5-9.5. The IEF gels were 

heated in a microwave oven during the staining process to 

minimize diffusion. 

517 521 

CONIDIAL DEVELOPMENT IN COLLETOTRICHUM GRAMINICOLA. 

D.G. Panaccione, L.J. Vaillancourt, Z. Yang, and R.M. Hanau. 

Dept. of Botany and Plant Pathology, Purdue University, West 

Lafayette, IN 47907 

Colletotrichum graminicola produced two types of conidia in 

culture and during lesion development on corn leaves. One was 

falcate and was produced from morphologically distinct 

conidiogenous cells. The second type was oval, variable but 

smaller in size than falcate conidia, and was from hyphae 

lacking distinct conidiogenous cells. Oval conidia were the 

only type produced by cultures grown In the dark whereas 

development of falcate conidia was light dependent. In vitro 

translation of poly(A)-RNA from vegetative and conidiating 

hyphae showed state-specific differences in mRNA at early 

stages in falcate conidial development. 

EFFECTS OF SOIL PHOSPHORUS (P) AND GLOMUS INTRARADICES ON 

GROWTH, CARBOHYDRATES, AND PHOTOSYNTHETIC ACTIVITY OF 

CITRUS AURANTIUM L. S. Nemec and J. C. V. Vu, USDA, ARS, 

Orlando, FL 32803. 

EVIDENCE FOR A COMPONENT OF RESISTANCE LIMITING NUMBER OF 

BACTERIAL BLIGHT INFECTIONS IN RICE. M.F. Koch, J. Parlevliet. 

International Rice Research Institute, Los Banos, Philippines 

and Agricultural University Wageningen, P.O. Box 386, 6700 AJ 

Wageningen, Netherlands 

Sour orange grown in low-P (9-12 ppm) and high-P (450 ppm) 

soil inoculated with or without Glomus intraradices (G.i.) 

were evaluated for biomass and various photosynthetic parameters. 

Growth of the low-P, no G.i. plants was lowest, 

with total dry biomass depressed up to 50% of the low-P, 

G.i. treatment. Leaf nonstructural carbohydrates (NSC) 

were 40% lower in low-P, no G.i. plants, compared to the 

Wher treatments. G.i. in low-P soil enhanced leaf 

CO uptake by 81%, chlorophyll content by 28%, and 

RuBP~ase activity by 34%, compared to the low-P, no G.i. 

treatment. Increased P-use efficiency by G.i. in low-P 

soillwas equally effective as high-P nutrition in improving 

CO uptake and NSC. PEPCase activity in low-P, 

no - G.i. eaves, however, was at least threefold higher than 

the other treatments, suggesting a possible alteration in 

Lesion size and lesion number were measured on cultivars of rice 

inoculated by clipping or by spraying with virulent isolates of 

Xanthomonas campestris pv. oryzae, the causal organism of 

bacterial blight. Correlations between the methods were high 

(r=0.82) but specific cultivars consistently deviated from this 

relationship. These cultivars had lower numbers of lesions 

following spray inoculation and slow disease progress during the 

first nine weeks after transplanting in a screenhouse bed. An 

increased resistance component inhibiting bacterial entry 

appears to be present in such cultivars. In order to select 

rice entries with high quantitative resistance to bacterial 

blight, based on both resistance to bacterial entry and to 

bacterial spread, two screenings, one for lesion length after 

clipping, and one for lesion number after spraying, are advised. 

organic acid metabolism due to P deficiency. 

518 522 

CHARACTERIZATION OF AN EXTRACELLULAR PROTEASE OF ERWINIA CARO- 

TOVORA. S. Kyostio and G.H. Lacy, Plant Molecular Biology, 

VPI, Blacksburg, VA 24061. 

Erwinia carotovora subsp, carotovora (ECI4) causes soft-rot on 

many plants by secreting several plant cell wall degrading 

enzymes. To clarifiy the role of protease in potato soft-rot we 

have characterized a proteolytic enzyme. The intra- and extracellular 

fractions of ECI4 were precipitated with 95% NHSO4. 

protease activity was detected only in the extracellular fraction. 

A protease inhibitory factor was detected in the intracellular 

fraction. The molecular weight (35 kd) and pI (4.8) 

of protease were determined. Protease activity was inhibited 

by EDTA, but not by PMSF or pepstatin suggesting that it is a 

metalloprotease. protease activity was detected in ECI4 cultures 

grown in mineral media supplemented either with polygalacturonic 

acid, gelatin, casamino acids, or tryptone. No protease 

activity was observed with glycerol as a carbon source. 

For further characterization a protease has been subcloned from 

cosmid pCA°/ into plasmid pSK- and is being sequenced, 

RESPONSES OF WHEAT SEEDLINGS TO LEAF RUST AT TWO TEMPERATURE 

REGIMES. Beatriz A. Perez and A. P. Roelfs, Department of Plant 

Pathology, University of Minnesota and Cereal Rust Laboratory, 

USDA/ARS, St. Paul, MN 55108. 

TcLrz34 is a near-isogenic wheat (Triticum aestivum) for this 

important adult plant resistance to leaf rust (Puccinia 

recondita f. sp. tritici). Seedlings of Tchr34, the near- 

isogenic lines used in race identification, selected cultivars, 

and Thatcher were tested against isolates of wheat leaf rust at 

5 C, and 20 C, with 8 h dark/16 h light cycle (15,000 lux). At 

20 C, seedlings of Tchr34 and cultivars postulated to have Lr34 

were susceptible to isolates with a wide range of virulence._At 

5 C, TcL r34 and the cultivars Chris, Era, Fletcher, Frontana, 

Klein Cometa, Marcos Juarez Inta, Polk, Rio Negro, Sonalika, 

Surpresa, Veranopolis, and Wheaton had low infection types to 

some of the isolates tested. The period required for full 

pustule development on the susceptible check varied from 23-25 

days and 12-14 days at 5 C and 20 C respectively. The ability to 

detect L r34 in the seedling stage would enable a more rapid and 

extensive testing program. 

1200 PHYTOPATHOLOGY

523 

AVIRULENCE OF WHEAT LEAF RUST TO BUCK MANANTIAL. Beatriz A. 

Perez and A. P. Roelfs, Department of Plant Pathology, 

University of Minnesota and Cereal Rust Laboratory, USDA/ARS, 

St. Paul, MN 55108. 

Buck Manantial is an Argentinian spring wheat (Triticum 

aestivum) derivative of Americano 25c through Buck Quequen, 

General Urquiza, and Klein San Martin. Buck Manantial has been 

resistant to leaf rust in Argentina since it was released in 

1965. The Canadian wheat Kenyon, postulated to have Lr13 and 

Lr16, was derived from Buck Manantial. Seedlings and adult 

plants of selected near-isogenic lines, wheat cultivars, and the 

susceptible check Thatcher were tested with isolates of Puccinia 

recondita f. sp. tritici with a wide range of virulence. 

Seedlings of Americano 25c, a land cultivar from Uruguay, and 

Kenyon were susceptible when tested against isolates virulent on 

Lr16. Seedlings of Buck Manantial were immune to all isolates 

evaluated; therefore, resistance beyond Lr16 is present. Adult 

plants of Buck Manantial were resistant to all cultures to which 

they have been evaluated. It is currently unknown if Lrl3 is the 

major source of this resistance. 

524 

GENES FOR POWDERY MILDEW RESISTANCE IN CULTIVARS OF SOFT RED 

WINTER WHEAT, S. Leath and M. Heun, USDA-ARS and Dept. of Plant 

Pathology, North Carolina State University, Raleigh 27695, and 

Lehrstuhl fur Pflanzenbau und Pflanzenzuchtung, D-8050 Freising- 

Weihenstephan, FRG, respectively. 528 

Twenty-two soft red winter wheat cultivars were inoculated with VARIATION IN FUSARIUM RESISTANCE IN SOMACLONES REGENERATED 

isolates of Erysiphe graminis f. sp. tritici to determine genes FROM ASPARAGUS OFFICINALIS CV. LUCULLUS 234. M.L. Smither, 

for resistance to the fungus. Cultivars were tested with T.L Wacker and C.T. Stephens. Dept of Botany and Plant 

isolates that had been characterized from reactions on P.t.g Mc higan.T StateUnisity, Es . Lans MI 4882 

differential host lines. Determinations were completed 

Pathology, Michigan State University, East Lansing, MI 48824 

independently in two laboratories with different isolates and 

results combined. Inoculations were done on intact 10-day-old Asparagus cv. Lucullus 234 is tolerant of Fusarium crown 

seedlings or on detached leaves on benzimidazole amended agar and in the root dark rot. at 27 Callus C on MS formed medium when supplemented seedlings with were 3% germinated sucrose, 

(50 ppm). Evaulations were done 10-14 days later and based on 1. me/L 2 7 1 mL N n me/L kithn3 Cals 

pustule number and type. Some cultivars were not fully 1.5 mg/L 2,4-D, o. mg/L NAA, and 0.5 mg/L kinetin. Callh s 

characterized while results also indicated other cultivars had was subcultured on the sauemeedium without 2,4-D. Shoos 

no genes for powdery mildew resistance. Gene Pm3a was 

identified as occurring most often in the cultivars tested. 1 mg/L kinetin at 27 C with a 16 hr. daylength, then rooted 

on MS medium containing 3% sucrose, 0.3 mg/L NAA, and 0.7 

mg/L kinetin. Somaclones were transferred to soil for 2 

525 wks. under plastic, then to Fusarium-infested soil. Disease 

development was least in the resistant control A. sprengeri, 

Screening methods to evaluate resistance to net and spot and greatest in susceptible UC 157. Disease expression in 

blotch diseases of barley. T. G. Fetch, Jr, B. J. Steffenson, Lucullus somaclones ranged from resistant to susceptible. 

J. D. Frankiowiak and V. D. Pederson. Dept. of Plant 

Pathology, North Dakota State University, Fargo, ND 58105. 529 

Net (Pyrenophora teres) and spot blotch (Cochliobolus sativus) INHERITANCE OF RESISTANCE TO RACES 0, 1, AND 2 OF FUSARIUM WILT 

are the most prevalent barley diseases in North Dakota. IN MUSKMELON LINE MR-I. F. W. Zink and C. E. Thomas. 

Disease control is best obtained by use of resistant Department of Vegetable Crops, Univ. of California, Davis, CA 

cultivars. Establishment of quick and reliable techniques to 95616 and USDA, ARS, U. S. Vegetable Laboratory, Charleston, 

evaluate barley lines is vital to the breeding program. sc 29414 

Several screening methods were tested for assessing resistance 

in barley. The best seedling test was the ragdoll germination In artificial inoculation studies, muskmelon (Cucumis melo L.) 

procedure using a leaf-dip inoculation and the best adult breeding line MR-i was resistant to races 0, 1, and 2; but not 

plant (early heading) testing method was a spray-gun 1,2y or 1,2w of Fusarium wilt incited by Fusarium oxysporum f. 

inoculation technique. Plants were rated 7 days after 

inoculation using a 9 point scale, and results were compared sp. melonis. Segregation of F1 , F 2 , and BC1 populations of 

crosses between resistant MR-i and susceptible Top Mark 

to field ratings. Correlation coefficients between seedling indicated that resistance to races 1 and 2 is conferred by a 

and field (0.43, 0.71), and adult and field ratings (0.64, single dominant gene. Linkage tests indicated that the genes 

0.76) were highly significant for net and spot blotch, for resistance to races 1 and 2 are not linked. Allelism 

respectively. These methods have proven successful for early tests showed that the single dominant genes in MR-i that 

detection of resistance in barley breeding material, confer resistance to races 0 and 2 and races 0 and 1 are the 

same genes or alleles of Fom-1 in differential cultivar 

526 Doublon and Fom-2 in differential line CM 17-187, respectively. 

RESPONSE TO S. RECURRENT SELECTION FOR MAIZE GRAIN YIELD INA, 

DISEASE-STRES ENVIRONMENT. M.L. Carson and Z.W. Wicks, III, 530 

SDSU, Plant Science Department, Brookings, SD 57007. 

Selection of genotypes that yield well in a disease-stress 

environment has been proposed as a way to increase disease 

resistance and yield potential in the absence of disease 

simultaneously. S1 recurrent selection was conducted for two 

cycles in the BS-19 maize synthetic for: 1) grain yield in 

the presence of northern leaf blight (NLB) caused by 

Exserohi lum turcicum, and Diplodia stalk rot (DSR) caused by 

_D. mads 2) grain yield in the absence of disease, and 3) 

resistance to both NLB and DSR. The original synthetic and 

the two advanced cycles from the three selection schemes were 

evaluated in 1988 in separate trials for yield in a NLB and 

DSR stress environment, yield in the absence of disease, and 

for NLB and DSR resistance. Selection for grain yield in the 

disease stress environment was effective in increasing NLB 

resistance and yield potential in the absence of disease. 

Direct selection of NLB and DSR resistance was also effective, 

EFFECTS OF HOST GENOTYPE ON INCIDENCE OF CERCOSPORA 

ARACHIDICOLA AND CERCOSPORIDIUM PERSONATUM ON PEANUT IN FIELD 

ISOLATION PLOTS. B. B. Shew and M. K. Beute, North Carolina 

State University, Box 7629, Raleigh, NC 27695. 

Six peanut genotypes, having various levels of resistance to 

Cercospora arachidicola (CA), Cercosporidium personatum (CP), 

or both pathogens, were planted in 9.8 m x 3.7 m plots in May 

1988. Each of the total 120 plots was separated by at least 

7.3 a of corn. A potted plant infected with CA, CP, or CA + CP 

was placed in the center of each plot on 2 August as a point 

source of inoculum. No infected plants were placed in control 

plots. AUJDPCs of CA, CP, or both leaf spots were calculated 

from % incidence on 9 rating dates. CA and CP were detected in 

all plots. AUJDPCs for CP were greatest in plots with a CP or 

CA + CP inoculum source, but genotype rankings varied depending 

on the source of inocutum. Inoculum source did not affect 

Vol. 79, No. 10,1989qR 1201

AUDPCs for CA or rankings of genotypes by AUDPC-CA. AUDPCs sions of pathogenic Pmm isolates. Disease ratings made 3 weeks 

were smalLest on NC 3033 for CA, on Southern runner for CP, and later were compared to greenhouse experiments. For both 

on GP-NC 343 for both Leaf spots, methods ratings of resistant cultivars were significantly 

lower than susceptible (P=0.05). Results indicate that the 

531 in-vitro technique is reliable and effective. 

REGENERATION OF TOLERANT SOYBEAN PLANTS TO SEPTORIA GLYCINES 

FROM ORGANOGENIC CALLI. H. S. Song, S. M. Lim, and J. M. Widholm. 

Dept. of Plant Pathology, University of Illinois, Urbana, 

IL 61801. 

535 

DISTRIBUTION OF GLOEOTINIA TEMULENTA, CLAVICEPS 

PURPUREA, AND ANGUINA AGROSTIS AMONG GRASSES IN THE 

Organogenic calli obtained from 225 immature embryos of soybean 

cv. BSR 201 were transferred to MSR medium ammended 

toxic 

with 

culture 

patho- 

filtrates of Septoria glycines (2:1, v/v). More 

than 90% of the calli died withi 0 48 hrs. The surviving calli 

were transferred again 4 times onto the ammended MSR medium and 

which survived were selected. Calli which developed shoots were 

transferred to MSR medium and evaluated for growth. After two 

months, the shoots were transferred to MSS basal medium ammended 

with the pathotoxin. Surviving shoots developed roots. Three 

regenerated plants were grown to maturity in the greenhouse and 

seeds were harvested. A detached leaf assay on R plants (second 

selfed generation) and control BSR 201 plants indicated that 

development of brown spot symptoms and pycnidia were significantly 

delayed on the leaves of th R2 plants when inoculated with 

spore suspensions of S. glycines. 

WILLAMETTE 

USDA/ARS, VALLEY 

3450 

OF 

S.W. 

OREGON 

Campus 

IN 1988. 

Way, Corvallis, 

S. C. Alderman, 

OR. 97331 

During the summer of 1988 a survey of commercial 

grass seed production fields in the Willamette 

Valley of Oregon was initiated to determine the 

incidence of the grass seed diseases, ergot, blind 

seed, and seed gall nematode. A total of 492 fields 

of various preselected cultivars were examined. 

Ergot was found in 52, 13, 1, 2, and 2% of the 

bluegrass, bentgrass, tall fescue, Italian ryegra 

and perennial ryegrass fields, respectively. Blind 

seed was detected in 26-30% of the tall fescue, 

annual ryegrass and perennial ryegrass fields. Seed 

gall nematode was found in 9% of the bentgrass 

fields. A survey of weed grasses indicated that 

532 

ergot was widespread throughout the valley on tall 

fescue, annual ryegrass, and quackgrass. 

SUSCEPTIBILITY OF FLORIDA SUGARCANE CULTIVARS TO 

MELANOCEPHALA. 

PUCCINIA 

R. N. Raid, University of Florida, 

Research 

Everglades 

and Education Center, Belle Glade, 33430 and B. R. 

Eiland, Okeelanta Corp., South Bay, 33493. 

536 

VERTICILLIUM WILT OF ALFALFA IN SOUTHERN CALIFORNIA. D. 

Erwin, R. A. Khan and Amy Howell. Dept. of Plant Pathology, 

Twelve commercial sugarcane cultivars were assessed for Univ. of California, Riverside, CA 92521. 

sugarcane rust severity resulting from natural infection during Verticilliun albo-atrun (Vaa), alfalfa strain, which causes a 

1988. A total of 36 top visible dewlap leaves from 3 vascular wilt of alfalfa, has been found in San Bernardino, Los 

replications were examined per cultivar for disease severity Angeles and Riverside counties and in the extreme south side of 

and host response. Significant differences (p < 0.05) were Kern county, but not in the Central Valleys. Optimal temperdetected 

among cultivars in both disease severity and response. atures for growth were 21 C and 24 C for different isola 

Cultivars were classified as being resistant, and moderately or Although maximum temperature for growth was 30 C, V. albohighly 

susceptible, exhibiting uredia with profuse sporulation atrum was isolated from infected plants in August when air 

and a mean disease severity of 39%. CP72-1210, CL73-239, temperatures were over 38 C. Pathogenicity of several isola 

CP7O-1257, and CP74-2005 were classified as moderately was tested on alfalfa by root dip inoculation (8 x 106 spores! 

susceptible with spore-producing uredia frequently being ml); all isolates reproduced wilt symptoms and Vaa was reisoobserved. 

Resistant cultivars exhibited only necrotic or lated. Germ plasms from southern California and the nondormant 

chlorotic flecking. Based upon these results, over 64% of cultivars CUFl0l, UC Cibola and Moapa 69 were susceptible; 

Florida's sugarcane acreage is currently planted to cultivars semidomant cultivar Vernema (Peaden, USDA-ARS) was moderately 

of moderate or high susceptibility, resistant and Vertus (France) was resistant. Cowpea (a new 

host) and cantaloup were highly susceptible. Beans, garbanzo 

533 beans, and peas were symptomless hosts. 

Use of phytotoxic components from Septoria nodorum and wheat 

callus cultures for in vitro germplasm screening. S. Leath and 537 

K. E. Papke, USDA-ARS, Department of Plant Pathology, 

Carolina 

North 

State University, Raleigh 27695-7616. AN EFFICIENT METHOD FOR INOCULATION OF SUBTERRANEAN CLOVER 

In an attempt to exploit possible somaclonal variation for WITH CERCOSPORA 

R. G. 

ZEBRINA 

Pratt, IN 

USDA, 

THE GREENHOUSE 

ARS, 

AND 

P.O. 

FIELD. 

resistance to 

Box 

Septoria 

5367, Miss. State, 

leaf 

MS 39762 

and glume 

culture 

blotch 

based screening 

of wheat, 

procedure was 

a 

developed. 

tissue 

Crude extract A mixture 

(CE) 

of 

was 

wheat 

obtained 

and oat 

from 

grains 

3-wk-old 

in flasks 

fungal 

was 

broth 

inoculated 

cultures 

with 

of two hyphal 

Septoria 

fragments 

nodorum 

of Cercospora 

isolates with 

zebrina 

three 

and 

successive 

incubated 

ethyl 

for 

acetate 

2 wk at 

25-30 C. 

extractions; 

The infested 

activity 

substrate 

was 

(WO) 

similar 

was 

to 

removed, 

that of 

air-dried, 

purified mellein. fragmented by 

Immature 

blending 

embryo 

while dry, 

callus 

and 

of 

sieved. 

six wheat 

Particles 

cultivars 

that 

resistance to S. nodorum was cultured on a modified ranging 

Murashige in passed terranean through clover a 40-mesh (Trifolium screen subterraneum) were dusted sprayed onto leaves with a of sticker. sub- 

and Skoo (MMS) medium with 1.0 mg 2,4-D/I. Cultures 

not show 

that 

leaf 

did 

or root differentiation 

Pat eeicbtdi 

after approximately 

auae 

two 

topeefr4dy 

subnsweque intly 

n 

in te amin saiuratda for shr 4- 

w 

days 53 

e e k s w 

at . 

e r e t r a n 

necoi 

s fe r r e d to m e d ia a m e n d e d w i t h CE. S in g l es u s 

challenges 

u e t y 

at 

n a m 

1000, 

i t 

750, 

a r f o 

and 

4 

500 

- d 

ppm 

y s 

and 

t 2 5 

repeated 

3 C . 

challenges 

N c t c 

at lesions that developed on leaves 

250 and 

were 

500 

identical 

ppm were 

to 

utilized 

those 

and 

ohthen 

cultures were transferred tamned by inoculations with 

to Ealutio MS 

spore 

wthot C. 

suspensions, 

of ermlas obaind 

and C. 

wth 

zebrina 

seetionM wtehoiut C.Eauationg s 

oth 

pofgermplasm 

was reisolated. 

obtaineaed 

Numbers 

wiath 

of lesions 

bot 

and percentage 

leaf tissue 

necrosis 

increased 

of 

slctioentechniun 

with inoculum 

esr 

concentration 

snwpoeyarmreeeatdpansi 

except 

at high levels. Resistant and susceptible reactions of 

currntlyundeway.cultivars 

were expressed with WO inoculum as with spore 

suspensions. Whole infested WO applied to field plots induced 

54 symptoms as efficiently as naturally infested plant debris. 

AN IN-VITRO TEHIQUE FOR SCREENING ALFALFA SEEDLINGS FOR 538 

RESISTANCE TO PHYTOPHTHORA ROOT ROT. M. J. Horstman and 

R. B. Carroll, University of Delaware, Newark, Delaware, INFECTION AND DEVELOPMENT OF THE SMUT PATHOGEN USTILAG0Q LYN- 

19717-1303 and E. R. Jones, Delaware State College, Dover, THERIsMAE IN LARGE CRABGRASS. 0. A. Johnson and A. B. A. N. 

Delaware 19901. 

Baudoin, Dept. of Plant Pathology, Physiology, and Weed 

Science, VPI&SU, Blacksburg, 

An in-vitro 

VA 24061. 

technique for 

sistance 

screening 

to 

alfalfa 

root rot 

seedlings 

caused 

for 

by Phytophthora 

re- 

megasperma f. sp. Ustilago svntherismae, 

medicaginis 

which causes 

(Pmm) 

loose 

was 

smut 

evaluated. 

of large 

Microbe-free 

and 

germlings of smooth crabgrass, infects 

Agate, 

its host 

WL-318, 

systemically 

Iroquois 

and 

and 

prevents 

Saranac were grown aseptically in seed production. 

test 

In the 

tubes 

field, 

containing 

diseased 

an 

plants 

inverted 

were 

filter 

usually 

paper cone moistened not observed until late 

with 

in 

Hoagland 

the growing 

solution. 

season, 

Tubes 

well 

were 

after crab- 

placed in growth chambers grass had commenced 

in a randomized 

flowering. 

complete 

Greenhouse 

block 

experiments 

design with 10 

were 

replications, carried out to determine 

After 

the 

4 

mode 

weeks 

of 

seedlings 

infection, 

were 

the 

inoculated 

disease 

with zoospore suspen- incidence that can be obtained by artificial inoculation, and 

1202 PHYTOPATHOLOGY

the reasons for the late observance of the disease. _U. syn- was 0, 3, 48, 80, 68, 65, and 3 percent, respectively. 

therismae infected large crabgrass (Digitaria sanguinalis) by The optimum temperature for germination was 20C in two 

both inoculation of seed (vacuum-infiltration with a suspension experiments, but a previous study with a different isolate 

of teliospores or dusting with dry teliospores) and application gave equally good germination at 24C. Acid rain solutions 

of teliospores to the surface of potting mix containing germi- with pH 2.0, 3.0, 4.0, 5.0, and 5.6 were incorporated 

nating seeds. Disease incidence was high (80-100%) in some in 2% agar plates, seeded with conidia and incubated 24 

treatments. Emergence of the inflorescences of infected plants hr at 24C. Spore germination was 0 at pH 2.0, but did 

was delayed by 2-5 weeks compared to those of healthy plants. not differ significantly over the range of pH 3.0 to 5.6 

(mean = 53%). Conidia in sterile water (pH 5.6) failed 

to germinate after 5 d, but when transferred to WA, 49% 

of these germinated within 24 hr. 

539 

YIELD RESPONSES OF SOYBEANS TO FUNGICIDES APPLIED DURING 

VEGETATIVE GROWTH STAGES. P. A. Backman and J. C. Jacobi, 

Dept. of Plant Pathology, Auburn University, AL 36849-5409. 

543 

VARIATION IN MUTANTS OF SEPTORIA GLYCINES INDUCED BY ULTRA- 

Disease severity of anthracnose and Septoria brown spot and 

yield response of soybeans to benomyl applications were 

evaluated in replicated field trials during 1987 and 1988. 

Each treatment received a single benomyl application during a 

different week of the vegetative period. Responses were 

compared to treatments receiving benomyl during the currently 

recommended reproductive growth stages. Yields were 

significantly increased in each of the tests, though disease 

control did not always correlate with yield. Other diseases 

such as the Diaporthe-Phomopsis complex may also have been 

affected, but were not rated. These results offer the 

possibility of altered timings to control midseason diseases, 

and additionally indicate that suppression of latent infections 

caused by the D-P complex or anthracnose during vegetative 

growth stages may result in yield improvements similar 

to those seen from recommended reproductive stage sprays. 

VIOLET IRRADIATION. H. S. Song and S.. M. Lim. Dept. of Plant 

Pathology, University of Illinois, Urbana, IL 61801. 

A single spore isolate of Septoria glycines was grown on potato- 

dextrose agar plates and irradiated with ultra-violet light (260 

nm) for 7 mins at 24 C. Approximately 0.4% of the irradiated 

spores survived and exhibited morphological, physiological and 

pathogenic variation. Examples of variation included hyaline 

spores, a leucine auxotroph, and loss of virulence on soybean 

cv. Williams. Some avirulent mutants were identical to the wild 

type isolate except for pathogenicity. Culture filtrates of the 

avirulent mutants did not exhibit pathotoxicity when cotyledons 

of Williams were bioassayed whereas culture filtrates of the 

wild type isolate exhibited pathotoxicity. Also, soybean callus 

growth, which was inhibited by culture filtrates of the wild 

type isolate, was not inhibited by culture filtrates of the 

avirulent mutants. Thus, pathogenicity of S. glycines appears 

540 

to be related to pathotoxin production. 

IMPACT OF PECAN SCAB AND PECAN LEAF BLOTCH ON LEAF GAS 

EXCHANGE OF PECAN. A. B. Gould, J. H. Aldrich, and P. 

C. Andersen. University of Florida, AREC, Rt. 4 Box 

63, Monticello, FL 32344. 

544 

ASCOKEY - A COMPUTER SOFTWARE PROGRAM FOR IDENTIFYING GENERA OF 

ASCOMYCETES. R.T. Hanlin and D.D. Pope, Department of Plant 

The influence of pecan scab (Cladosporium caryigenum) Pathology, University of Georgia, Athens, GA, 30602. 

and pecan leaf blotch (Mycosphaerella dendroides) was ASCOKEY was developed to assist mycologists at all levels to 

assessed on leaf gas exchange and chlorophyll 

concentration of pecan (Carya illinoensis) ova. 'Cape 

Fear' and 'Choctaw'. Scab lesions occupying ca. 5% of 

the surface of expanded summer-flush leaves reduced 

net CO2 assimilation (A) and transpiration rates (E), 

leaf conductance to water vapor (gl) and chlorophyll 

concentration (Chl) by 20 to 55% for both cultivars. 

Leaf blotch lesions covering 50% of the expanded 

summer- and spring-flush leaf surfaces decreased A by 

65 to 85%, but had a lesser impact on E, gl, and Chl. 

Intercellular CO2 concentration increased and Chl 

decreased in leaves affected by either disease. These 

data indicate that pecan scab and leaf blotch have a 

direct impact on the photosynthetic apparatus. 

identify important genera of ascomycetes. The software is coded 

in PASCAL and is completely menu driven; it can be run on any 

IBM-compatible computer. It currently contains keys and 

descriptions to 100 genera, but it can be readily expanded. The 

program contains three main modules. The first module consists 

of a dichotomous tree structure containing appropriate couplets 

which the user traverses to reach a particular genus. This key 

is also reversible. The second module provides a synoptic key 

for identifying genera on the basis of morphological 

characteristics. The third module contains descriptions of each 

genus; these descriptions can be accessed from either key or 

directly from the opening menu. Illustrations of each of the 

100 genera are available in the published version. The key will 

be a useful aid both for training mycology students and for field 

mycologists. The key is under copyright to APS Press, which is 

541 

also publishing the book on which the key is based. 

DETECTION OF ISOLATE DIFFERENCES IN PERONOSPORA TABACINA UTILIZING 

A CORESTA LEAF DISK BIOASSAY. M. D. Wiglesworth, W.C. Nesmith, C.E. Main, 

M.R. Bonde, G.L. Peterson, and M.R. Siegel. University of Kentucky, Lexington, Kentucky 

40546; North Carolina State University, Raleigh, N.C.; and USDA, Ft.Detrick, Md. 

Isolates of the blue mold pathogen, Peronospora tabacina, were maintained in liquid nitrogen at 

the University of Kentucky and the U.S.D.A. laboratory at Ft.Detrick, Maryland. This collection 

included domestic (Texas 1983-1988, Kentucky 1985-1987, and western North Carolina 

1986-1987) and international (Mexico 1987 and Bulgaria 1988) isolates of P. tabacina. To detect 

whether differences existed between these isolates, a leaf bioassay was developed utilizing 

cultivars of the CORESTA trap collection, acetone-dipped leaf disks (4mam in diarneter), and 

flourescent microscopy. Measurements of sporangiospore germination and penetration on inoculated 

disks indicated that isolates of domestic origin were similar within a particular year of 

collection. Comparison between years indicated a significant difference (PR>0.0001)arnong 

domestic isolates. Within a particular year, comparisons of locations indicated the isolates collected 

in Texas and Kentucky were similar in the years 1985-1987. Isolates collected from 

North Carolina were similar to the Texas and Kentucky isolates in 1987. Mexican and Bulgarian 

isolates were significantly different (PR>'0.0001) from all other isolates. For each of the 

isolates, significant (PR>0.O001) cultivar/ isolate interaction was observed with the nine cultivars 

of the CORESTA collection. These data are consistent with the hypothesis that the Texas 

isolates may be the source of inoculurn for epidemics in Kentucky and North Carolina. Differences 

noted in the foreign isolates may indicate the presence of different fungal stralins, 

545 

RHIZOCTONIA SPECIES ASSOCIATED WITH BEDDING PLANTS, 

NURSERY PLANTS AND SOME VEGETABLE CROPS IN SOUTH 

AUSTRALIA:IDENTITY AND PATHOGENICITY. D.A. Schisler, S.M. 

Neate, and G. Masuhara, CSLRO Division of Soils, Adelaide. 

Diseased plants and soil were collected from 30 nurseries and four vegetable 

crops in South Australia. Forty-nine isolates of Rhizoctonia were obtained from 

diseased plants, soil and soil baited with seedlings of wheat, radish, brussel 

sruonmna uizni n elppe.Tev sltswr 

sruonmna uizni n elppe.Tev sltswr 

multinucleate and 37 binucleate. When the multinucleate isolates were tested for 

anastomosis with isolates from AG-i to AG-8, four were AG-2- 1, seven were 

AG-4 and one could not be identified. All isolates were then grouped according 

to origin and morphology and representatives were fruited by the soil over agar 

method. Five isolates formed the Thanatephorus cucumeris teleomorph, six were 

Ceratobasidiurm cornigerum, one was Ceratobasidium pseudocornigerum and one 

was a Ceratobasidium sp. Electrophoretic patterns of pectic enzymes produced 

by the isolates showed differences between genera and species. Isolates were 

tested in growth cabinets for their pathogenicity on a representative from the 

Grarnineae, Cruciferae, Leguminosae, Compositae and Solanaceae. Host plant 

and temperature significantly influenced the pathogenicity of isolates. 

542 

TEMPERATURE, PH AND FREE WATER EFFECTS ON IN VITRO GERMINATION 546 

OF CONIDIA OF A DISCULA SP. ISOLATED FROM DOGWOOD ANTHRACNOSE 

LESIONS. Kerry 0. Britton, USDA Forest Service, SEFES 

Carlton St., Athens, GA, 30602 

RELATIONSHIP OF CELL WALL COMPONENTS TO QUIESCENT INFECTION OF 

STRAWBERRY. W. S. Conway, USDA, ARS, Hort. Crops Quality Lab, 

Beltsville, MD 20705, B. 0. Bruton, USDA, ARS, SCARL, Lane, OK 

In vitro germination of a Discula sp. isolated from dogwood 74555, K. C. Gross, USDA, ARS, Hort. Crops Quality Lab, and 

anthracnose lesions from north Georgia was tested. Conidia J. L. Mass, USDA, ARS, Fruit Lab, Beltsville, MD 20705. 

were sprayed on pH 5.6 water agar (WA), and incubated 

24 hr at 5, 10, 15, 20, 25, 30, and 35C. Mean germination In order to determine the possible effect that various cell 

Vol. 79, INo 101989 :l~~ 12031"

wall components have on quiescent infection of strawberry phaseoli by homology with the heterologous cDNA. The 3' region 

caused by B. cinerea, cell walls were extracted from fruit of the F.solani gene has been sequenced. It revealed an intron 

harvested at different stages of maturity from several of 48 bases. The 5' and 3' splice sites as well as internal 

strawberry cultivars. Cell wall neutral sugars rhamnose, consensus sequences of the intron matched well with those of 

arabinose, galactose, xylose, mannose, and glucose all Neurospora crassa. The 3' region of the gene showed a very 

decreased by varying amounts on a g fresh wt basis as the fruit high degree of homology (86%) to the EIN cDNA of F.oxysporum. 

matured. Both total calcium and cell wall bound calcium The promoter of this highly expressed gene will be used to 

decreased with maturity on a g fresh wt basis as well. Since construct a transformation vector for Fusarium spp. 

both sugar and calcium content of the host tissue have been 

shown to affect fungal growth and pectolytic enzyme production 

and activity, the possible relationship that these cell wall 551 

components have to quiescent infection will be discussed. IMPROVED TRANSFROMATION OF COLLETOTRICHUN GRAMINICOLA 

PROTOPLASTS AND ITS USE IN CLONING THE PYRI GENE. 

J.B. Rasmussen, D.G. Panaccione, and R.M. Hanau. Dept. of 

547 Withdrawn Botany and Plant Pathology, Purdue University, West Lafayette, 

IN 47907 

Transformation of C. graminicola was improved by adding 

spermidine (8 mM) and heparin (I pg/pl) to DNA prior to 

transformation and by osmotically stabilizing the polyethylene 

glycol with KCl (600 mM). With these modifications, over I000 

benomyl-resistant transformants were recoverd per pg of pCG7 

DNA which encodes a mutant Q-tubulin gene from the fungus. 

The procedure was used to screen a C. graminicola cosmid 

library for sequences that would complement C. graminicola 

strain M2001 which is a uracil auxotroph isolated by selection 

for resistance to 5-fluoroorotic acid. PYRI, the gene 

responsible for complementing the mutation in M2001, was 

rescued and subcloned as a 3 kb Hind III/Sal I fragment 

creating vector pJR70. Transformation with pJR70 yields in 

excess of 1000 PYR+ transformants per pg of plasmid. 

552 

EXPRESSION OF MBC (BENOMYL) RESISTANCE IN ASPERGIFLUS FLAVYJS 

TRANSFORMED 548 WITH A HOMOLOGOUS B-TUBULIN GENE. E. R. Seip, C. P. 

Woloshuk, G. A. Payne and C. R. Adkins. Dept. Plant Pathology, 

CLONING AND SEQUENCE ANALYSIS OF GENE(S) ENCODING O-TUBULIN IN North Carolina State University, Raleigh, 

THE Gold HEMIASCOMYCETE and 

NC 

N. 

27695. 

T. Keen. PLANT Dept. PATHOGEN of Plant GEOTRICHUM Pathology, CANDIDUM. University S. E. of Aspergillus flavus was transformed 

California, 

with plasmid 

Riverside, 

pBRG4 

CA 92521. 

containing tubulin gene both from the pyr4 gene of Neurospora crassa 

Sequences A. and 

homologous 

flavus conferring resistance to 

a B- 

to 8-tubulin were cloned with the goal of MBC (the 

active uracil component prototrophy 

of at beWnomyl). a frequency Transformants of 15 per ug were transforming 

obtaining taxonomic 

selected for 

data and developing a transformation system 

for the hemiascomycete 

DNA. Trtytrormat 

Geotrichun candidun 

wereqteste 

(G.c.). 

for 

Additionally, 

resisanctornB 

wild 

DNA. Thirty transiormants 

inudrtnigtesrcuryfteegns type G.c. is resistant were 

to benomyl 

tested 

adding 

for aesistance 

to the interest 

to MBC1 

in 

Six 

understanding sn were as sensitive 

the 

h as 

structure 

ug/ml MBC), 19 were intermediate the recipient 

of (grew on strain 

these 1 or (sensitive 

genes. Using 2.5 ug/ml to 

the and 5 were resistant 

MBC) 

cloned Neurospora crassa Tub (Bnr) 

to 5 ug/ml 

from 

MBC. 

plasmid 

Vector 

pSV5G 

DNA 

, 

appeared 

two 

to 

groups of 

integrate 

clones 

at 

were 

the 

isolated 

homologous 

from 

locus 

an 

in 

EMBL3 

all 

G.c. 

phenotypic 

genomic 

classes. 

library. 

Selection 

The two 

of 

groups 

transformants 

differed 

on 

in 

0.75 

intensity 

ug/ml MBC 

of 

resulted 

hybridization 

in an 

signal and restriction 

average frequency 

pattern. 

of 2.8 

A 

per 

2.8 

ug 

kb 

transforming 

fragment including 

DNA. The lower 

550 base pair 

frequency 

(bp) 

as 

5' 

compared 

and 600 

to 

bp 

uracil 

3' to 

selection 

the coding 

was 

region 

presumably 

has 

caused 

by the failure to select 

now 

transformants 

been sequenced from 

that 

one of these 

were 

clones. 

sensitive 

Salient features 

o 

relevent 

inter ea te 

to 

in 

taxonomy 

sel e t o a w 

will 

e i 

be presented. The second group of intermediate in 

clones 

resistance 

has yet 

to NBC. 

to be characterized. Transcriptional 

using 

fusions 

noncoding regions of the sequenced 0-tubulin 

flank 

gene 

antibiotic 

to 

resistance 

a 

gene(s) 

transformation 

are being 

system 

tested 

for 

to 

the 

develop 

fungus. 

553 

ISOLATION AND FUSION OF PROTOPLASTS FROM FUSARIUM 

OXYSPORUM f.sp CONGLUTINANS AND f.sp RAPHANI 

5E.A. Momol, F. N. Martin, and H. C. Kistler. Plant Pathology 

University 

Department, 

of Florida, Gainesville, FL. 32611. 

PROTOPLAST 

TUM. 

FORMATION 

Dr) Griffin, M. DeVit, 

AND 

and R. 

TRANSFORMATION 

Tuor. SUNY, College 

OF 

of 

HYPOXYLON 

Environmental MAMMAand 

Forestry, Syracuse, NY 13210. Science Protoplasts were obtained from strains of two formae speciales 

wilt 

(f.sp) 

pathogen 

of the 

Fusarium oxvsporum using cell wall degrading preparation 

A transformation system for Hypoxylon mammatum has been accomplished using Novozyme 

protoplasts 

234. Protoplasts 

obtained from 

of 

mycelium 

isolates 

of 

722 

40 hr 

(f.sp 

cultures 

conglutinans, 

in defined 

a 

medium 

cabbage 

inoculated 

with 

pathogen) 

mycelial fragments. 

and 724 (f.sp 

Protoplasts 

raphani 

were 

pathogenic 

released 

of 

using 

radish) 

Novozym® 

were 

234 

labelled 

in 1 .2M 

with 

MgSO 

fluorescein isothiocyanate and hydroethidine, respectively. 4 . Addition of 8-mercaptoethanol increased the yield of protoplasts. Proto- 

Labelled 

protoplasts 

plasts were 

were 

purified 

fused 

from 

in 

hyphal 

the presence 

fragments with 

of 

a 

PEG 

sorbitol 

and 

step 

sorted 

gradient, 

based 

but 

on 

sorbitol 

size 

the regeneration 

in 

medium 

and fluorescence 

was inhibitory, 

using 

as were 

flow 

the 

cytometry. 

osmoticants 

Regeneration 

mannitol and KCl. 

frequency of 

forma 

inter- 

Optimal viability of 20-25% 

.specialis 

was obtained 

fusion 

with 

products 

sucrose 

was 

or glucose 

approximately 

at 0.8M with 

0.5%. 

bovine 

Mitochondrial 

nuclear 

and 

backgrounds 

serum 

and the 

albumin 

presence 

treated 

of mitochondrial 

protoplasts. 

plasmids 

Transformation 

in fusion 

CaCl2 in the presence 

efficiency 

of aurantricarboxylic 

was optimal 

acid. Stable 

at 

transformants 

50 mM 

were obtained products were examined 

using pBT6 

using 

carrying 

DNA probes 

the Neurospora 

specific for 

crassa 

each 

benAr 

forma specialis. 

gene. Characterization 

transformants 

of the 

will 

Fusion 

be discussed. 

products were obtained having non-parental combinations of nuclear 

and mitochondrial markers; the influence of this on host specialization will be 

550 

determined. 

554 

CLONING AND CHARACTERIZATION OF THE EIN GENE OF FUSARIUM ELECTROPHoRETIC CONFIRMATION OF CHROMOSOME REARRANGEMENTS IN 

SOLANI f.sp. PHASEOLI. G.H. Choi, E.T--- Marek, C.L. Schardl COCHLIOBOLUS .HETEROSTROPHUS - -- TRANSLOCATION AT TOXl. Tzeng. 

and D.A. Smith, Department of Plant Pathology, University of T., H. Chang, C. R. Bronson, Department of Plant Pa-thology, 

Kentucky, Lexington, KY 40546-0091. 

Iowa State University, Ames, IA 50011. 

The EIN gene of Fusarium solani f.sp. phaseoli and We are confirming linkage group assignments in our RFLP map of 

Fusarium.oxysporum f.sp. cucumerinum is induced by various C. heterostrophus by hybridizing the probes used for 

stress factors, including ethanol. Previously, a cDNA clone of constructing the map to electrophoretically separated 

EIN mRNA from F. o xysporum was sequenced. The EIN gene was chromosomes. To date, several differences in chromosome 

isolated from a cosmid genomic library of F. solani f.sp. arrangement between the isolates used to make the map have been 

1204 PHYTOPATHOLOGY

verified. A translocation linked to Toxl has also been appearance of A 2 detected. To determine the tightness of this linkage, probes 

around Toxl were hybridized to chromosomes of near-isogenic 

race T (TOX_ ) and race 0 (toxl) strains. Results support our 

hypothesis that race T and race 0 differ by a reciprocal 

translocation with its breakpoint at or near Toxl. 

mating type of P. infeatans in Europe may have 

resulted from the com mercial application of metalaxyl for control 

of late blight of potato and tomato in that region. 

59 

A TECHNIQUE FOR PRODUCING PROTOPLASTS AND RECOVERING INTACT CELLS 

FROM THE PATHOGENIC FUNGUS USTILAGO HORDEI. K.E Duncan and D.D. 

Pope, Department of Plant Pathology, University of Georgia, 

Athens, GA 30602. 

A technique was developed to obtain high yields of Ustilago 

hordei protoplasts and to recover intact cells from protoplasts 

as a prerequisite for transformation experiments. Protoplasts 

were obtained from log-phase, haploid, sporidial cells by 

degrading fungal cell walls with a mixture of enzymes (Novozyme- 

234) dissolved in a KC1 buffer solution. After 24h treatment 

with the enzyme solution, no intact cells were found; only 

protoplasts remained. Healthy colonies with intact cell walls 

were recovered with an efficiency of 5.6% by incubating 

protoplasts in liquid complete medium amended with KC1 prior to 

plating on nonamended complete medium. Efficiency of 

regeneration of cell walls was measured by comparing colony 

counts from KCl incubated protoplasts with counts from 

protoplasts incubated in water. Recovery of healthy colonies was 

fivefold greater for KCl incubated protoplasts versus the water 

treated control. 

EVIDENCE FOR HORMONAL REGULATION OF SEXUAL 

REPRODUCTION IN PYTHIUM SPLENDENS. L. Y. Guo and W. 

H. Ko, Department of Plant Pathology, University of 

Hawaii, Hilo, Hawaii 96720 

When + and - mating types of Pythium splendens were 

paired on opposite sides of a polycarbonate membrane 

(0.2 um, 90 mm diam.) for 6 days at 24 C in 

darkness, + but not - isolate produced oospores by 

selfing. When newly inoculated (O-hr) culture of - 

isolate was paired with 0-, 6-, 12- or 24-hr culture 

of + isolate, the 12-hr culture of + isolate 

produced the largest amount of oospores. The - 

isolate did not produce oospores in any of the 

experiments involving different age combinations. 

Our results show that sexual reproduction in P. 

splendens requires hormone produced by - isolate to 

initiate oospore formation by + isolate. This 

represents a new phenomenon of hormonal 

heterothallism in the biological world. 

560 

MOLECULAR CHARACTERIZATION OF A B-TUBULIN GENE FROM ERYSIPHE 

556 

GRAMINIS. John E. Sherwood' and Shauna C. Sommerville'. 'Dept 

Plant Pathology, Montana State Univ., Bozeman 59717, and DOE- 

Plant Research Lab, Michigan State Univ., E. Lansing 48824 

OCCURRENCE OF DNA PLASMIDS IN ISOLATES FROM ANASTOMOSIS GROUPS 

(AG) 2,3,4, AND 5 OF RHIZOCTONIA SOLANI. T. Syminis and S.M. 

Tavantzis, Department of Botany and Plant Pathology, University 

of Maine, Orono, ME 04469. 

Fourteen Rhizoctonia isolates members of AG-4 were examined for 

the presence of DNA plasmids. Five isolates were found to contami 

two plasmid bands which were associated with the mitochondrial 

fraction. The buoyant density of these molecules was 

lower than that of the nuclear DNA of R. solani. These plasmids 

were sensitive to pancreatic deoxyribonuclease I and resistant 

to pancreatic ribonuclease A, lambda exonuclease and 

exonuclease III. Southern blot hybridization analysis indicated 

the presence of homologous sequences between the two plasmids 

within an isolate and plasmids in the other AG-4 isolates. 

There was no homology between the plasmids and nuclear or mitochondrial 

DNA. The presence of the above plasmids in AG-4 

isolates is not correlated with a certain phenotype. Furthermore, 

this study showed that a number of different DNA plasmids 

are present in R. solani isolates from AG-2,-3, and -5. 

A B-tubulin gene from Erysiphe graminis f.sp. hordei, an 

obligate fungal pathogen of barley, was cloned from a genomic 

lambda library prepared from conidial DNA, using the Neurospora 

crassa tub-2 gene as a probe. The E. graminis structural gene, 

which encoded a polypeptide of 446 amino acids, had six introns 

with positional and splice sequence homologies to tub-2 from N. 

crassa. The predicted amino acid sequence also showed a high 

degree of homology with other fungal B-tubulins. A major 

difference between the 1. graminis gene and the majority of 

other fungal B-tubulin genes was the lack of codon usage bias 

observed with the E. graminis gene, in which 60 of the 61 sense 

codons were used. While N. crassa tub-2 hybridized to only one 

sequence of the 1. graminis genome, the j. graminis 8-tubulin 

clone hybridized to an additional genomic sequence. This result 

suggests that there is a second 8-tubulin gene whose sequence 

diverges considerably from that of the N. crassa gene. 

56 

ISOZYME PHYLOCENETIC RELATIONSHIPS AMONG ANAST(IMOSIS AND INTRA- 

SPECIFIC GROUPS OF RHIZOCTONIA SOLANI KUEHN. Z. Liu, D. L. 

557 

OCCURRENCE OF MITOCHONDRIAL 

Nickrent, and J. B. Sinclair, Departments of Plant Pathology 

and Plant Biology, University of Illinois, Urbana, IL 61801 

PLASMIDS IN ENDOPHYTIC FUNGI. K.L. 

Mogen, C.L. Schardl, and M.R. Siegel. Department of Plant Pathology, University of 

Kentucky, Lexington, Kentucky 40546-0091. 

Protein extracts of mycelium from 28 isolates of Rhizoctonia 

solani belonging to 12 anastomosis (AG) and intraspecific (ISG) 

Acremonium spp., sect. Albo-lanosa, are fungal symbionts of various grasses. They are 

.closely related to, and in some hosts the anamorph of, the choke pathogen Epichloo" 

;and typhina insect (Clavicipitaceae). resistance, as well Grasses as more infected vigorous with growth, the endophytes compared 

exhibit 

to non-infected 

greater drought 

plants. 

We are studying the genome structures and gene expression in the endophytes. Total and 

snitochondrial DNAs from several isolates were analyzed. The mitochondria of one E. 

syphina isolate (from Lolium perenne) contained at least three plasmids with sizes of 7.5 

kb, 2.1 kb and 2.0 kb. These three plasmids are linear DNA species with no detectable 

somology to the mitochondrial chromosomes. Interestingly, all other isolates of E. 

!yphina and the various Acremonitsm spp. contained one or both of the smaller plasmids. 

Exonuclease digests showed that they were double-stranded and had blocked 5'-termini. 

P'lasmid sequence analysis is underway, 

groups (AG-l, AG-2-1, AG-2-211IB, AG-2-21V, AG-3, P13-4, AG-5, 

P1-6, PC3-7, 143-8, AG-9 and AG-BI) were analyzed electrophoret- 

ically for isozyrre variation. Eleven enzyme systems were used: 

ACO, ACP, EST, GPI, GSR, HXK, 1DM, LAP, MDH, P(I, anrld 6-PGD. 

For each enzyme, bands with the same relative nubility were 

treated as distinct characters, hence the metrix reflected 

the presence or absence of each enzyme variant. Relationships 

amrong all ISGs were inferred using PAUP. Each ISG occupied a 

separate position on the cladogram -which provided an indication 

of their genetic distinctiveness. Within each ISG, representative 

isolates clustered together regardless of geographic 

origin. Isolates from PA3-2-1 and PAG-2-2 clustered separately. 

558 

INDUCTION OF MATING TYPE CHANGE IN PHYTOPHTHORA 

Within P13-2-2, AG3-2-2III8 and AG3-2-2IV were distinguished by 

this isozyme analysis. 

INFESTANS BY METALAXYL. T. T. Chang and W. H. Ko, Department 

of Plant Pathology. University of Hawaii, Hilo, Hawaii 96720 

15 AILBLN FSIOLSACTISRAEPOEN 

l"~RDOAEIGO PRPAM IR UFC RTIS 

Fletcher, Department of Plant Pathology, Oklahoma 

J" 

State 

laolates of P. infestans from vacious sources were grown on University, Stillwater, OK 74078-9947. 

medium containing 0, 20, or 50 ug/ml metalaxyl to determine if 

this fungicide can cause mating type change. Among 70 At and 53 

A 

Recently we used proteases and surface immunoprecipitation to 

2 

tested, 1 A 1 and 1 A 2 formed oospores in sectors after growing 

on medium containing 20 or 50 ug/ml metalaxyl for 6 wk, 

indicating the appearance of the opposite mating type. The teat of 

the isolates tested did not form oospores after 3 ,months on the 

same medium. Single-zoospore cultures obtained from the oospore 

sector of isolate 902 (A 

identify five surface proteins of the wall-less mollicute S_. 

citri (Phytopathology 77:1726). Only one of these, spiralin 

(29 KDa), was previously shown to have a surface location. In 

the present study, radiolabeling was used to enhance detection 

of surface proteins, Surface-exposed proteins of 5. citri 

1 ) consisted of 108 A 1 , 61 A 2 and 1 A 1 A 2 , were iodinated using Na 1 2 5 and those obtained from the oospore sector of isolate 920 (A 

I and Iodobeads. Autoradiograms of 

2 ) 

consisted of 47 A 

labeled proteins had fewer bands than did profiles of total 

t and 113 A 2 . Our results suggest that recent cell protein. When surface-iodinated spiroplasmas were used 

562) 

Vol. 79,kl INo 10,1989 1205

for surface immunoprecipitation, autoradiograms showed twelve MLOs; nor with Spiroplasma citri or S. kunkelli. The two 

bands, five of which correspond to those of our previous probes hybridized with three type strains of Western AY-MLO, 

studies and seven additional polypeptides, which were two AY-MLO field isolates from northern California, and AY-MLO 

interpreted as surface proteins. 

strains from other geographic regions in the U.S. and Canada. 

Hybridization patterns of EcoRI or HindIII-digested DNA from 

many of the AY-MLO strains differed. Such RFLPsma buseful 

563 in characterization of geographical strains of AY ML 

INFLUENCE OF PRUNUS ROOTSTOCKS ON FEEDING PREFERENCE 

OF PHONY PEACH DISEASE VECTOR (HOMALODISCA COAGULATA) 567 

AND AMINO ACID CONCENTRATIONS IN XYLEM FLUID. W. JMYCOPLASMA 

J 

French, 16S 

J. 

rRNA 

H. Aldrich, 

SEQUENCE SHOWS THAT 

A. B. Gould, 

PLANT PATHOGENIC 

B. V. Brodfeck, R. 

F. Mizell, 

ORGANISMS" 

and 

ARE 

P. 

EVOLUTIONARILY 

C. Andersen. DISTINCT 

University 

FROM ANIMAL 

of Florida, 

MYCOPLASMAS. 

P.O. Limr and B.B. Sears1, 2 . IGenetics Program and 2 AREC, Rt. 4 Box 63, Monticello, 

Department 

FL 32344. of Botany and Plant Pathology, Michigan State University, 

Phony peach disease (PPD) is caused by the xylem East Lansing, MI 48824. 

limited bacterium Xylella fastidiosa (Xf) and vectored 

by the leafhopper Homalodisca coagulata. Although The plant pathogenic "mycoplasma-like organisms" (MLOs) are 

prevalent in the southeastern U. S., PPD has not been so-named because they lack cell walls. In order to establish 

observed in South America. Three rootstocks, domestic a definitive phylogeny, the 16S rRNA gene from a representative 

peach ('Nemaguard') and plum (1I-i'), and a peach of this group has been cloned and sequenced. Sequence 

rootstock from Brazil ('A-82'), were budded with comparisons indicate that this MLO is related to Mycoplasma 

'Flordaking' peach. The effect of rootstock on vector capricolum and that these two bacteria share their phylogenetic 

feeding preference was assessed during 1985 to 1988. origin with Bacillus subtilis. Furthermore, a low G+C content 

Xylem fluid amino acid levels were assessed in 1986 to of this gene and its presumed secondary structure indicate 

1988, and Xf concentrations were assessed in 1987 and that MLOs belong in the same class as the animal mycoplasmas, 

1988. Leafhopper counts and asparagine and arginine the Mollicutes. However, the presence of certain 

concentrations were rootstock dependent. Interactions characteristic oligonucleotides indicates a closer relationship 

among leafhoppers, amino acids and PPD are discussed, to another family of this order, the Acholeplasmataceae. 

565 propiconazole. 

568 

EFFICACY OF TRIAZOLE FUNGICIDES ON FOLIAR DISEASES COMMON TO 

WHEAT IN OKLAHOMA. E. Williams Jr., K. E. Jackson, and P. W. 

Pratt. Plant Pathology Department, Oklahoma State University, 


Winter wheat plots were established in north central and east 

central OK from 1984-1987 to determine efficacy of 

propiconazole and tebuconazole to foliar fungal infections. 

Plots were 2.4 X 9.1 m, replicated 4 times in randomized 

complete block. Fungicides applied in water by ground sprayer 

(187 1/ha) at growth stage 9 (Feekes scale). Cited significance 

represents LSD tests (P< 0.05). In 1987-88, significant 

reductions in Puccinia recondita f. sp. tritici severity 

resulted from both fungicides; however, tebuconazole provided 

significantly lower severities than propiconazole. Both 

fungicides significantly reduced Erysiphe graminis f. sp. 

tritici, Septoria tritici, Septoria nordorum, and pyrenophora 

tritici-repentis. Significant grain yield increases were 

obtained in 37.5% of tests for tebuconazole and 25% for 

DETECTION AND DIFFERENTIATION OF MYCOPLASMA-LIKE ORGANISMS 

USING MLO-SPECIFIC RIBOSOMAL RNA OLIGONUCLEOTIDE SEQUENCES. 569 

B. Pathology, C. Kirkpatrick University and J. of D. California, Fraser, Department Davis, CA of 95616. Plant 

ERADICANT AND PROTECTANT ACTIVITY 

BOTRYOSPHAERIA 

OF FUNGICIDES AGAINST 

OB[USA ON APPLE FOLIAGE. 

Computer-assisted comparisons 

L. 

of 16S 

F. 

ribosomal 

Arauz 

RNA (rRNA) 

and 

Sutton. 

T. 

Dept of Plant 

8. 

sequences of the 

Pathology, 

X-disease MLO 

North 

(X-MLO), 

Carolina 

plant 

State 

chloroplasts, 

and culturable Mycoplasmas 

University, 

were 

Raleigh, 

used 

27695-7616. 

to identify several oli- Reduction 

gonucleotide 

of radial growth 

sequences of Botryosphaeria 

which could be 

obtusa 

used 

was 

as specific 

deter- 

or mined 

general 

on 

probes 

fungicide-amended 

to detect MLO 

PDA. 

but 

EC 

not 

50 values 

plant nucleic 

(ug/ml) 

acids. 

were: 

One benomyl 

oligonucleotide (be), 0.032; 

(WXl) 

bitertanol 

hybridized (bi), 

with 

0.043; 

nucleic flusilazole 

acids from 

(fl), 

plants 

0.045; manoozeb (ma), 10.26; myclobutanil 

bunch infected with X-, aster yellows (AY), walnut/pecan 

(my), 

(W/PB) 

0.426; 

NLOs, and culturable Mollicutes. Another oligo- penconazole (te), 0.036. Six (pe), selected 0.132; pyrifenox fungicides (py), were 1.77; tested terbutrazole 

nucleotide (WX2) hybridized with nucleic 

on apple 

acids seed- 

from plants in- lings for protectant 

fected with 

and 

the 

eradicant 

X- end activity. 

WP/B-M4LOs When 

but not 

plants 

to culturable 

were 

Nollicutes 

or AY-MLO-infected 32 inoculated 

plants. 

7 days aftefugcdaplatomndias 

P-labelled WXl and WX2 sevenities (lesions/cm 

provided ) 500 

were: 

to 

be, 

800 times 

0.046; 

more 

bi, 0.046; 

sensitive fl, 

detection 

0.207; 

of MLOs 

when 

ma, 

used 

0.004; 

in rRNA:DNA 

pc, 0.116; 

as 

te, 

compared 

0.009; 

to 

control, 

DNA:DNA 

1.02. 

hybridizations, 

Percent leaf 

area diseased 

These results 

for fungicides 

suggest that 

applied 

MLO-specific 

48 hr after 

rRNA 

inoculation 

oligonucleotides was: be, 5.6; 

cen 

bi, 

provide 

4.2; fl1, 

very 

3.2; 

sensitive 

ma, 3.9; 

and 

pe, 

specific 

2.7; te, 

detection 

2.9. The 

of plant length of the period of eradicant activity to achieve a 

pathogenic 

2/3 

MLOs. 

reduction in disease severity as compared to the control was 30, 

45 and 48 hr for fl, te and pc, respectively. This reduction was 

not obtained with the other fungicides. 

IDENTIFICATION AND DIFFERENTIATION OF ASTER YELLOWS MLO STRAINS 570 

USING ASTER YELLOWS NLO-SPECIFIC DNA PROBES AND RFLP ANALYSIS. 

C. R. Kuske, B. C. Kirkpatrick, Department of Plant Pathology, PERSISTENCE OF FUNGICIDES FOR CONTROL OF ALTERNARIA 

University of California, Davis, CA 95616 LEAF BLIGHT OF MUSKQMELON. H. Suheri and R.X. Latin, Department 

of Botany and Plant Pathology, Purdue University, West Lafayette, IN 

DNA extracted from celery infected with the severe strain of 47907. 

aster yellows MLO (AY-NLO) was digested with EcoRI and BamHI, 

ligated into pUC18, and cloned in E. coli JM109. Two transformants, 

containing inserts of 2.0 kb (pAYC3) and 4.1 kb The objective of this research was to determine the relative persistence of two 

(pAYC4), were identified by DNA hybridization analyses as frag- fungicides, chlorothalonil and mancozeb that are used againstA. ai 

ments of the MLO chromosome. In Southern blot analyses of Eco- cucumerina, the causal agent of Ahternaria leaf blight of muskmelon. A 

RI-digested DNA from plants infected with a number of Molli- cellophane bioassay with A.cucumeiina as the test organism and inhibition of 

cutes, pAYC3 and pAYC4 did not hybridize with DNA from healthy spore germination as the response variable was used to determine persistence. 

plants; plants infected with elm yellows, western-X, or BLTVA Subsequent to treatment, plants were exposed to both wet (12 h) and dry 

1206 PHYTOPATHOLOGY

egimes and were sampled at 1, 3, 5, 7, and 10 days after fungicide 

application. A linear relationship between fungicide loss and time resulted for 

all treatments. Under the wet regime, initial loss and the rate of fungicide 

loss were significantly greater for mancozeb than chlorothalonil. No 

significant differences occurred under the dry regime where the bioassay 

575 

EXPRESSION OF STRIPE RUST RESISTANCE GENES IN DIFFERENTIAL 

EATRESSIOOFSR RUST ReSan Ce U.S .DeptIAL 

WHEAT CULTIVARS. R.F. Line, and X .n. Chen, U.S. Dept. of 

Agriculture and Washington State Univ., Pullman, WA 99164-6430 

showed a relatively slow decrease in effectiveness of both fungicides for 9 Seedlings of parents and Fl. F and BC progeny from crosses 

days. 

among differential cultivars were inoculated with specific 

races of Puccinia striiformis. Chinese 166, Heines VII, 

Yamhill, T. spelta alba, Fielder, Heines Kolben, Heines Peko, 

571 Lee, Compair, Riebesel 47/51, Clement, and Moro have genes 

EFFECTS OF FUNGICIDE SEEDPIECE TREATMENTS ON EMERGENCE AND_,r 

YIELD IN POTATO. D. J. allenberg, SDSU, Plant Science 

Department, Box 210--9,Brookings SD 57007. 

Lee, Compair, Rieee 47/51, Clement and Noro hav genes Y , 

-- 'Y 2 2Y5 Yr Yr =_ Yr Yr and2 Yr-. 7 , -r 8 , Y--=- 9 , -:-' 

anA Yr 10 , respectively. Druchamp, Stephens, and Yamhill have 

genes-sa the Yr or Yr locus. Lemhi, Tyee, Spalding Prolific 

Moro, Druchamp, Lee, Fielder, and Compair each have a newly 

Several fungicide seedpiece treatments were used in field identified gene. Paha has three genes. Yr, Yr, Yr, Yr 8 

trials during 1986 - 1988 to determine the effects of these Yr 9 ,Yr and -Yrl, the Lemhi gene, and one Paha gene were dominant. 

materials on plant emergence and tuber yield. Trials were Depending upon race that was used and/or parent in the cross, 

conducted at Watertown, SD (early planting) and Brookings, SD one Compair gene was either dominant or partially dominant, and 

(late planting), and utilized two cultivars each season. Yr Yr^, Yr., Yr, the Tyee gene, one Lee gene, and two Paha 

Stand counts were made approximately four weeks and six weeks genes were either recessive or dominant. Epistatic patterns 

after planting, and total tuber yield was determined at the also varied depending upon race and parent. 

end of the season. Yield differences between cultivars were 

evident at both locations in all three seasons. Within 

cultivars, there were no differences among treatments in 576 

stand count or yield in any season at Watertown. At 

Brookings, all fungicide treatments increased stand count and INHERITANCE OF LATENT PERIOD OF PUCCINIA RECONDITA IN WHEAT. 

yield over the check for one variety only in 1987 and 1988. Greg Shaner and George Buechley, Purdue University, West 

Lafayette, IN. 

Long latent period, a major component of slow leaf-rusting 

resistance in wheat, was studied in F 7 families derived from CI 

572 13227 x Suwon 92 There were highly significant differences in 

FUNGICIDE RESISTANCE IN BOTRYTIS CINEREA ISOLATES pROm PENNSYLlatent 

period among families. The distribution of F family 

7 

means was skewed toward long latent period, and the population 

VANIA GREENHOUSES. G. W. Noorman and R. J. Lease, Department of mean was significantly below the mid-parental value. These 

Plant Pathology, The Pennsylvania State University, University data, along with data from analyses of earlier generations from 

Park, PA 16802 CI 13227 x Suwon 92, suggest that three loci with epistatic 

effects control latent period. Because approximately three- 

Twenty Botrytis cinerea isolates from infected greenhouse floricultural 

crops in Pennsylvania were grown on a range of concentrations 

of benomyl, chlorothalonil, cupric hydroxide, mancozeb, 

thiophanate methyl + mancozeb, vinclozolin, and zineb in vitro, 

Five isolates were resistant to only benomyl and 14 were resistant 

to both benomyl and vinclozolin. Isolates with fungicide 

reitant toibothfeno e 

and vinozolin olateo ised wnitfun e 

resistance infected and sporulated on excised geranium 

fourths of the F families had values below the parental 

7 

midpoint, we reasoned that a gene at one of the three loci 

exerted a major effect on latent period. If the three loci are 

symbolized as A, B, and C, with the capital letter indicating 

the allele for short latent period, then A can be considered the 

major gene. B is considered to have greater effect than C in 

the absence of A. C in the absence of A and B has only a small 

effect. Genotype aabbcc conditions the long latent period of CI 

(Pelargonium) leaf disks that had been treated with the label 13227. 

rate of the fungicide to which they were resistant. Linear 

growth rates and sclerotium formation in vitro and sporulation 

in vivo, used as saprophytic and parasitic fitness parameters, 

were compared among isolates. 

577 

MAPPING LEAF RUST RESISTANT GENES, Rph3 AND Rph7, ON BARLEY 

573 CHROMOSOME 3. 

Pathology, J. 

Y. Jin, G. D. Statler, Department of Plant 

D. Franckowiak, Department of Crop & Weed 

Somoclonal variations in root development of Maricongo suscep- Science, North Dakota State University, Fargo, ND 58105. 

tible to "plantain decline." L. J. Liu, E. Rosa-Marquez and E. 

Lizardi, University of Puerto Rico, Rio Piedras, PR 00927. Barley lines having the leaf rust resistant genes Rph3 (Pa3) 

and Rph7 (Pa7) were crossed with the lines having 1, 2 or 3 of 

Drastic reduction in ratoon productivity on Maricongo, a major the following recessive marker genes located on chromosome 3: 

plantain cultivar, is of great concern to growers in Puerto al (Albino lemma), gs2 (glossy sheath 2), Int (low number of 

Rico. Since such decline in ratoon crops has not occurred on tillers), msg5 (male sterile 5), sld (slender dwarf) and uz 

Grand Naine (a banana cultivar) nor on Harton (a plantain 

cultivar) which seem to have a large root system, efforts were 

(semi-brachytic). Four to six hundred F 2 plants from each 

cross were tested for reaction to Puccinia hordei Otth. at the 

made to examine root development of somaclonal selections of seedling stage and scored for genetic markers. Results 

Maricongo with the objective of screening for resistance to indicated that R and Rph7 are not linked with any of these 

the "plantain decline." Results obtained indicate that some 

of the somaclonal selections (Sa of Maricongo showed greater 

genetic markers (X1 = 1.0 to 7.0). 

between Rph3 and Rph7 (X 

Also, no linkage was found 

2 = .01). These two major resistant 

root development than others (S2' Sl4 Sl arnd S ) when 

cultured in a modified Murashige and 4 Skoog's 1' basal 4previous medium2' 

genes probably are not located on chromosome 3, 

workers. 

as suggested by 

supplemented with 0.5 mg! 1 kinetin and 0.5 mg/i 6 benzyl amino 

purina and supported with 3 layers of filter paper. Such a 

phenomenon was also observed in greenhouse tests when57 

plantlets derived from calti were planted in glass window57 

boxes containing promix. PATHOGENICITY GROUPING OF LEPTOSPHAERIA MACULANS ISOLATES BASED 

574 

ON THREE CULTIVARS OF BRASSICA NAPUS. A. Mengistu, S.R. Rimmer, 

E. Koch and P.N. Williams, 1630 Linden Dr., University of 

TRANSFORMATION OF TOBACCO AND POTATO WITH PEA DISEASE 

RESISTANCE RESPONSE GENE (DRRG) PROMOTERS. C. C. Chiang, N. 

Wisconsin, Madison, WI 53706. 

The pathogenicity of 39 isolates of Leptosphaeria maculans from 

N. Chang and L, A. Hadwiger. Department of Plant Pathology, N. America, Europe and Australia against a range of Brassica 

Washington State University, Pullman, WA 99164. napus var oleif era (oilseed rape) cultivars was studied. Isolates 

were categorized into four groups based on differential 

Pea tissue is able to inhibit many of the organisms that are pathogenicity on cotyledons of Westar, Quinta and Glacier. P01 

pathogens of tobacco and potatoes. This resistance is isolates can be distinguished by lack of virulence to Westar. 

associated with the enhanced activity of some specific pea PG2 isolates are virulent only on Westar but give slightly more 

genes. Transfer of the pea genes active in the pea's disease susceptible interaction phenotypes on Quinta than on Glacier. 

resistance response to tobacco or potatoes is potentially a PG3 isolates are virulent on Westar and Glacier and intermediate 

new source of disease resistance. The promoter (5') region on Quinta. PG4 isolates are virulent on all three cultivars. 

of the pea gene, DRRG-49, constructed in line with a CAT Using these cultivars as differentials we have examined about 70 

(chloramphenicol acetyl transferase) marker gene has been single ascospore isolates from rape seed debris from Saskatcheshown 

to be expressed in tobacco and potato. In the wan and Manitoba, Canada and from Western Australia and New 

transformed tobacco tissue this promoter directed function is South Wales, Australia. All Canadian isolates tested were PG2 

expressed following the challenge of tobacco leaves with whereas isolates from Australia were characteristic of PG2, PG3 

Fusarium solani f. sp. pis, a pathogen of peas. and PG4. Only Western Australian isolates produced pseudothecia. 

Vol. 79, No. 10.,1989 1207

579 583 

CHARACTERIZATION OF ADHESIONLESS MACROCONIDIAL MUTANTS OF 

NECTRIA HAEMATOCOCCA. M.J. Hickman, M. B. Buurlage, L. 

Epstein. Department of Plant Pathology, University of 

California, Berkeley, Berkeley, CA 94720 

The macroconidia of N. haematococca attach to host 

nonhost 

and to 

tissues, and to inert hydrophilic and hydrophobic 

surfaces. To determine the role of adhesion in pathogenicity, 

we isolated adhesionless mutants. Uninucleate microconidia 

were mutagenized in 2.7 mM N-methyl-N'-nitro-N-nitrosoguanidine 

for 50 min (90-95% kill). Using an enrichment procedure, 

populations of macroconidia were repeatedly selected for the 

adhesionless phenotype. Six mutant strains were obtained that 

produced macroconidia with approximately a 50% reduction in 

wild-type adhesion. Mutant macroconidia germinated at a 

similar rate and obtained a maximum percentage of germination 

(approx. 80% in 4 h) as wild-type macroconidia. The mutants 

will be used in disease assays to assess the role of adhesion 

in pathogenesis. 

580 

HISTOCHEMISTRY AND ULTRASTRUCTURE OF INDUCED AND NONHOST 

RESISTANCE IN CUCURBITS TO COLLETOTRICHUM LAGENARIUM. B.D 

Stein, K. L. Klomparens, and R. Hammerschmidt. Michigan State 

University, E. Lansing, MI 48824. 

Cucumber plants (cv. SN R 58) were injected with Pseudomonas 

syringae pv syringae to induce systemic resistance against 

Colletotrichum lagenarium race 1. The third leaves of induced 

plants and controls were detached, inoculated with C. 

lagenarium, incubated in a moist chamber for 24-72 hours, and 

prepared for transmission and scanning electron microscopy. 

Seedlings of pumpkin (cv. Spookie), resistant to C. lagenarium, 

were inoculated with C. lagenarium and incubated for 48 hr. 

prior to sampling. Ultrastructural localization of lignin with 

KMnO and 

42 

Br was carried out via TEM and X-ray analysis, respectively. 

Lignin was found to accumulate in the outer epider- 

mal wall of resistant pumpkin and induced resistant cucumber. 

Peroxidase activity, as localized with diaminobenzidine, was 

also correlated with resistance. Appressoria on cucumbers with 

induced resistance were frequently found to be necrotic. 

PATTERNS OF INTRAPOPULATION ISOZYME VARIATION IN THE BEAN RUST 

FUNGUS. J. W. McCain and J. V. Groth, Dept. of Plant Path., 

Univ. of Minnesota, St. Paul, MN 55108. 

Mass field collections of Uromyces apoendiculatus from Phaseolus 

vulgaris often are mixtures of races. We surveyed 

single-uredinium 

several 

isolates from each of 12 field collections, to 

seek similar intrapopulation variability of isozyme bands. 

Proteins extracted from urediniospores were assayed by 

polyacrylamide gel electrophoresis. Of 10 putative loci in five 

enzyme systems, seven loci varied in most collections. Among the 

66 isolates, 25 isozyme phenotypes (races) were identified; 32% 

of the isolates belonged to the most common race, but no other 

race accounted for more than 9%. Two collections made 4 years 

apart from one location differed significantly in Shannon 

diversity index. The earlier collection was among the most 

variable, but the latter was the least, with mostly homozygouslike 

band patterns. This study confirms that multiple samples 

are necessary to determine the genetic heterogeneity of rust 

spore collections. 

584 

GENOTYPE DEPENDENT RESISTANCE OR SUSCEPTABILITY TO ROOT-KNOT 

NEMATODE IN ROOTS GROWING FROM THIN CELL LAYER CULTURES OF 

TOMATO. D. N. Radin* and J. D. Eisenback**, Depts of Agronomy* 

and of Plant Pathology**, VPI & SU, Blacksburg, VA 24061. 

A " 

A new aseptic root-culture system has been developed for the 

parasitic nematode, Meloidegyne incognita and tomato, Lycopersicon 

esculentum. Epidermal thin cell layer explants from floral 

stems of tomato produced up to 20 adventitious roots per culture 

in 7 to 14 days on MS medium plus 3% sucrose and the hormones 

kinetin and IAA. Rooted cultures were transferred to Gamborg' 

B-5 medium with 1% sucrose and inoculated with infective second- 

stage juvenile nematodes surface sterilized with hibitane. 

Gall initiation was apparent five days after inoculation in the 

susceptable (mi-/mi-) tomato cultivars, Rutgers and Red Alert. 

Resistant (Mi7+-Mi+- genotypes, LA655, LA656, LA1022, exhibited 

a characteristic hypersensitive resistance response. This cul- 

ture system has potential for use in nematode propagation or 

for experimental studies on the molecular basis of the plantnematode 

relationship. 

581 

HETEROKARYOSIS AMONG SUBSPECIES OF COLLETOTRICHUM 

GLOEOSPORIOIDES. R.J. Chacko, G.J. Weidemann and D.O. 

TeBeest. Department of Plant Pathology, University of 

Arkansas, Fayetteville, AR 72701. 

585 

REDUCED PECTINASE ACTIVITY OF ASPERGILLUS FLAVUS IS ASSOCIATED 

WITH REDUCED VIRULENCE ON COTTON. T. E. Cleveland and 

P. J. Cotty, USDA, ARS, Southern Regional Research Center, 

P.O. Box 19687, New Orleans, LA 70179. 

Several subspecies of Colletotrichum gloeosporioides have An infective strain of 

potential 

Aspergillus 

as bioherbicides 

flavus was shown 

including 

to produce 

C. gloeosporioides f. several pectolytic 

sp. aeschynomene 

enzymes during 

(CGA) 

invasion 

and C. 

of 


cotton bolls 

f. sp. (Cleveland 

jussiaeae 

and McCormick. 

(CGJ). 

1987. 

Both subspecies 

Phytopathol. 

lack 

77:1498-1503). 

a known teleomorph, Sixteen strains 

therefore, 

of A. flavus, 

genetic 

varying 

variability 

in their 

may 

ability 

arise through 

to 

hetero- invade boll tissues 

karyosis 

(Cotty. 

and mitotic 

1989. Phytopathol. 

recombination. 

In press), 

In vitro 

were 

studies with examined for their 

nutritionally 

ability to 

deficient 

produce 

mutants 

pectinases. 

of GCA 

Aggressi 

yielded hetero- ness of strains 

karyotic 

during the 

colonies 

infection 

at 

process 

a rate of 

was 

lxl0-5. 

correlated 

Conidial 

from 

isolations 

several heterokaryotic 

with 

colonies 

their ability 

yielded 

to 

both 

secrete 

parental 

pectinase(s) on 

cottonseed 

sterilized 

and 

isolates 

in pectin-containing 

but no putative diploids 

liquid media. 

or recombinant 

When pec 

genotypes. nases of the various 

Crosses 

strains 

between 

were 

compatible 

analyzed by 

strains 

isoelectric 

of CGA and CGJ mutants focusing, it was 

failed 

demonstrated 

to produce 

that 

heterokaryons. 

four fungal strains 

Studies 

with 

of heterokaryosis lowest ability to infect boll tissues lacked a major 

may indicate the 

pectolyi 

potential for genetic exchange between l 

related subspecies of C. 

I 

release of bioherbicides. 

gloeosporioides following field activity which was always present in 

These 

highly 

results 

aggressive 

suggest 

isolates. 

that certain pectolytic enzymes produced 

by A. flavus during host infection are required for fungal 

virulence. 

582 586 

EARLY CELLULAR RESPONSES DURING BACTERIA-INDUCED IN PLIAN\TA GROWTH OF PSEUD(3M[D4S SYRINGA~E 

HYPERSENSITIVITY 

PV SYRINGAE HRP 

IN ALFALFA AND TOBACCO. N.R. O'Neill, MUTANTS. I. Yucel and 

C.J. 

S. W. 

Baker, 

H-utchescn. 

and 

Dept. 

L.D. 

of 

Keppler. 

Botany, 

USDA, Agricultural Ukniversity of M$aryland, College Park, MD 

Research 

20742. 

Service, Beltsville, MD 20705. 

In an effort to study the role of nutrition in the growth 

The bacteria-induced hypersensitive response (HR) in behavior of hro mutants, P seudomonas svrinqae pv. syrinoae 

alfalfa and tobacco is characterized as a rapid, strains 61 (WFF), 87 (hro: :Tn5) and 61-145 (a deletion mutant in 

localized necrotic response to pathogenic bacteria, a hro regulatory determinant) were suspended in 10 rrt NaPi buf- 

We are investigating the nature of early interactions fer containing 10 mg/mi proteose peptone (PrP) or buffer alone 

with compatible and incompatible bacteria and their and infiltrated into surface-sterilized, detached Nicotiana 

association with defense gene activation in suspension tobacum var. Samsun leaves. Leaves were incubated under high 

cells. The addition of bacteria which induce the HR humidity and were reinfiltrated with fresh buffer three times 

initiated a net uptake of extracellular H+ and net during the initial 6 h of the interaction. Bacterial 

increase in extracellular K+. A transient and populations were monitored during the initial 48 h. A~dded PrPm 

prolonged pH change was observed, and this response had little effect on WIF growth but delayed onset of the 

correlated with the evolution of active oxygen, hypersensitive response 6-12 h. Growth of 87 was only observed 

determined by luminol-mediated chemiluminescense. when PrP= was present, but was (10%. the WI level. No growth of 

Conductivity and bacterial cell death were also 61-145 was observed under either condition iqn oata. P. 

monitored. The physiological responses of various fluorescens 55 grew to WI levels in the presence of-PrP. These 

plant-bacteria combinations correlated with host- results suggest that the limited growth of hro mutants inn 

pathogen compatibility. olanta may not be due to nutritional limitations. 

1208 PHYTOPATHOLOGY

587 591 

MEASURING VIRULENCE ASSCIATIONS IN MASS UREDINIAL COLLECTIONS OF 

THE BEAN RUST FUNGUS. -J. V. Groth, E. V. Ozmon, and D. C. Linde, 

Department of Plant Pathology, University of Minnesota. St. 

Paul, MN 55108. 

SELECTIVE ISOLATION OF GLIOCLADIUM VIRENS AND G. ROSEUM 

FROM SOIL. Y-. H. Park and J. P. Stack. Department of Plant 

Pathology and Microbiology, Texas A&M University, College Station 

77843. 

A method was devised to directly measure departures from 

randomness of specific virulence traits in mass samples of 

urediniospores of Uromyces appendiculatus. Differential bean 

lines are used on which avirulent reactions are countable and 

virulence is polymorphic. Reciprocal serial inoculations are 

made on paired lines and on a universally susceptible host line. 

The random expectation is that the frequency of virulence on the 

differential is the same whether the inoculum has been screened 

through the other differential or taken from the susceptible 

line. Deviations from identity of frequency provide a measure of 

the degree of virulence coupling or repulsion association. On 

two lines tested with a single field rust collection, the two 

twolines testedno with s , a l bt 

oSelective 

virulences were not random, with about half of the expected 

coupl ing associations being absent. This method should be useful 

for rapidly surveying or comparing multiple virulence 

associations in many collections. 

Semi-selective media were developed for the isolation and enumeration 

of Gliocladium virens and G. roseum from soil. The basal medium 

consisted of 3.0 g glucose, 0.2 g MgSO 4 "7H2 0, 1.0 g K2HPO 4 , 0.5 g KCI, 

1.0 g NaN0 3 , 0.01 g FeSO 4 *7H 0, 50 mg chloramphenicol, 50 mg rose 

bengal, propionate, 50 mg and streptomycin 20 g Bacto agar sulfate, in 1.0 500 L distilled pg benomyl, water. 500 The mg pH sodium of the 

medium was adjusted to 6.0 with 25% phosphoric acid before autoclaving. 

By the dilution plate method, selective isolation (ca. 100% recovery) 

of G. virens from natural soil infested with Rhizoctonia solani, Fusarium 

oxysporum f. sp. vasinfectum, and Pythium ultimum was achieved by 

adding 20 mg gliotoxin to 1.0 L basal medium. At 3 days, no other fungi 

grew on this medium, and G. virens colony size was optimal for quantifi- 

cation. This medium is being used for population studies of G. virens. 

isolation (ca. 35% recovery) of G. roseum from the same soil was 

achieved by adding 10 mg gliotoxin, 20 mg pentachloronitrobenzene, and 

30 mg acriflavin to 1.0 L basal medium. The G. roseum selective medium 

is useful for isolation of the fungus but not for quantification, while 

that for G. virens is adequate for both. 

588 

AN EFFICIENT AND RAPID IMMUNIZATION FOR GENERATION OF HYBRIDOMA 

ANTIBODIES SPECIFICALLY REACTING WITH PLANTS INFECTED BY AN EFFECT OF AGAR BRAND ON THE EFFICACY OF A SELECTIVE MEDIUM. 

UNCULTURED MYCOPLASMA-LIKE ORGANISM. H. T. Hsu, I. M. Lee*, M.L. Courtney and J.C. Rupe, Department of Plant Pathology, 

and R. E. Davis*, USDA-ARS, Florist & Nursery Crops and University of Arkansas, Fayetteville, AR 72701. 

*Microbiology & Plant Pathology Laboratories, Beltsville, Md. 

The results of certain microbiological assays may depend 

A method to produce target-specific hybridomas for antigens upon the brand of agar used in the preparation of the 

which are difficult to purify is described. Mouse hybridomas medium. Five brands of agar were used in the preparation of 

secreting antibodies to a plant pathogenic mycoplasma-like modified Nash and Snyder's medium, selective for Fusarium 

organism (MLO) associated with tomato plants with symptoms of solani. Samples of either F. solani conidia or soil 

big bud disease were successfully produced. Neonatal mice were infested with F. solani were diluted in 0.1% water agar and 

injected with extracts from normal host plants twice, once each spread on plates of the selective medium made with each of 

on days 1 and 7. When they were 8 wk old, mice were immunized the agars. Colony counts from media containing Sigma and 

with an enriched MLO preparation in which about 10% were target Difco Bitek agars were significantly lower than from media 

antigen determined by dot-blot immunoassays. There were 589 containing Moorhead, Difco Bacto, and Northeast agars. 

hybrids produced from 20 96-well tissue culture plates seeded Colony counts on Moorhead, Difco Bacto and Northeast agars 

in 2 fusions. Tests by indirect ELISA selected 20 hybridomas were not significantly different. 

that secreted antibodies specific to the plant pathogenic 

MLO. The dot-blot immunoassay was a sensitive method for 

detection of MLO infection. 

593 

589 DETECTION OF OPHIOBOLIN IN CULTURE FILTRATES OF Bipolaris oryzae 

AND USE IN A WILD RICE ROOT ELONGATION ASSAY. D. R. Johnson and 

LONG-TERN LIQUID NITROGEN STORAGE OF CRONARTIUM QUERCUUM F. J. A. Percich, Department of Plant Pathology, University of 

SP. FUSIFORNE BASIDIOSPORES AND MYCELIUM. Pauline C. Spaine, Minnesota, St. Paul, MN 55108. 

USDA, Forest Service, SEFES, Carlton St., Athens, GA 30602. 

Ophiobolin was detected in cultures of Bipolaris oryzae, the 

Basidiospores and mycelium underwent slow chilling (l°C/min) causal organism of fungal brown spot of wild rice (Zizania 

in a Cryo-Med programmable freezer to -50'C, and were then palustris L.), and tested in a wild rice root elongation assay. 

plunged into liquid nitrogen. Several cyro-protectants were Aqueous culture filtrates were extracted with chloroform 1:1 v/v 

tried in initial studies including DMSO, glycerol, sucrose for 24 h, the organic fraction was reduced to near dryness in a 

and milk. Germination of basidiospores from mass gall isolates rotary evaporator and crystallized at 24 C. Ophiobolin was 

frozen for seven months ranged from zero to 63%. A glycerol- confirmed by 2-dimensional thin layer chromatography (2-D TLC) 

milk combination favored spore viability, while 6% sucrose with an authentic ophiobolin standard. A rapid I-D TLC detection 

provided equally effective mycelial regeneration. Basidiospores method was devised for purified ophiobolin using plastic silica 

derived from single aecial isolates frozen for 3 months had a gel plates developed in toluene-ethyl acetate-formic acid 

mean germination of 83 and 90% for two isolates and 0 and 2.0% (6:3:1). Developed chromatograms were sprayed with 1for 

two other isolates receiving the same treatment. Pine anisaldehyde or phosphomolybdic acid, and the limit of detection 

seedlings inoculated with spores derived from a single gall was 50 ng. Wild rice primary root elongationf was ignificaptly 

isolate in long-term storage showed 10% infection. Regeneration inhibited by ophiobolin concentrations of I0", I0- ~and1I0-°M, 

occurred in 96% of frozen mycelial plugs from single and mixed but a I0-" M solution was not different from H 2 0 controls. 

gall isolates. 

590 

USE OF RESTRICTION FRAGMENT LENGTH POLYMORPHISMS TO DETECT 

PHENOTYPIC DIVERSITY IN THE BEAN RUST FUNGUS. D. C. Linde, L. J. 

Szabo, and J. V. Groth, Dept. of Plant Pathology, Univ. of 

Minn., and USDA/ARS Cereal Rust Laboratory, St. Paul, MN 55108. 

Virulence and isozyme markers have been used previously to 

detect phenotypic diversity in the bean rust fungus, Uromvyces 

appendiculatus. In a sample of 27 geographically diverse 

isolates, three fairly distinct clusters of isolates were 

identified with the virulence and isozyme markers. Restriction 

fragment length polymorphisms (RFLPs) are being developed as a 

new class of marker to 1) compare the overall phenotypic 

diversity detected by RFLPs with that detected by virulences and 

isozymes, and 2) confirm the presence of the three clusters in 

the same sample of 27 isolates. Random genomic clones derived 

from bean rust as well as clones of several known genes from 

other organisms will be used as probes in the RFLP analysis. If 

the presence of three clusters is confirmed with RFLP analysis, 

these clusters probably represent gene pools between which there 

is limited gene flow. 

Vol. 79, No. 10,1989R~ 129(.:

595 599 

CLONING AND CHARACTERIZATION OF PATHOGENICITY GENES FROM EVALUATION OF TN4431-INDUCED PROTEASE MUTANTS OF 

XANTHOMONAS CAMPESTRIS PV. GLYCINES 8ra. I. Hwang, S. M. Lim, XANTHOMONAS CAMPESTRIS PV. ORYZAE FOR GROWTH IN 

and P. D. Shaw. University of Illinois at Urbana-Champaign, PLANTA AND PATHOGENICITY. G. -W. Xu and C. F. Gonzalez. 

Urbana, IL 61801. Department of Plant Pathology and Microbiology, Texas A&M University, 

College Station, TX 77843. 

N-methyl-N-nitro-N'-nitrosoguanidine was used to generate 

nonpathogenic mutants, and fifteen nonpathogenic and five Studies to determine the role of extracellular enzymes in pathogenesis of 

reduced pathogenic mutants were isolated from two thousand Xanthomonas campestris pv. oryzae (Xco), the causal organism of bacterial 

colonies examined. A cosmid clone that complemented four leaf blight of rice were conducted. Transposon Tn4431, containing the 

nonpathogenic mutants was isolated from a genomic library, and promotorless luciferase (lux) operon and the tetracycline resistance gene 

it contained about 30 kb insert DNA. This suggests that the (Tcr) was introduced into Xco strain XI-5-R using the suicide vector 

pathogenicity genes consisted of at least two complementation pUCD623. Tcr exconjugants were screened in vitro for production of 

groups. A restriction map of the fragment was constructed, and extracellular enzymes. Southern blot analysis showed protease deficient 

a 10 kb HindIII fragment was subcloned into pLAFR3. That mutants that were Tcr had Tn4431 inserted into their genome. The 

fragment, which also complemented the mutants, was mutagenized bioluminescence of the mutants was confirmed photographically. The 

with lac fusion transposon, Tn3-HoHol. Complementation tests protease mutants were reduced in pathogenicity and populations in 

indicated a minimum of three pathogenicity genes in the 10 kb planta were 10 to 100 fold lower than those observed for the parental 

fragment. The lac fusions also indicated that these genes may strain. The results indicate that protease may play an active role in 

not be expressed in vitro. disease caused by Xco. 

596 600 

CHARACTERIZATION OF A PATHOGENIC DETERMINANT FROM PSEUDOMONAS 

PLASMID-ASSOCIATED STREPTOMYCIN RESISTANCE OFXANTHOMONAS SYRINGAE PV. TOMATO. D.P. Jackson', D.A. Cuppels 2 , and V.L. 

CAMPESTRIS PV. VESICATORIA. G.V. Minsavage, B.I. Canteros, and R.E. Morris] . Univ. of Western Ontario, Dept. Microbiol.a 

Stall. Plant Pathology Dept., Univ. of Florida, Gainesville, FL 32611. Immunol., London, ON, N6A 5C1, Canada and 2 Agriculture Canada 

Res. Centre, 1400 Western Rd., London, ON, N6G 2V4, Canada. 

Streptomycin -resistant strains of Xanthomonas campestris pv. vesicatoria The prototrophic Tn5-induced mutant P. syringae pv. tom 

(Xcv) from pepper and tomato grew variably on media containing Dhe istnophic ondtoedtoupant ye s stilleable to 

streptomycin sulfate at concentrations of 50 to 1000 /sg/ml. A DNA library incite a hypersensitive response on tobacco leaves. Although 

of resistant strain BV 5-4a was constructed in the cosmid vector pLAFR- it is the result of a single TnS insertion, it also cannot use 

3. A clone of 17 kbp of Xcv-DNA conferred resistance to streptomycin after tartrate as a carbon source. Clone pDJ208, containing 30 kb 

complementation in a sensitive strain of Xcv. A 5 kbp subclone encoding of wild type DNA, restored the Path'Tar' phenotype to 3481. 

wild-type resistance to streptomycin was used as a probe in Southern Complete EcoRl digestion of the insert DNA produced seven 

hybridization analyses. The locus for the streptomycin-resistance gene(s) EcoRl fragments: 13, 6.2, 4.2, 3.7, 2.5, 0.94, and 0.8 kb. A 

in strain BV 5-4a occurs on a plasmid of 45 MDa. The subclone hybridized subclone containing the 4.2-kb EcoR1 fragment restored 

with EcoR1 -digested genomic DNA of three of twelve other resistant strains pathogenicity but at a reduced level; it did not restore the 

of Xcv as well as the plasmid DNA from streptomycin-resistant strains Psp Tar- phenotype. Subsequent restriction enzyme mapping 

demonstrated tbat Tn5 bad inserted into the 3.7-kb EcoRl 

34 and Psp 36 of Pseudomonas syringae pv. papulans. No hybridization fragment and that it was in close proximity to the 4.2-kb 

occurred with DNA from Erwinia amylovora strain UCBPP 829 or P. cichorii fragment. 

strain Pc 83-1, which are also resistant to streptomycin. 

597 

CONSTRUCTION AND ANALYSIS OF ERWINIA CHRYSANTHEMI MUTANTS 

Genetic analysis of a cloned DNA sequence required for DEFICIENT IN MULTIPLE PECTIC ENZYMES. R. G. McGuire, S. Y. He, 

coronatine production by Pseudomonas syringae pv. tomato. S- A. D. Brooks and A. Collmer, Department of Plant Pathology, Cornell 

W. Ma', V.L. Morris 2 , and D.A. Cuppels'. 'Agriculture Canada University, Ithaca, NY 14853-5908. 

Res. Centre and 2Dept. of Microbiol. & Immunol., Univ. of 

Western Ontario, London, ON, N6G 2V4, CANADA. E. chrysanthemi EC16 produces multiple pectic enzymes, including exo-poly- 

A 30-kb EcoRl fragment of P. syringae pv. tomato DC3000 DNA ct-D-galacturonosidase (exoPG), exopolygalacturonate lyase (exoPL), pectin 

contains sequences that restore coronatine production to methylesterase (PME), and four isozymes of pectate lyase (PL). The 

coronatine-negative TnS-induced mutants of this pathovar. For bacterium can also utilize isolated plant cell walls or polygalacturonate as sole 

further analysis, this cloned region was mutagenized with the sources of carbon, cause maceration and cell killing in a wide variety of plants, 

transposon Tn3-Spice. Tn3-Spice carries the ice nucleation and multiply in plant tissues. Mutations were introduced by marker exchangegene 

inaZ which acts as a "reporter" of the transcriptional eviction mutagenesis, using previously cloned genes, into E. chrysanthemi 

activity of the genes into which it is inserted. Based on the AC4150 (Nalr derivative of EC16) or CU1006 (opelA, B,C,E derivative of 

map location of the Tn3-Spice mutations, the ice nucleation AC4150) to produce mutants with combinations of deficiencies in exoPG, 

activity of these mutants, and the results of functional exoPL, PME, and PL. Our results indicate that, although PL is responsible 

complementation tests with the Tn5 mutants and subclones of for most of the maceration, exoPG has an important role in the utilization of 

wild type DNA, the coronatine genes contained within this 30kb 

region are organized into two large transcriptional units pectate, and factors other than these pectic enzymes can contribute to the 

(operons) which are transcribed in opposite directions, ability of E. chrysanthemi to multiply and cause maceration in plant tissues. 

602 

598 IDENTIFICATION AND REGULATION OF CELLULOSE SYNTHESIS GENES IN 

Identification of a genetic locus required for 

of leaf and fruit necrosis by Pseudomonas 

the induction 

syringae pv. 

AGROBACTERIUM TUMYEFACIENS. R.T. Lightfoot and A.G. Matthysse. 

Dept. of Biology, Univ. of North Carolina, Chapel Hill 27599 

tomato. R. L. Schwartz, V. L. 

Dept. of Microbiol. & Immunol., 

and Agriculture Canada, London, 

Morris, and D. A. Cuppels, 

University of Western Ontario 

Ontario, N6A 5CI, CANADA. 

A subclone of a cosmid which complemented a transposon In5 in- 

duced cellulose synthesis deficient mutant of Agrobacterium 

Five P. syringae pv. tomato mutants able to induce leaf 

chlorosis but not necrosis (Nec-) were generated uaing Tn5 

mutagenesis. All grew poorly on leaves and did not infect 

fruit; four did not induce a hypersensitive response (IIR) on 

tobacco. Southern blot analysis showed tbat Tin_ bad inserted 

into a single unique site for each mutant. A pl.AFRl library 

of P. syringae pv. tomato wild type DNA was mated en masse 

into Nec- mutant DC3162 and the resulting transconjugants were 

screened for leaf necrosis. One Nec÷~ clone, p007, which 

contained three EcoRI fragments (2.7, 6.2, and 12.5 kb) of 

wild type DNA, restored necrosis north.oly to 3162 but also to 

three of the other Nec- mutants. It also restored the H]R, 

fruit necrosis, and a better growth rate on leaves. 

tumefaciens has been identified. Multiple insertions of a Tn3 

transposon containing a promoterless beta-galactosidase gene 

(Tn3HoHol) into this clone have delineated the boundaries of a 

cellulose production region and indicate that several gene pro- 

ducts are necessary for synthesis. Synthesis was increased by 

the addition of plant extract to the bacterial culture medium. 

A Tn5 insertion into a nearby gene resulted in cellulose over- 

production, raising the possibility that this mutant was in a 

repressor gene. Tn3HoHol insertions into this gene indicate 

that its expression is insensitive to plant extract. Studies 

are underway to determine whether expression of these genes is 

regulated by the plant extract, and to explore the expression 

and regulation of cellulose synthesis in Agrobacterium tune- 

faciens. 


601

603 

PHYSICAL CHARACTERIZATION OF MUTATIONS IN PLASMID pPT23A WHICH 

RESULT IN THE CORONATINE-DEFECTIVE PHENOTYPE. C. L. Bender and 

S. A. Young, Dept. of Plant Pathology, Oklahoma State Univ., 

607 

CLONING AND PRELIMINARY CHARACTERIZATION OF AN HRP 

GENE CLUSTER FROM ERWINIA AMYLOVORA. R. J. Laby; C. H 

Zumoff; B. J. Sneath; D. W. Bauer; and S. V. Beer. Department of Plant 

Pathology, Cornell University, Ithaca, NY 14853 


We have recently demonstrated that plasmid pPT23A is involved 

in coronatine biosynthesis in Pseudomonas syringae pv. tomato 

PT23.2 (J. Bacteriol. 171:807-812). Nine coronatine-defective 

mutants of P. syringae pv. tomato PT23.2 were found to contain 

either Tn5 insertions or deletions in plasmid pPT23A. In the 

present study, the Tn5 insertions in five mutants were mapped 

to two EcoRI fragments, which were 7.9 and 18.5 kb in length. 

Several transposon-induced mutants of E. amylovora, deficient in 

pathogenicity to pear and unable to elicit the hypersensitive response 

(HR) in tobacco (Hrp-) could not be complemented with previously 

identified cosmids and plasmids. To complement these mutants, a 

cosmid library of wild-type E. amylovora DNA was screened in two 

ways. After conjugating the library into an Hrp- mutant, cosmid 

pCPP430 was identified by restoration of pathogenicity on immature 

pear fruit. Cosmids pCPP440 and pCPP450 were identified by 

In four mutants which contained deletion derivatives of pPT23A, 

the extent of DNA deleted from the 101 kb plasmid pPT23A ranged 

from 8 to 30 kilobases. These four deletion derivatives of 

pPT23A were missing variable amounts of DNA from the two 

EcoRI fragments. These results strongly suggest that at least 

some of the genes for coronatine biosynthesis reside on two 

EcoRI fragments on pPT23A. 

hybridization with subclones of the previously identified plasmids. 

These two cosmids complement for pathogenicity all but three Hrp- 

mutants, while pCPP430 complements all (20) Hrp- mutants. Each of 

the three cosmids enables E. coli to elicit the HR and has ca. 40 kb of 

DNA in common. Mutagenesis with Tn5-uidA is underway to further 

elucidate the organization and regulation of the transcriptional units. 

604 

CLONING OF A DNA REGION OF PSEUDOMONAS SOLANACEARUM STRAIN AWl THE CLONING OF AN AVIRULENCE GENE IN XANTHOMONAS CAMPESTRIS 

THAT AFFECTS BOTH THE HYPERSENSITIVE RESPONSE ON TOBACCO AND pv. VESICATORIA THAT DETERMINES HYPERSENSITIVITY IN TOMATO. B. 

PATHOGENICITY ON TOMATO AND EGGPLANT. L. A. Macol and T. P. I. Canteros, G. V. Minsavage, and R. E. Stall. Plant Pathology Department, 

Denny, Dept. of Plant Pathology, UGA, Athens, GA 30602. University of Florida, Gainesville, FL 32611. 

The suicide plasmid pSUP2021 was used to introduce Tn5 into 

Pseudomonas solanacearum strain AWl. This strain elicits a. 

hypersensitive response (HR) on tobacco and a disease response 

on tomato and eggplant. Four HR- mutants were isolated by 

screening over 6,000 Kmr colonies for loss of phenotype on 

tobacco. All HR- mutants had also lost pathogenicity on tomato 

and eggplant. This suggested that a hrp gene(s) had been 

inactivated. Southern blot analysis and genomic transformation 

confirmed that loss of phenotype was due to Tn5 insertion. The 

Tn5 element plus flanking DNA was subcloned from a HR- mutant, 

designated AWl-101, and used to identify seven cosmid clones from 

a genomic library of AWl. Further characterization of this DNA 

region will involve complementation analysis, in planta growth 

assays, and physiological studies. 

Several strains of Xanthomonas campestris pv. vesicatoria (Xcv) from pepper 

failed to cause disease in leaves of tomato cultivar Bonny Best when 

inoculated along with abrasion by carborundum. A DNA library of one of the 

strains was constructed in pLAFR-3. One fragment in the library encoded 

fo a h 

for a hypersensitive reaction in tomato after strains that were compatible with 

tomato were complimented with the fragment. Mutagenesis by Tn-5 

insertion into a 1.7 kbp subclone abolished its function. The cloned gene 

(avrBsp) was located by Southern hybridization analysis on a plasmid of 

about 40 kbp that is present in many strains of Xcv. The avrBsp-encoding 

fragment hybridized to DNA of several Xcv strains that were avirulent to 

tomato but not to DNA of several pathogenic strains. The avrBsp gene was 

linked to another avirulence gene (avrBs 3 ) on the plasmid in most strains. 

609 

605 MOLECULAR CLONING OF AN EXO-PECTATE LYASE GENE FROM ERWINIA 

GENETICS OF XANTHOMONADIN PRODUCTION IN XANTHOMONAS CAMPESTRIS 

PV CAMPESTRIS. A. R. Poplawsky, M. D.IKawalekXT OandCN. W. 

Schaad2. P1. Path/PSES, University of Idaho Moscow, ID 83843. 

1 2 

Current addresses: USDA-ARS, Corvallis, OR 97330; Harris 

Moran Seed Co., San Juan Bautista, CA 95045. 

Fourteen white mutants of the yellow pigmented X 

strain B-24 were shown by spectrophotometric methods to be 

gretlyredcedin 

antomoadn pgmets.Twocosidsfro a 

greatly reduced in xanthomonadin pigments. Two cosmids from a 

pLAFR3 cosmid clone bank of strain B-24 were selected which 

complemented (restored) pigment production in the fourteen 

mutants and a naturally occurring X.c.c. white strain (B-122). 

muthse cosmids, re speivel pI~l~ and pig2 Xcntained 32 aind 2 b-DNA 

and ri'contedonu digestiN 

inserts, respectively, and restriction endonuclease digestion 

indicated that they contained a common 18 kb sequence. A five 

enzyme, were constructed. restriction Cmplementatin endonuclease map analysis of the region of the and fourteenand subclones 

mutants and strain B-122 with the subclones has identified five 

c~mpemetat~n wthi a rous cntiuou reionof bou 25 

copeettokgopbihna.otgosrgino bu 5equivalent 

CHRYSANTHEMI AND CHARACTERIZATION OF THE GENE PRODUCT. Alan D. 

Brooks , IBoany Alan Collmer2, and Steven Hutcheson . 

Dept., 

University of Maryland, College Park, MD 20742 and Plant 

Pathology Dept., Cornell University, Geneva, NY 14456. 

Erwinia chrysanthemi EC16 mutant UM1O05 produces no 

extracellular endo-pectate lyase (PL) because of deletions in 

h nonpigeebtch utn tllmcrte.ln 

the known pel genes, but the mutant still macerates plant 

tissues. In an attempt to identify the remaining macerating 

factor(s), a gene library of UM1005 was constructed in E. coli 

fanctr ened fo p r oy y acivty a structua gne for 

and screened for pectolytic activity. A structural gene for 

exo-pectate Lyase (exo-PL) was identified in this library 

(pPNL5). Exo-PL was purified to apparent homogeneity from E. 

coli DHS5 pPNL5 and found to have an apparent molecular weight 

of 76,000 daltons and an isoelectric point of 8.6. Purified 

exo-PL had optimal activity between pH 7.5 - 8.0, and could 

e ptat citr petin h highl , y and methl-esterified 

y e d 

Link pectin as substrates. A PL- exo-PL- mutant of ECI6 was 

constructed that retained pathogenicity on chrysanthemum 

to that of UMI005. 

606 

TRANPOSN T5 MTAGNESS 

O XANHOMNASCAMESTIS vs. MANGANESE DETECTION BY ELECTRON SPIN RESONANCE DURING WOOD 

TRANPOSN T5 MTAGNESS O XANHOMNASCAMESTIS VS. DECAY BY BROWN- AND WHITE-ROT FUNGI. B. L. ILLMAN. U.S.D.A., 

CITRUMELO AND TRANSLUCENS. M.T. Kingsley, y.R. Waney and D.W. Forest Service, Forest Products Laboratory, One Gif ford Pinchot 

Gabriel. Plant Pathology Dept., University of Florida, Gainesville, FL 32611. Drive, Madison, Wisconsin 53705-2398 USA. 

Suicide vector pSUP1011 was used to transfer Tn5 from E. coil strain SM10 

to X. campestris pv. citrumelo strain 3048 (spr); pRK600::Tn~uidA was used 

Changes in manganese were detected in fungal degraded wood by 

electron spin resonance spectroscopy (ESR). Samples of wood 

to introduce the transposon to X~c pv. translucens strain 216.2 (Spr). 

Kanamycin resistant (25 pg/mI) exconjugants of both strains were obtained 

on complete media with spectinomycin (35 pg/mI) at frequencies of 10were 

colonized by one of two white-rot fungi, Coriolus 

versicolor or Phanerochaete chrysosporium, or one of two brown- 

7 to 

10-8 per recipient. By Southern blot analyses, the transposons appeared to 

be randomly dispersed in both genomes. Strain 3048 causes disease on 

rot fungi, Gloeophyllum trabeum or Postia placenta. ESR 

spectra for the paramagnetic divalent manganous ion, Mn( II), 

were collected at weekly intervals for six weeks from wood 

citrs ad Axotophi cmmonbea. muant ofstran 348 ere 

citrs ad Axotophi cmmonbea. muant ofstran 348 ere 

recovered at afrequency of 2.6%. Nonauxotrophic mutations in strain 3048 

affecting pathogenicity on both hosts (Path) or only one host (host species 

samples of cottonwood (Populus deltoides), Douglas-fir 

(Pseudotsuga menziesii), sweetgum (Liquidambar styraciflua) and 

redwood (Sequoia sempervirens), with and without the presence 

of the fungi. Changes in the degree of the Mn( II ) ESR spectra 

specific, Hss~) were recovered at frequencies of ca. 0.36% and 0.33%, of each wood species were dependent upon the presence and the 

respectively. X.c. pv. translucens strain 216.2 causes disease on barley, 

oats, rye, triticale, and wheat. Pathogenicity tests of 216.2 insertional 

species of the decay fungus. In general, the magnitude of the 

change in the manganese signal correlated with susceptibility 

derivatives are in progress, 

of wood species to decay by the fungi. 

608 

610 

Vol. 79, No. 10, 1989 1211

611 615 

INHIBITORY EFFECT OF TRICYCLAZOLE AND OTHER MELANIN INHIBITORS 

ON GREEN PIGMENTATION OF PENICILLIUM SPP. AND ASPERGILLUS SPP. INFLUENCE OF FERTILIZATION AND PLANT AGE ON DETECTION OF 

Wheeler, MN H. and M. A. Klich, USDA, ARS, Cotton Pathology ACREMONIUM COENOPHIALUM. B. L. Randall-Schadel, K. D. Gwinn, 

Research Unit, Rt. 5, Box 805, College Station, TX 77840 and A. M. Gavin, R. A. Shelby, and L. W. Dalrymple. NC Dept. of 

USDA, ARS, SRRC, P. 0. Box 19687, New Orleans, LA 70179. Agric., P.O. Box 27647, Raleigh, NC 27611, Univ. of TN, Knoxville, 

TN 37901, Auburn Univ., Auburn, AL 26849. 

Five compounds that inhibit melanin synthesis in a number-of 

brown to black imperfect and ascomycetous fungi were used to Samples from 4 tall fescue seed lots were evaluated in a 

treat fungi that produce green conidial pigments. Tricyclazole, regional referee for viability of A. coenoilum by 7 

chlobenthiazone, pyroquilon, fthalide and 2 , 3 , 4 ,5,6-penta- laboratories. Results had an unacceptable level of variation. 

chlorobenzyl alcohol, added to malt extract agar at 8 or 30 Since fertilization and the age of the plant varied among 

pg/ml, prevented normal green pigmentation in nine Penicillium laboratories, this study was designed to examine the effects 

spp. and in three of six Aspergillus spp. The compounds did of these variables on endophyte detection. Treatments were 

not prevent green pigmentation in Gliocladium virens or 3 levels of fertilization, 3 ages of plants and 3 seed lots. 

Trichoderma harzianum. Those fungi whose pigment was affected The experiment was performed twice. Each treatment was 

by the five inhibitors were lighter green or brown in color, evaluated at two locations using either ELISA or microscopic 

Chlobenthiazone at 30 jg/ml was the only treatment that assay. ELISA and microscopic assay were highly correlated 

strongly inhibited fungal growth and sporulation. These (r = .93131, P = .0001). Within individual seedlots, no vaniresults 

suggest that many Penicillium spp. and Aspergillus spp. tion in endophyte infection was found among nitrogen levels or 

may contain reductase enzymes similar to those in the melanin plant ages. Endophyte infection levels of seed lots were 

pathway of many brown to black fungi. statistically different (P = .0001). 

612 616 

COMPARISONS OF DEVELOPMENT AND MORPHOLOGY BETWEEN SCLEROTIA DEVELOPMENT OF AN ANTISERUM TO DETECT GREENING DISEASE OF 

AND BASIDIAL CYMES IN RHIZOCTONIA SOLANI KUHN ANASTOMOSIS GROUP CITRUS. R-J Chippindall and V H Whitlock, Department of 

ONE. X_Yana., C.S. Kousik, G.T. Berggren, and J.P. Snow. Dept. of Plant Path. Microbiology, University of the Witwatersrand, P 0 

& and Crop Physi. La. Ag. Expt. Sta., LSU Ag. Center, Baton Rouge, LA 70803. 2050, South Africa. 

Initiation and growth of sclerotia and basidial cymes in Rhizoctonia solani 

Kuhn AG1 were studied using light and scanning electron microscopy. The AG- 

1-1B and AG-I-IC intraspecific groups produced both microsclerotia and the 

basidial stage on water agar. Growth of sasakii-type sclerotia of AG-I-IA 

intraspecific group is a result of interweaving of mycelium and is referred to 

as 'loose type'. Microscrerotia of intraspecific groups IB and IC are initiated 

from a lateral point, develop by lateral branching, and are referred to as 

lateral types. The initiation and development of basidial cymes and 

microsclerotia is morphologically similar. Lateral growth of microsclerotia 

may be related to sexual reproduction of the fungus (Willetts 1972, Bio. Rev. 

47:515-536). 

Antisera raised against laboratory cultures of 

initially 

a bacterium 

thought to be the causative agent of citrus greening 

disease were found to be unreliable in distinguishing between 

healthy and greening-infected material in various assays. 

In order to avoid using the cultured organism as 

a extract polyclonal of the 

immunogen, 

antiserum greening was organism raised against from citrus. a partially-purified 

Extracts were 

prepared by homogenization and enzymatic digestion of plant 

material followed by differential and gradient centrifugation. 

After exhaustive cross-adsorption against healthy plant 

material, the antiserum was able to detect greening using 

the ELISA and immunoblot assays. Preparation 

was 

of samples for 

blotting found by to alkali be particularly leaching of effective. bacteria from This infected antiserum tissue is 

613 

currently being used to identify greening-infected material 

using the immunoblotting procedure. 

Comparison of pepper isolates of Ebybtophthora §apsici from New 

Mexico to other solanaceous and non-solanaceous isolates. ". Y. 

Ulbbd.a and M. Aragaki. Department 

University 

of Plant 

of Hawaii, 

Pathology, 

Honolulu. 

Thirty isolates of ejbjltbphQhorA gsici obtained from pepper in 

the Rio Grande Valley near Las 

culture 

Cruces, 

originated 

New Mexico 

in 

where 

1922, 

the 

were 

type 

compared to isolates from 

tomato and pepper from Hawaii and Mexico. Fifteen other 

isolates obtained in Hawaii from other hosts were also studied, 

P-hytopQthora capsici cultures from pepper and tomato from New 

Mexico and elsewhere: (1) did not produce chlamydospores; (2) 

varied in the production of sporangial fans; (3) were Al, A2, or 

homothallic; (4) produced sporangia variable in L:D ratios with 

long, variable pedicels; and (5) virulent to pepper. 

ytophthora Qapic i cultures from other hosts formed a distinct 

group 

to form 

which: 

gametangia; (1) produced 

(3) promptly 

chlamydospores; 

produced sporangial 

(2) frequently 

fans; failed 

(4) 

produced narrow sporangia with long pedicels; and (5) were 

avirulent to pepper. 

617 

A SEMI-SELECTIVE MEDIUM FOR PHOMA MACDONALDII (PMSA). P.A. 

Donald and J. R. Venette. Department of Plant 

Dakota 

Pathology,-North 

State University, Fargo, ND 58103. 

A technique for detecting Phoma macdonaldii in its ecosystem 

has been Lacking. f. m acdonaLdii conidia and ascospores are 

formed on sunflower stalk debris. P. macdonaldii is difficult 

to recover from debris because it competes poorly with other 

organisms in culture. Soil extract medium selective for P. 

betae was modified to support growth of P. macdonaldii and 

reduce growth of contaminants. To test The medium, infected 

sunflower stalk debris was finely ground and the powder was 

mixed 1:1 to 1:10 (stalk volume:nonsterile field soil volume). 

Portions was recovered of the 

from mixtures 

all ground were plated stalk assays on PMSA. 

and "from P. macdonaldii 

all 

mixtures through 1:4. The pathogen was recovered from 80% of 

the assays with stalk:soil ratios of 1:8 and 1:10. .P. 

macdonaldii was not recovered from field soil alone. When 

conidia from culture were plated on PMSA, at least 1X10 3 

614 

conidia/ml were needed for growth to be observed. 

MITOCHONDRIAL AND PLASMID DNAS IN TRICHODERMA. Robert J. Meyer. 

SBML, USDA-ARS, Beltsville, MD 20705. 

61 8 

The Trichoderma species aggregates have been difficult to subdivide into 

biological species by morphological characters. The nucleic acids of these 

species are being examined for new characters that could help define the 

biological species. Analysis of mitochondrial DNA (mtDNA) 

revealed 

preparations 

two possible characters, highly varied mtDNA restriction patterns and 

mitochondrial plasmids. Despite the variability in the mtDNA restriction 

patterns, it was possible to subdivide the strains into groups. Two of these 

groups correlated with two new morphological subgroups of the Trichoderma 

viride species aggregate. The plasmid DNAs were unique to each strain and the 

pattrnsgenratd no coresondto dd he goup baed n mDNAppm mDNAresistant 

restriction patterns. This result suggested that the mtDNAs and plasmid DNAs 

evolved independently. Nuclear ribosomal DNA restriction maps are being 

examined to see if they give a corroborative or dissimilar grouping of these 

strains. 

DIAGNOSTIC MEDIA FOR THE DETECTION OF FUNGI (BOTRYTIS CINEREA) 

RESISTANT TO VINCLOZOLIN AND BENOMYL. T.R. Bardinelli, E.J. 

Butterfield, and T.L. Jones. BASF Corporation, Agricultursl 

27709. etr PO o 158 Rsac Tinl PrN 

279 

A diagnostic medium was developed for the detection of Botrytis 

cineres strains resistant to vinclozolin and benomyl. The 

medium contains 0.04% (w:v) brom cresol purple, 10% 0. IN NaOH, 

and 2% agar. After autoclaving, filter-sterilized dextrose is 

added to 4% then 40 ppm vinclozolin or 10 ppm benomyl and 50 

streptomycin 

spores 

sulfate are 

causes 

added. 

a 

Germination 

color change 

and growth 

from 

of 

red to yellow 

18-48 hours 

in 

after inoculation. Laboratory and field teats 

demonstrated selectivity against fungal contaminants, making 

the medium useful for field monitoring of resistance. 

Comparisons between this method and other techniques such as 

1212 PHYTOPATHOLOGY

agar diffusion tests and spore germination on fungicide dahliae in stem bases at 105 DAP. Main effects of irrigation 

ammended media (PDA, MA or WA) showed excellent correlations, and V. dahliae on tuber dry weight were significant at final 

This medium has also been used for the detection of resistant harvest (145 DAP), but not at 48, 77, or 105 DAP. Yield 

strains of Monilinia fructicola to benomyl. reductions for both effects were due to lower tuber specific 

gravities and a smaller yield of grade A tubers. The 

irrigation x V. dahliae interaction was not significant. 

619 

SEROLOGICAL COMPLEXITY IN PAV-LIKE BARLEY YELLOW 623 

DWARF VIRUSES. R.E. Klein, G.N. Webby, and R.M. Lister. Dept. 

of Botany & Plant Path., Purdue University, West Lafayette, IN 47907 SUCCESSFUL PRODUCTION OF MONOCLONAL ANTIBODIES TO THREE 

CARNATION VIRUSES USING AN ADMIXTURE OF ONLY PARTIALLY PURIFIED 

In experiments to develop panels of antibodies for detecting and VIRUS PREPARATIONS AS IMMUNOGEN. Ramon Jordan. USDA-ARS, 

discriminating isolates of barley yellow dwarf virus, leaf samples of and NS CS Labo Rator Jordan. MDA20705 

several cereal species from various sources were screened by ELISA with 

polyclonal (PAbs)ormonoclonal (MAbs)antibodies to aPAV-likeisolate. One of the stated advantages of hybridoma produced monoclonal 

Four MAbs were PAV-specific and three reacted with PAV as well as an antibodies (McAbs), when compared with conventional ly produced 

MAV isolate. Most samples which reacted positively with PAV PAbs also polycl onal antibodies i s the ability to produce and select 

reacted with all seven MAbs in subsequent tests. However, several target-specific McAbs even when impure antigen or antigen 

samples failed to react with one or more of the PAV-specific MAbs and mixtures are used as immunogens. This 'advantage' was tested in 

quantitative differences were also found among samples with MAbs that a project designed to generate McAbs to carnation necrotic fleck 

reacted with both PAV and MAV. Based on these results, PAV-like (CNFV), carnation mottle (CarMV) and/or carnation latent (CarLV) 

isolates could be separated into at least three groups. The results viruses. An admixture of partially purified extracts from 

emphasize that, as with MAV (Phytopathology 78:766-770), no single singly- and doubly-infected carnation plants was used as immuno- 

MAb can reliably detect all PAV-like isolates. gen and individually as ELISA screening antigens (along with a 

similarly prepared extract from healthy plants). Extracts contained 

5-20 protein bands as determined by SDS-PAGE. Forty-one 

620 antibody-secreting virus-specific (4 to CarMV, 4 to CarLV and 

33 to CNFV) hybridoma clones were selected. Virus specificity 

MODELS TO ESTIMATE YIELD LOSSES IN BELL PEPPER CAUSED BY TOBACCO was confirmed by Western-blot analysis. Selected McAbs have been 

ETCH VIRUS EPIDEMICS. F. W. Nutter. Jr., C. W. Kuhn, and *J. N. used routinely in ELISA for detection of virus in sap extracts. 

All, Departments of Plant Pathology and *Entomology, University 

of Georgia, Athens, GA 30602. 

Field experiments were conducted in northeast Georgia during the 624 

years 1985-1988 to quantify the effect of tobacco etch virus WHITEFLY-MEDIATED SILVERING OF SQUASH LEAVES. N. Bharathan, 

(TEV) epidemics on yield of Yolo Wonder B bell pepper. TEV was 

found to reduce both fruit number and average fruit weight/plant. R. Graves, K. R. Naray anan H. nter, and R. T. NcMillan, Jr. 

Early season infection reduced yield 74% in 1986 and 73% in 1987 Tropical Research and Education Center, University of Florida, 

while late season infection reduced yield 5 and 7% in 1986 and 

1987, respectively. Pepper yield increased in a logistic fashion A disease of unknown etiology resulting in silvering of squash 

with respect to the delay in time (days after transplanting) that leaves has become very prevalent in South Florida. Typical 

individual plants exhibited TEV symptoms. Critical point (CP), 

multiple point (NP), and area under the TEV disease progress symptoms include vein clearing and subsequent silvering of 

p pleaves. These symptoms are observed 2-3 weeks after whitefly 

curve (AUDPC) models had coefficients of determination ranging (Bemisia tabaci) infestation. Squash leaves were analyzed fo 

from 52-85%. An additive multiple point model based on the double standard RNA (dsRNA) which are known to be foot printa 

weekly change in TEV incidence as a function of days after of viral infection. Double stranded RNA bands of approximate 

transplanting pepper explained more of the variation in yield (83 of al infetion.iDoubleystranded in can d o , p itely 

to 94%) than traditional CP, MP, and AUDPC models. Yearly yield 4.6 and 4.2 Kb were consistently observed in caged, whiteflylosses 

due to TEV ranged from 15 to 50% during the 4 yr period, infested plants. In contrast, no dsRNA bands were observed in 

caged, whitefly-free plants. Further characterization of the 

whitefly-mediated silvering in squash leaves is in progress. 

621 

EFFECTS OF NET BLOTCH ON YIELD OF MOREX BARLEY. Y. Jin, V. D. 625 

Pederson. Department of Plant Pathology, North Dakota State PARTIAL CHARACTERIZATION OF MONOCLONAL ANTIBODIES TO 

University, Fargo, ND 58105. ZUCCHINI YELLOW MOSAIC VIRUS (ZYMV) AND WATERMELON MOSAIC 

VIRUS-2 (WMV-2). G. C. Wisler, C. A. Baker, D. E. Purcifull, and E. 

The relationship between disease sevenrties of barley net Hiebert. Department of Plant Pathology, University of Florida, Gainesville, FL 

blotch (incited by Pyrenophora teres Drechs.) and yield of 32611. 

Morex barley was investigated by inoculating barley plants at 

different growth stages (GS) in the greenhouse. Inoculations Monoclonal antibodies (MAbs) to ZYMV and WMV-2 purified virions were 

at GS 32, GS 48 and GS 75 reduced yield 48.7, 39.4 and 13.5 developed by a modified in vitro immunization procedure by fusing BALB/c 

percent respectively. Early inoculations significantly reduced spleen cells with SP2/o cells MAbs of potential diagnostic value were 

the number of seeds per head, height of plants and dry weight evaluated by an indirect ELISA with homologous rabbit polyclonal antiserum 

of plants. A yield loss model was developed based on disease as the trapping antibody. All virus isolates were propagated in Cucurbita 

sevenities in the top three leaves. Collinearity among the peo L. One MAb to ZYMV (MAb-Z) detected all 10 ZYMV isolates from 

independent variables was diagnosed using a condition number Florida and all four from Europe. One WMV-2 MAb (MAb-W) detected all 

index and variance of decomposition proportion. It was removed 18 WMV-2 isolates tested, including 14 from Florida, two from California, 

using a ridge regression technique with a ridge trace K -- 0.04 and one each from New York and New Zealand. No cross-reactions were 

(SAS-PROC RIDGEREG). The model accounted for 92.1 percent of detected between any of the ZYMV and WMV-2 isolates and their 

the total variation. A model using inoculation stages as heterologous MAbs, nor were reactions obtained with leaf extracts from 

qualitative independent variables accounted for 93.2 percent of uninoculated plants or plants infected with papaya ringspot, squash mosaic, 

total variation. or cucumber mosaic viruses. Thus, MAb-Z and MAb-W appear to be 

useful for detection of these ZYMV and WMV-2 isolates, respectively. 

622 

EFFECTS OF VERTICILLIUM DAHLIAE AND DROUGHT STRESS ON POTATO 

GROWTH. R. L. Bowden, 0. I. Rouse, T. T. N. Tiirinlahti, and 

B. 0. Bowen, Dept. Plant Pathology, University of Wisconsin, 

Madison, WI 53706. 

Effects of Verticillium wilt and drought stress on 'Russet 

Burbank' potato grown in Plainfield loamy sand were studied in 

a split-plot experiment in 1988. Main plots were overhead 

irrigation treatments (1 or 3 irrigations/week) and subplots 

were levels of V. dabliae inoculum (0, 9, or 39 propagules/ 

gram of soil). Canopy radiation interception was reduced by 

drought stress between 88 and 97 days after planting (OAF) and 

by V. dahliae between 110 and 124 OAF. Level of V. dahliae, 

but not irrigation, had significant effects on incidence of V. 

Vol. 79, No. 10, 1989 1213

(T), all of which had been obtained from nurseries outside the 

state. These isolates were designated TmRSV-WR5, -TSI, and 

-TKBI, respectively. The isolates had similar host ranges but 

varied in symptom severity on cucumber. TmRSV-WR5 induced very 

severe mosaic and leaf curling, and often plant death, but 

TmRSV-TS1 and TmRSV-TKB1 were much less severe with no plant 

death. A single band of Mr 60,000 was obtained upon polyacryla- 

627 mide gel analysis of proteins isolated from purified 

TmRSV-WR5 

virus. 

was serologically similar to typical eastern North 

American TmRSV isolates, and 

ANTIGENIC 

reacted 

DIVERSITY 

identically 

OF PAPAYA RINGSPOT 

with 

VIRUS 

six 

(PRSV) 

TmRSV 

ISOLATES antisera. one of the However, six antisera TmRSV-TS1 indicating and TmRSV-TKBI, serological formed differences. spurs with 

DETECTED BY MONOCLONAL ANTIBODIES TO PRSV-W. C. A. Baker and 

D. E. Purcifull, Dept. Plant Pathology, University of Florida, Gainesville, FL 

32611 631 

Six monoclonal antibodies (MAbs) were obtained after the fusion of SP2/0 CHARACTERIZATION OF A GEMINIVIRUS OF PEPPER. D. C. 

myeloma 

Stenger, 

cells 

J. 

with spleen cells from BALB/c mice immunized with a Florida E. Duffus, USDA-ARS, Salinas, CA, 93905, 

isolate 

and 

of PRSV 

B. Villalon, 

type W (PRSV-W) or its capsid protein. The MAbs were Texas Agricultural Experiment Station, Weslaco, TX, 78596. 

tested by indirect ELISA against 15 isolates of PRSV-W, one isolate of PRSV 

from papaya (PRSV-P), 

M~b- al 

and 

17PRS recte 

the Tigre 

wih islats. isolate (PRSV-T) from Guadeloupe. A geminivirus causing leaf curl and distortion 

MAb-1 ~b-2reatedwit al 15 isolated from pepper (Capsicum annuum) grown in symptoms 

reacted Texas. was 

with all 17 PRSV isolates. MAb-2 reacted with 

The 

all 15 Texas pepper geminivirus (TPGV) was transmitted persistently 

isolates of PRSV-W and with PRSV-P. MAb-3 reacted only with the 15 by Bemisia tabaci, and mechanically, to 

PRSV-W 

species of 

isolates. 

the 

MAb-4 reacted with 13 PRSV-W isolates and with Solanaceae. Electron microscopy of purified virions 

PRSV-P. 

revealed 

MAb-5 reacted with 12 PRSV-W isolates and with PRSV-P and typical geminate particles. Extracts from infected 

PRSV-T. 

plants 

MAb-6 reacted only with the PRSV-W isolate used for contained a supercoiled replicative form (RF) 

immunization. 

DNA species of 

None of the MAbs reacted with the Moroccan isolate of 2.6 kilobase pairs. RF DNA was digested with EcoR 

watermelon 

I or 

mosaic virus. These results provide evidence for at least six Hind III and cloned into 

different 

pUC8. 

epitopes 

Analysis 

on PRSV, 

of recombinant 

five of which varied among the PRSV isolates plasmids indicated that two distinct species were cloned 

that were 

from 

tested. RF DNA. One TPGV DNA hybridized with DNA A of 

mosaic 

tomato 

virus 

golden 

(TGMV). No hybridization was observed between 

TPGV DNAs and TGMV DNA B. Both cloned TPGV DNAs were required 

628 for systemic infection of plants. TPGV is a typical whitefly 

transmitted, bipartite geminivirus not previously known to 

occur in the United States. 

RAPID METHOD FOR MAIZE STRIPE VIRUS IDENT- 

IFICATION. 0. E. Bradfute and J. H. Tsai*, Ohio State 

University, OARDC, Wooster, OH 44691 and *University of 632 

Florida, IFAS, Ft. Laud. REC, Fort Lauderdale, FL 33314 SEROLOGICAL RELATIONSHIPS BETWEEN BARLEY YELL 

Maize 

DWARF 

stripe 

VIRUSES. 

virus (MStpV) 

G.N. Webby 

was identified 

and R.M. 

by 

Lister, 

direct 

Purdue 

observation 

Univ., W. 

Lafayette, IN 47907. 

sap in phase-contrast from symptomatic light microscopy leaf areas of of maize needle-shaped (Zea mays crystals L). Crystals in 

Serological differentiation indices (SDI's) 

range 

for five isolates representative 

of barley 

of a 

yellow 

were similar 

dwarf 

to crystals of 

virus 

MStpV noncapsid 

serotypes 

protein (NCP) in 

were 

ELISA with 

measured 

rabbit polyclonal antisera to each 

by 

isolate 

an 

and using 

indirect 

purified 

morphology and in differential solubility and reacted with viruses coated to microtiter plates in 0.1 M phosphate, pH 7.0. The isolates 

antiserum to NCP in immunofluorescence microscopy. Crystals were: PAV (non-specifically transmitted by Sitobion avenae 

could 

and 

be readily found in naturally and experimentally MStpV- Rhopalosiphum p.adi), MAV (specifically transmitted by S. avenae), and 

infected maize plants with all types of symptoms and throughout SGV 

serotypes, 

(specifically 

and RPV 

transmitted 

(specifically 

by Schizaphis 

transmitted 

graminum), 

by R..a..a) 

regarded 

and 

as Group 

RMV 

disease development, but not in maize infected by other maize (specifically transmitted by R. maidis), regarded as Group 2 serotypes. 

viruses or mycoplasmas. This simple test should be useful in Group 1 isolates were all closely related (SDI's 1-3), MAV being 

disease surveys and in monitoring breeding programs. intermediate between PAV and SGV. RPV and RMV (Group 2) were 

distantly related to Group 1 isolates (SDI's 5-8), but only moderately related 

to each other (SDI's 3.5-5.5). SDI's were reduced, particularly for distantly 

related isolates, when antisera produced by intradermal injection were used, 

perhaps indicating that such antisera contained antibodies to a greater variety 

of epitopes. 

630 

633 

THE V G OF TOBACC 9 ETCH VIRUS (TEy) J. F. Murphy, R.E. 

Wk2 Aepartments a aw . 

of Plant 

Pathology , Biochemistry and Agronomy , University of Kentucky 

Lexington, KY. 40546. 

TEV RNA, purified by SOS-sucrose gradient centrifugation, was 

digested with RNase A and analyzed by SOS-PAGE. Silver staining 

revealed proteins of 49, 32 and 24 k~a. The 49 and 24 k~a 

proteins were detected with anti49K antibody, while the 32 k~a 

protein was detected with anticoat protein antibody. Further 

purification of the RNA removed all traces of coat protein, but 

not the 49 and 24 k~a proteins. Furthermore, the 49 and 24 k~a 

proteins did not migrate into an SOS-polyacrylamide gel when 

TEV RNA was not digested with R~ase A. These results indicate 

that 1) the VPg of TEV is 24 k~a, 2) it is immunologically 

related to the 49K nuclear inclusion, and 3) some viral RNAs 

contain covalently bound 49K protein, presumably due to 

incomplete cleavage of the VPg. 

VARIATION IN TOMATO RINGSPOT VIRUS ISOLATED FROM GRAPE. D.C. 

Bays and S.A. Tolin, Department of Plant Pathology, Physiology 634 

and Weed Science, Virginia Polytechnic Institute and State University, 

Blacksburg, VA 24061. PROPERTIES OF THE RNA AND COAT PROTEIN OF SPRING BEAUTY LATENT 

VIRUS AND COMPARISON WITH THOSE OF FOUR BROMOVIRUSES. R. A. 

A comparison was made of tomato ringspot virus (TmRSV) isolated Valverde. Dept. of Plant Pathology and Crop Physiology, 

from three sources of grape, Vidal 256 in a Virginia vineyard Louisiana Agricultural Experiment Station, Louisiana State 

(WR), and rootstocks S04 and Kober 5BB in a rootstock nursery University Agricultural Center, Baton Rouge, LA 70803. 

1214 PHYTOPATHOLOGY

Spring beauty latent virus (SBLV) has been proposed as a new was found in winter wheat infected with Sclerophthora 

member of the bromovirus group. Four dsRNAs (MW: 3.1, 2.6, 1.8, macrospora (Sacc.) T. S. & N. from central Arkansas. The virus 

and 0.6 x 106), with a profile similar to but not identical with was extracted from infected wheat using 1 part tissue, 2 parts 

those of four bromoviruses, were obtained when plant tissue 0.05 M phosphate buffer, pH 5.5, and 2 parts chloroform 

infected with SBLV was analyzed. Molecular hybridization using followed by alternate low and high speed centrifugation. 

cDNA to SBLV RNA did not show any detectable relationship with Negative staining of both leaf dips and purified preparations 

other bromoviruses. Infectivity tests using SBLV ssRNAs revealed uniform particles, and Model E analysis indicated a 

indicated that it has a tripartite genome requiring RNAs 1, 2, single 134S component. An antiserum made to purified virus 

and 3 for infectivity. Translation of RNA 4 yielded a protein reacted with sap of virus-containing downy mildew-infected 

(MW=23,000) which comigrated with the coat protein in SDS- wheat but not with sap from wheat infected with downy mildew 

polyacrylamide gels. Western blot analysis of the coat proteins alone. Reciprocal serological tests indicate that this virus 

of SBLV and those of four bromoviruses revealed different is related to the type B virus of downy mildew from rice in 

degrees of homology. Results presented here provide further Japan (Honkura, R., et al. 1983. Ann. Phytopath. Soc. Japan 

evidence that supports placing SBLV in the bromovirus group. 49:653). This is the first report of this virus occurring in 

the U.S.A. 

635 639 

MOLECULAR CLONING OF GENOMIC RNAS OF SWEET CLOVER NECROTIC Carbohydrate degrading enzymes of Enterobacter cloacae D.P 

MOSAIC VIRUS (ALFALFA STRAIN). H.R. Pappu, J.A. Williams*, 

C. Hiruki and J.B. Bell*. Departments of Plant Science and 

Genetics*, University of Alberta, Edmonton, Alberta T6G 2P5, 

CRborate dgd S i ene s of Enter tericloacae D.P" 

Roberta and C.J. Sheets. USDA, ARS, Beltsville, MD 20705. 

Enterobacter cloacae strain E6 is a potential biocontrol agent 

Canada. for Pythium ultimum. Production of carbohydrate degrading 

Complementary DNA (cDNA) copies of the two genomic RNAs of enzymes of E6 was studied to understand how E6 establishes and 

sweet clover necrotic mosaic virus (alfalfa strain) were cloned maintains itself in the rhizosphere. No xylanase, carboxyinto 

Lambda gtlO. Using viral RNA-specific nucleic acid probes, methylcellulase, amylase, laminarinase, chitinase, arabinothe 

cDNA library was screened and two clones with inserts of galactanase, polygalacturonase, or pectate lyase activities 

1.8 kb and 1.4 kb were selected that were specific to RNA-l and were detected in culture supernatants from E6 grown in basal 

RNA-2 respectively. The specificity of each clone was further salts + 0.1% (w/v) glycerol (BSM), BSM + 0.2% (w/v) lettuce 

confirmed by Northern analysis. Both cDNA clones were separa- roots, or BSM + 0.2% (w/v) of the substrate polymer of the 

tely sub-cloned into Blue Scribe and M13 vectors. Single- enzyme. In contrast, a number of glycosidase activities includstranded 

DNA templates prepared from recombinant Ml3mpl8 and ing -D-glucosidase, ,-D-n-acetylglucosaminidase, 8-D-xylosi- 

Ml3mpl9 containing the 1.4 kb fragment specific to RNA-2 are dase, ct-L-arabinopyranosidase, o-D-galactosidase, 8-D-galactobeing 

sequenced using the dideoxy chain termination method. sidase, and c-D-glucosidase were detected in extracts from E6 

Analysis of the sequence information and in vitro transcription grown in basal salts + 0.5% glycerol. ,-D-glucosidase was the 

studies are in progress. 

major glycosidase detected in extracellular and membrane 

fractions and preliminary experiments suggest that these 

fractions contain different ý-D-glucosidase enzymes. 

636 

BYDV-PAV virioas contain readthrough protein. Waterhouse, P.M., 2 Martin, 640 

R.R. 2 and 'Gerlach, W.L. 1CSIRO Div of Plant Industry, Canberra, 

Agriculture Canada, Vancouver, B.C. 

ACT and FIELD POPULATION DYNAMICS OF AGROBACTERIUtM SPP. ASSOIATED WITH 

?HERRY SEEDLINGS. V.O. Stockwell , LW. Moore , M.D. Kawalek J.. Loper, 

The PAV strain of barley yellow dwaft virus (BYDV-PAV) hs bn s 

the coat protein ORF shown to end with an amber termination codon (MR 

16:6097). Following the amber codon there is an 0PF that could encode a 

protein of 50 Kd, referred to as the readthrough protein. This readthrough 

proteins ofth0re ferred to, ans the roadt protein u O . Terahis ucleaditho 

ORF, sinus the first codon, and the coat protein ORF were 6 each ubclon) d into 

Department of Botany & Plant Pathology, Oregon State University, and USDA-ARS, 

HRCL, Corvallis, OR 97331. 

Agrobacterium radiobacte K84 has been employed as a biocontrol agent of crown 

ArbceimrdoatrK4hsbe mlyda lcnrlaeto rw 

gall disease for the past decade, yet little is known about its survival and dissemination 

in agricultural fields. Population dynamics of the biocontrol agent, strain K84, and the 

the &maa site of the GEX-2 expression vestor (Gene 67:31-40). Fusion 

proteins were purified by affinity chromatography on glutathione-agarose 

beads. In Westerns, BYDV-PAV antiserum prepared to whole virus reacted with 

the readthrough and coat protein fusion proteins. tsunoglobulins fr BiDh 

Phe viriton h antiserm tat erotei afsioni purifiedns. onreadthifrough fi 

PAV viricn antiserum proteindfomi that reacte g wthg not 

protein suggesting that the readthrough protein is present w rified c 

aroteind di ggstinc thecoat the protein. ilated 

and distinct from the coat protein. 

crown gall pathogen, A. tumefacien$ strain B49c, were evaluated in cherry crown galls 

and rhizospheres. Following inoculation of cherry seedlangs the rhizosphere popula- 

tion size of B49c was stble ovgr an 18 week period (10 -10 cfu/g fw root), while that 

of K84 declined frm 10 to 104 cfu/g. The population size of B49c in crown gall tissue 

increased from 10 to 10 cfu/g over 8e 8 weeks, whereas whereis that of K84 Kith remained remine stable soatb05at 

105 cfu/g. Strain K84 was not detected in soil cores taken more than 5 cm from Inocu- 

cherry seedlings. The population size of strain K84 was stable in crown gall 

tissue, but declined in rhizosphere and bulk soil. 

637 641 

SEROLOGICAL GROUPING OF THE CYTOPLASMIC INCLUSIONS OF 6 STRAINS 

OF SUGARCANE MOSAIC VIRUS. S. G. Jensen and J. L. Staudinger, 

USDA and University of Nebraska, Lincoln, NE 68583. 

Antisera to the 66kd cytoplasmic inclusion protein of 5 strains 

NOVEL CHEMICAL AND BIOLOGICAL METHODS TO CONTROL POSTHARVEST 

BROWN ROT OF NECTARINES. J. L. Smilanick, M.-Y. Ho, 0. Henson, 

and P. Anderson. USDA, ARS, HCRL, 2021 South Peach Avenue, 

Fresno, CA 93727 

of sugarcane mosaic virus (SCMV) or maize dwarf mosaic virus Nectarines were fumigated with sec__-butylamine (SBA), acetalde- 

(MDMV) were reacted in western blots against antigens of 6 hyde (ACH), or benzaldehyde (BZH); or treated with fungi or 

virus strains. The concentration of each antigen was adjusted bacteria to reduce postharvest decay by Nonilinia fructicola. 

to a constant stain density in PAGE. The intensity of the blot All work was conducted at 20-25°C. Fumigation 1 hr with 2% ACH 

reaction, which was linear With concentration, was measured or 0.3% SBA reduced decay lesion area more than 90% after 3 

using a scanning digital densitometer (Bio Image, Visage 110). days at 20'C. Phytotoxicity was minor and transient with SBA 

These tests divided the 6 strains into 3 serological groups, but moderate and persistent with ACH. BZH was phytotoxic and 

Strains MDMV-A and Minn-ll formed one group, MDMV-B and 1-188 did not reduce decay. Aureobasidium pullas Expil 

the second group and KS-I and MDMV-O the third. Inclusions mansonii, Hormonema prunorum, an unidentified pink yeast, and 

within groups cross reacted strongly. Cross reactions between Pseudomonas cepacia significantly reduced decay when 10,000 or 

the first and second groups were moderate (equivalent to an more propagules of these organisms were placed into 3x3 mm 

antigen concentration of about 1/3). Group 3 did not cross wounds at the same time as or as long as 7 hr before 100_N. 

react with the other two groups. Serogroups based on inclusion fructicola spores were inoculated. Precolonization of wounds 

proteins agree with serogroups based on capsid proteins, with candidate organisms improved decay suppressiveness, but 

decay control efficacy was variable. All but P. cepacia were 

638 

isolated from the surface of nectarines. 

CHARACTERISTICS OF A VIRUS ISOLATED FROM DOWNY MILDEW-INFEcTED 642 

WHRAT PLANTS. R. C. Gergerich and K. S. Kim, Dept. of Plant 

Pathology, University of Arkansas, Fayetteville, AR 72701. 

An isometric virus measuring approximately 30 nm in diameter 

BIffiONTROL OF BOTRYTIS ROT OF APPLE: ELECTRON MICROSCOPY OF 

EFFECTIVE AND INEFFECTIVE STRAINS OF THE YEAST, DEBARYOMYCES 

HANSENII. M. Wisniewski, R. McLaughlin, C. W. Wilson, and E. 

Vol. 79, No. 10, 1989 1215

Chalutz. USDA-ARS, 45 Wiltshire Rd., Kearneysville, WV 25430 Callan, 

and Volcani 

D. E. 

Center, 

Mathre, 

Bet 

and 

Dagan, 

J. B. Miller, 

Israel. 

Western Agricultural 

Research Center, Montana 

and 

State 

Dept. 

University, 

Plant Pathology, 

Corvallis, 

MSU, Bozeman, 

MT 59828; 

MT 59717. 

A histological study was conducted using an effective (87) and 

an ineffective (118) strain of D. hansenii. Greater mycelial Sweet corn with the 

development 

sh2 gene for 

was 

enhanced 

observed 

sugar 

in 

content 

samples 

is 

treated with 118 than with highly 

87. 

susceptible 

87. This was associated 

to 

with 

Pvthium 

more extensive 

ultimum 

wound colonization 

preemergence 

Corn 

damping. 

and greater 

Thseaser 

production 

asodciatewith 

of an 

mo r 

extracellular 

extensivewound cooiai byand on 

were rhizosphere antagonistic bacteria to which 

matrix the adhered growth to of hyphae 

by this pathogen 

of 

87. 

P. ultimum 

isolated from Bitterroot Valley soils. An isolate were of 

Attachment of yeast cells on mycelia was more apparent with Psed fuoreses aB wen applie to s ovide 

87. After 48 hr, mycelia in wounds treated with 87 appeared Pseudomonas 

collapsed 

fluore$¢ens, 

and 

AB254, 

convoluted, 

when applied to 

while 

seed prov 

mycelia appeared normal in significant protection 

wounds extensive treated 

from 

tissue with 118. By 72 hr, 

P. 

ll8-treated 

ultimum 

degradation wounds 

damping-off 

while showed naturally-infested 

in 

87-treated wounds appeared seed of AB254 was needed cold to to warm achieve soils. maximum At least 

protection. loglo 7 cfu 

In 

per 

a 

similar to unchallenged wounds. Results indicate biocontrol by process we have termed "bio-priming," 

87 may 

dry 

be 

seed 

mediated 

was coated 

by more 

with 

effective colonization, attacmrent AB254 and 

and/orycopiousiptoductionoofaffectivecolonizatrion, 

then allowed to imbibe moisture 

and/or attemperatures 

under 

copious 

warm 

production of an extracellular matrix. Bio-priming provided 

until a 

equal 

40% 

or 

moisture 

better 

content 

protection 

was achieved. 

against P. 

ultimum damping-off than did metalaxyl treatment when the seeds 

643 were planted into cold soil. 

EVALUATION OF PSEUDOMONAS FLUORESCENS FOR SUPPRESSION OF 647 

SEEDLING DISEASE IN COTTON. C. Hagedorn and N. Nelson 

Virginia Polytechnic Institute and State University, Blacksburg, EFFECT OFLr GENE COMBINATIONS ON RESISTANCE TO WHEAT LEAF 

VA 24061-0331 

RUST. S. German and J.A. Kolmer, University of Manitoba and 

Agriculture Canada, Winnipeg, MB R3C 2M9, Canada. 

A strain of Pseudomonas fluorescens (EG1O-53) antagonistic to 

Pythium ultimum and Rhizoctonia solani, the seedling pathogens Wheat lines isogenic for leaf rust resistance were crossed 

of cotton (Gussypium hirsutum L) was evaluated in a peat-based with TcLr34 to determine if the adult resistance gene Lr34 

carrier for disease suppression. Both disease severity (root interacts with other Lr genes to condition enhanced 

lesions) and incidence (infected plants) declined with either resistance. An average of 300 F 2 seedings from each of two 

higher application rates or with larger numbers of cells at any families of Lr34 x seedling genes LrB, Lr2c, Lr3ka, Lr11, 

one rate. Low application rates resulted in plants that were Lrl6, LrI7, Lrl8, and Lr2l were inoculated sequentially with 

no better than non-inoculated controls; higher rates produced two cultures of leaf rust avirulent and virulent to the 

plants equal or superior to fungicide treatments. Colonization seedling gene in each cross. F 2 progenies from Lr34 x Lr2c 

studies using an antibiotic-resistant mutant and a direct- segregated in a 3:1 ratio for resistance: susceptibility to 

observation staining technique indicated rapid growth and the culture avirulent to Lr2c; and segregated in a 9:7 ratio 

colonization of EGIO-53 on cotton roots. Bacterization of for resistance to the culture virulent to Lr2c. A ratio of 

cotton with EGIO-53 enhanced both plant stands and dry weights, 9:3:4 was observed when segretation to both cultures was 

and reduced disease severity on surviving plants. combined. Resistance gene Lr2c apparently interacted with 

Lr34 to condition enhanced resistance in the seedling stage. 

F 2 progenies from the other crosses did not display the same 

644 level of resistance as seedlings. 

USE OF AN INSECT VECTOR TO OVERCOME THE FREE WATER REQUIREMENT 648 

OF A MYCOHERBICIDE. J. A. Liebman and R. P. Wrubel, Department 

of Plant Pathology, 147 Hilgard Hall, University of California, 

Berkeley, CA 94720. PATHOGENICITY, VIRULENCE AND CORN INBRED REACTION TO ROOT ROT 

Most plant pathogenic fungi require free 

CAUSED 

water 

BY 

for 

BIPOLARIS 

germination ZEICOLA RACE 

Williams, 

3. P. 

Dept. 

E. Lipps 

of Plant 

and L. 

Pathology, 

E. 

Ohio 

and 

State 

infection. 

Univ., Wooster 

This limits the effectiveness of fungi as weed OH 44691. 

biocontrol agents. In greenhouse experiments, we investigated 

the use of the weevil Hypurus bertrandi to vector the fungus Isolates of B. 

Dichotomophthora 

zeicola, obtained 

portulacae 

from corn 

into 

roots, 

the 

crowns 

leaf tissue 

and 

of common stalks wereidentified 

purslane as race 

(Portulaca 

3 by inoculation 

oleracea). onto 

On plants 

leaves 

sprayed with 

of 

an inbreds B73, Pr, and 

aqueous 

Prl. 

suspension 

Regression analysis 

of the fungus, 

indicated 

2.1 ± 

a 

1.6% (mean ± sd) of significant (E

SOYBEANS. Barry M. Cunfer and Juju B. Manandhar, Department of 

Plant Pathology, Georgia Station, University of Georgia, 

Griffin, GA 30223. 

Following harvest of 'Hunter' wheat, soybeans were planted 

654 

THE USE OF REPLACEMENT SERIES 

TEUEO 

TO 

ELCMN 

STUDY 

EIST 

COMPETITION 

TD 

BETW 

PYRENOPHORA 

OPTTO EWE 

TRICITI-REPENTIS AND SEPTORIA NODORUM IN THE WHEAT 

LEAF. S. R. AdeeI, W. F. PfenderI, and D. C. Hartnett 2 without tillage into the standing wheat stubble. After wheat 

, Dept. 

of Plant PathologyI and Division of Biology 2 , Kansas State 

volunteers emerged, the leaf area with Septoria nodorum 

colonization and the disease incidence were quantified by 


plating all leaves from randomly selected plants on Bannon's 

medium. Despite dry and hot weather during early and 

mid-summer, S. nodorum became established immediately after 

volunteers emerged. The wheat seed was not infected with S. 

nodorum. Therefore the inoculum source was the wheat 

stubble. Disease incidence increased from 45 to 78% and the 

Replacement series experiments, adapted from plant ecological 

studies, were uaed to quantify suhstrate partitioning and 

interspecific competition between Pyrenophora tritici-repentis 

and Septoria nodorum For these greenhouse experiments, spore 

suspensions of the pathogens were sprayed either separately or 

in combination (at various ratios) onto the leaves of wheat 

percentage of diseased leaf tissue increased from 5 to 12% as plants at anthesis. Plants were placed under moist conditions 

the summer progressed. The absolute area of wheat leaf with to initiate infection, then returned to the greenhouse. 

S. nodorum increased 6-fold between July and September as the Following ripening of the wheat seed, the senescent top three 

volunteer plants grew. Volunteer wheat in no-tillage systems leaves were removed and placed in moist chambers; after three 

is a reservoir for S. nodorum throughout the summer, weeks the number of fruit bodies was counted. The results 

indicate that S. nodorum is more sensitive to interspecific 

competition than is P. tritici-repentis. 

651 

SURVIVAL OF THE RICE BLAST PATHOGEN IN THE NILE DELTA OF EGYPT. 

A.P.K. Reddy and O.A. Bastawsi, Rice Research and Training RHIZOCTONIA ROOT ROT OF BARLEY AFFECTED BY TIMING OF 

Center, Sakha, Kafer El-Sheikh, Egypt. GLYPHOSATE APPLICATION. R.W. Smiley, W. Uddin and K.E.L. 

Rhinhart, Oregon St. Univ., Columbia Basin Agr. Res. Ctr., 

No information is available on the mode of off-season survival P.O. Box 370, Pendleton, OR 97801. 

of Pyricularia oryzae in the Nile Delta. Seed of the 1988 

crop and straw of the 1987 crop (varieties Giza 171 and Giza Timing intervals between glyphosate application and planting 

172) were sampled at random in 1988. Samples were incubated on of no-till spring barley were evaluated. Volunteer grasses 

a moist blotter for 48-72 hr and observed for sporulation of and cereals infected by Rhizoctonia solani and Pythium spp. 

the fungus. Transmission tests from seed and straw of the 1987 were present when glyphosate (1.1 kg/ha) was applied to 

crop were made in a glasshouse. P. oryzae was recovered from replicated 6 x 230m plots 21, 14, 7, 3, or 0.5 day prior to 

67 of 127 seed lots and from 14 of 24 straw piles. The degree seeding, and 1 day after seeding. Barley height and weight, 

of seed infection within a lot varied from 1-11%. Transmission tillering, leaf and root numbers, and root rot index were 

tests gave 0.5-2.5% diseased seedlings. The pathogen was also evaluated 50 days after emergence. Height and grain yield 

transmitted to seedling from infested straw of the 1987 crop. were measured at harvest. Rhizoctonia root rot increased, 

The results indicate the fungus readily overwinters in seed and and plant growth decreased, as the time interval between 

infested straw. Methods to reduce primary inoculum could be application and seeding decreased. Yield was reduced 0, 14, 

highly effective for blast management under Egyptian conditions, 19, 22, or 24% by applications of glyphosate 21, 14, 7, 3, 

as rice is grown in small, noncontiguous areas in a 3-year crop or 0.5 day before seeding and by 29% 1 day after seeding. 

rotation. 

652 

656 

Cylindrocladium leaf spot of ijtrlc.itzia in Hawaii. M. Aragakt, 

SURVIVAL OF PUCCINIA RECONDITA AND P. GRAMINIS UREDINIOSPORES 

IN THE ATMOSPHERE. M. G. Eversmeyer, C. L. Kramer and L. E. 

P. S. Yahata, and J. Y. Uchida. Department of Plant Pathology, 

University of Hawaiii, Honolulu, Hawaii 96822. 

Browder, USDA-ARS, Dept. of Plant Pathology and Division of 

Biology, Kansas State University, Manhattan, KS 66506 

In December, 1987, a severe leaf spot of potted seedlings of 

Strelitzia nicolai (bird-of-paradise tree) was reported in a 

Urediniospores of Puccinia recondita and P. g raminis were 

nursery 

appeared 

in 

on 

Hilo, 

nursery 

Hawaii. 

seedlings 

A month later, 

of a. rginae 

a similar 

(bird-of-paradise). 

disease 

exposed to the atmosphere to determine the survival rate under Isolations from a. nlG.Qjlaj yielded C Wlindrocladium theae 

various environments throughout the year. Freshly collected (teleomorph = Ca1onectria theae), whereas Q. pteridis was 

spores of four cultures of both P. recondita and P. graminis obtained from leaf spots of a. reginae. Isolates of Q. Theag 

were placed in 10 X 10 cm nylon hangers at 3 m above the ground from a. nicolaij, Metrosideros Qo]]i.ous, and tiowea forsterana 

at Plant Pathology plots near Manhattan, Kansas in 1987-89. were similar in pathogenicity to seedlings of a. nicolai, but 

Spores were collected daily from the hangers and assayed for less virulent than Q. pteridis. On a. reginae seedlings, Q. 

germination on water agar. Significant differences in survival, "_ri_ was also more virulent than all tested isolates of C. 

as measured by germination percentage, were found between theae. An isolate of Q. pteridis obtained from leaf spots o 

cultures and between species. As temperature decreased from rtentifobtine beathnrsleat fer Rumofhr. 

fro 

July-August 

lafso s tof 

highs of 30-35 C, spore survival, measured as germi- leather-lre~aferand B.wnibcola, a1andi~ml)was pathoguenice to 

nation greater than 5%, decreased from 25-30 days to 3-5 days Dteridis obtained from 5. relna~e. 

when December-January temperatures were below -10 C. " 

65365 

A BRIGHT-FIELD AND SEN STUDY OF ZOYSIAGRASS AND 

ILLUSTRATIONS OF MYCOSPNAERELLA GRAMINICOLA AND SEPTORIA PAEDERIA SCANDENS INFECTED WITH PUYCCINIA ZOYSIAE. 

TRITICI. P.R. Scott, F.R.Sanderson, and P.W.Benedikz. N. M. Kulik and P. D. Dery. USDA-ARS, BELTSVILLE, 

CAB International, Wallingford, Oxon OX10 8DE, UK. MD 20705. 

Photographs are presented of: (1) lesions on leaves from UK Puccinia zoysiae grows intercellularly through zoysiagrass 

wheat crops with pycnidia and conidia of Septoria tritici; leaf mesophyll, invading parenchyma, and forming haustoria. 

(2) asci and ascospores of Mycosphaerella graminicola from It does not enter vascular bundles or bulliform cells; 

weathered leaves and leaf sheaths. Both conidia and ascospores conseq]uently, it forms longitudinal lesions, usually 

germinate on agar to produce, by a budding process, colonies adaxially. Echinulate urediniospores form with paraphyses 

consisting mainly of conidia and short hyphae. These cultured soon after infection, and are rapidly exposed. Teliospores 

conidia are not produced in pycnidia and are shorter, broader form many we~eks later, first within uredinia, then in telia 

and more irregular than pycnidial conidia from leaves. When appearing on bo~th leaf surfaces. They are two-called, 

inoculated onto wheat, they produce normal symptoms, pycnidia persistent, and pedicellate. Inconspicuous spermogonia 

and conidia of S. tritici, irrespective of whether they (pycnia) develop intercellularly within leaves of Paederia 

originated from ascocarpa or pycnidia, thus proving the scandens, the alternate host. Aecia also form interconnection 

between perfect and imperfect states. A-scospores cellularly and eventually erupt through the lower leaf 

are found mainly in the winter and presumably contribute to epidermis. Aeciospores with pulvinulate ornamentation are 

the primary inoculum for epidemics on wheat crops. produced in basipetal succession. 

655 

Vol. 79, No. 10, 1989 1217

658 662 

DRESCHLERA CLADOPHYLL BLIGHT OF CHRISTMAS CACTUS. Robert D. LEAF RUST POPULATIONS IN THE UNITED STATES IN 1987 AND 1988. 

Raabe, Department of Plant Pathology, University of California, David L. Long, A. P. Roelfs, B. D. Potter and M. E. Hughes, 

Berkeley, CA 94720. Cereal Rust Laboratory, USDA-ARS, Department of Plant Pathology, 

University of Minnesota, St. Paul, MN 55108. 

A fungus on the cladophylls of Christmas cactus (Schlumbergera Surveys from 1987 and 1988 of Puccinia recondita f. sp. tritici 

truncata) in a California nursery was determined to be Dreschlera were analyzed to determine ecological areas in which specific 

cactivora (Helminthosporium cactivorum) based upon a description virulence/avirulences were common. Race occurrences are often 

of the fungus including conidial measurements and host range. In correlated with the cultivars grown, as well as other factors. 

addition to a dark, soft rot on the cladophylls, infected Overwintering rust populations often outcompete exogenous 

cladophylls frequently dropped from the plants. Inoculations of populations. Different overwintering areas for races TBB, MBB 

Christmas cactus plants were made using conidia produced on pea and MBG were indicated by comparing virulence frequencies in 

straw natural media. Inoculations were not successful on both years. Lr24 virulence frequency was 10% or more both years 

uninjured surfaces but were when tissues were injured by in the potential overwintering areas of the southern and central 

pinpricking. The cultivar Maria was most susceptible, cultivar Great Plains, where acreage of cultivars with Lr24 resistance 

Annette was less susceptible and cultivars Rita and Majestic were has increased. An avirulence frequency to Lr2a greater than 50% 

the least susceptible. Although Christmas cactus previously was occurred in the eastern USA and central Great Plains. 

reported as being inconsistently infected when inoculated with D. Overwintering cultures in the east lack Lr2a virulence. 

cactivora under experimental conditions, this is believed to be Cultivars in the southern and central Great Plains lack Lj2a, 

the first report of the disease occurring naturally under however, Lr2a virulence is common. Some northern plains 

greenhouse conditions. cultivars possess Lr2a but virulence is low. 

663 

USTILAGO SCITAMINEA SORUS PRODUCTION AND TELIOSPORE 

DISSEMINATION AND DEPOSITION IN LOUISIANA. J. W. Hoy, M.P. 

Grisham, and C. P. Chao, Dept. Plant Pathol. and Crop Physiol., 

Ag. Exp. Sta., La. State Univ. Ag. Center, Baton Rouge, LA 

70803, and USDA-ARS, Sugarcane Research Unit, Houma, LA 70361. 

Production of smut sori, or whips, on infected sugarcane stalks 

in inoculated tests began during May, increased sharply during 

June and July, and continued at a lower rate through October. 

Whip production in resistant, moderately and highly susceptible 

clones was compared. Smut spore counts from spore traps 

indicated spore concentrations increased during May and were 

high from June - September. Spore concentrations per cubic 

meter of air decreased with distance and were less than 0.5% of 

levels above the crop at a distance of 135 m. A consistent 

diurnal pattern of spore dissemination was not detected. Spore 

numbers deposited at the base of plants decreased sharply with 

distance from an inoculum source. Heavy concentrations were 

deposited below and directly adjacent to plants containing smut 

whips. 

660 664 

MODELING THE GROWTH AND FUSION OF LESIONS OF 

XANTHOMONAS CAMPESTRIS PV. DIEFFENBACHIAE ON ANTHURIUM. HOST SPECIALIZATION OF PYRENOPHORA TERES ISOLATES FROM HORDEUM 

JE. Yuen, Department of Plant Pathology, 3190 Maile Way, Honolulu, HI MURINUM SSP. LEPORINUM. M. P. Brown and R. K. Webster. Depart- 

96822. ment of Plant Pathology, University of California, Davis, CA 

95616. 

Modeling the increase of diseased area on individual leaves requires Hordeum murinum sap. leporinum is-the only known alternativ 

information about the distribution of individual lesions and their respective Hor Pyrenophora tere nurally o ng in aliorna. 

growth rates. For bacterial blight of anthurium (caused by Xanthomonas host for Pyrenophora teres naturally occurring in Californi 

campestris pv. dieffenbachiae), the increase of lesion radii was found to be a Twenty-five isolates of P. teres from this wild relative of 

linear function of time, and rates in the greenhouse were approximately I Hordeum vulgare exhibit resistant lesion types when inoculated 

mm/day following artificial inoculation. This information was integrated in a onto a barley differential set in the greenhouse. These isocomputer 

simulation model that divides the entire leaf surface into either lates also differ in virulence to the wild host, cultural 

diseased or healthy 'leaf elements'. Simulation runs with increasing numbers of characteristics, and mating compatibility with isolates ofP. 

lesions, placed either randomly on the leaf surface or randomly on the leaf teres from barley. No morphological distinctions between the 

margin produced disease area increase curves that were initially linear over isolates of P. teres exists. This evidence for host specialitime, 

but then showed decreasing disease increase rates as the amount of zation in P. teres suggests that the role of alternative hosts 

healthy leaf area became limiting. Modifications were made to the model to in the epidemiology of barley net blotch is greatly restricted 

enable generation of disease assessment keys. in California. 

665 

661 PHYSIOLOGICAL SPECIALIZATION OF LEAF RUST ON DURUM WHEAT. J. 

EFFECT OF GENOTYPE MIXTURES ON THE DEVELOPMENT OF Huerta-Espino and A. P. Roelfs, USDA-ARS, Cereal Rust 

SORGHUM LEAF BLIGHT. J. A. Sifuentes, and R. A. Frederiksen, Laboratory, Department of Plant Pathology, University of 

Department of Plant Pathology and Microbiology, Texas Agricultural Minnesota, St. Paul, MN 55108. 

Experiment Station, Texas A&M University, College Station 77843. TeNrhAeia ifrnil eeo iie au nrc 

We conducted experiments to evaluate the effect of cultivar mixtures on studies of Puccinia recondita Rob. ex Desm. f. sp. tritici 

the development of sorghum leaf blight caused by Exserohilum turcicum, collected from certain populations of Triticum turoidum L. 

Three locations with different disease potential were chosen. Seed of (durum wheat). To distinguish among isolates avirulent on the 

resistant and susceptible sorghum hybrids were mixed in 1:0, 1:3, 1:1, 3:1, Thatcher near isogenic lines, other wheats were evaluated. 

and 0:1 ratios. We used a randomized complete block with 8 x 8 m plots Selected durum wheats were evaluated using 30 single pustule 

in eight replications. Maize was planted around each plot. Diseased isolates representing the leaf rust populations on durum wheats 

area of the leaf was measured for the top 5-6 leaves. We evaluated in Ethiopia, Burundi, Italy, Turkey, Tanzania, Bolivia and 

resistant and susceptible plants separately. Disease severity on the Mexico. Glossy Hugenot, DZO4-118, Maruccos 9623, Kunduru, 

resistant hybrid was not affected by the mixtures. In contrast, there Berkman and Local Red were susceptible to all isolates. Those 

were significant differences in the amount of diseased tissue on the with a differential response were: Mindum, Rojal de Almeria, 

susceptible hybrid (P=.O1). An addition of 25% resistant plants to the Boohai, ELS 6404-145-2, Gaza, Haig Mouiline, Qued Zenati 2909, 

susceptible plant population significantly reduced disease development Peliss and Pentad. Local Red and Berkman were susceptible to the 

(P=.Ol). Susceptible plants had a nonsignificant tendency towards six isolates of leaf rust from bread wheat (_I. aestivum L.), 

higher yield in mixtures. while others responded differentially or were resistant. 

1218 PHYTOPATHOLOGY

666 670 

COMPUTER-ASSISTED WHITE MOLD MANAGEMENT ON SNAPBEANS USING A 

PREDICTIVE MODEL. Jana Stewart and W. 

Plant Pathology, University 

R. Stevenson, Dept. 

of Wisconsin-Madison, 

of 

WI 53706. 

ROLE OF IRRIGATION, RAINFALL, AND 

THE DEVELOPMENT OF' PHYTOPHTHORA 

INITIAL INOCULUM DENSITY 

ROOT AND CROWN ROT EPIDEMICS 

IN 

AND YIELD IN BELL PEPPER. J. B. Ristaino, Department of Plant 

Two computerized models were developed to predict white mold Pathology, North Carolina State University, Raleigh, NC 27695. 

incidence 

parameters 

on and snapbeans field history. 

based on 

A 

cultural, 

database environmental 

derived from 171 

fields planted to beans in Wisconsin during the period 

1984-18 waanaed toubeansing stisconn dinghea pegreiod oeither 

1984-1987 was analyzed using stepwise linear regression of 

continuous and indicator variables. Derived models predict 

whit asedon mod inidece ielddisaseand ropingmoderate 

white mold incidence based on field disease and cropping 

history, irrigation frequency, row width, evapotranspiration, 

Peppers were grown in two fields in which mainplots were drip 

irrigated 3 times/wk, weekly, or not irrigated. Subplots were 

not infested or infested with graded levels of inoculum 

(lx1 .lX1 .01X) of Phtoptora capsici. When rainfall was 

mdrt duIng during diseaseodeveop disease development casc. (6.5 in in), disease ons onset 

occurred earlier (26 DAI) and at a faster rate in plots that 

were irrigated 3 times/wk than weekly or nonirrigated plots 

heat units, canopy density, rainfall/irrigation and stand (39 DAI). At high inoculum densities (11 cfu/gm), disease 

density. One model, based on data collected during the 7 reduced yield by 43, 21 and 37 percent in plots irrigated 3 

days prior to 10% bloom, has an R 2 value of 27.4 (P

674 678 

DELINEATION OF CLONES OF HETEROBASIDION ANNOSUM IN A RED 

PINE-WHITE PINE STAND. D.M. Rizzo and T.C. Harrington, Dept. of 

Botany and Plant Pathology, Univ. of New Hampshire, Durham, 03824. 

SEASONAL VARIATION IN THE DISPERSAL AND PATHOGENICITY OF 

SPHAEROPSIS SAPINEA IN SOUTH AFRICA. W.J. Swart and M.J. Wingfield, 

Departments of Plant Pathology and Micriology, University 

of the Orange Free State, Bloemfontein 9300, South Africa. 

Heterokaryotic isolates and single basidiospore strains of the P-type 

(pine-type) of Heterobasidion annosum were collected from a 60 X 60 m 

plot in a diseased Pinus resinosa-P. strobus stand in Durham, NH. 

Heterokaryons were isolated from decay in 24 trees. Isolates were 

paired on Hagem's medium, malt extract agar (MEA) and MEA with 

white pine twigs to test for vegetative compatibility. Only the twig 

medium was useful for differentiating clones; a sparse zone formed 

between heterokaryons of differing genotype. Thirteen clones were 

identified from 26 heterokaryotic isolates; the largest clone was approx. 

10 m in diameter and colonized nine trees. Two single-basidiospore 

strains etrachnof(30pbasidiocarpse(fromat25g-trpee)adpidi 

(representing the two mating-type alleles) were 

alleles) 

collected from 

e comntiron, 

each of 30 basidiocarps (from 25 trees) and paired in all combinations 

Twenty-four alleles were identified, suggesting 12 clones. These clones 

were generally consistent with the clones identified by vegetative 

compatibility. Electrophoretic markers of four isozymes supported the 

delineation of clones based on vegetative and sexual compatibility, 

Infection of Pinus spp. by Sphaeropsis sapinea through pruning 

or hail wounds results in cankers and bTluestain of timber. Stu- 

dies were undertaken to determine the time of year when S. sapinia 

conidia are dispersed and woody pine tissue is most suscep- 

tible were trapped to infection on microscope and colonization slides coated by this with pathogen. petroleum Conidia 

jelly. 

Trapping was conducted over a period of two years in summer, 

winter, and whole-year rainfall regions of South Africa. Al- 

though conidial dispersal was strongly related to the occurren- 

ce of rainfall, maximum conidial dispersal was more closely related 

to increased temperature than to maximum rainfall. Pinus 

radiata trees were artificially inoculated during autumn mna 

spring over a period of two years. Six months after inocula- 

tions, cambial lesions were significantly longer on trees inoculated 

in spring than on those inoculated in autumn. Results of 

these studies show that season must be taken into account when 

pruning trees, testing pathogenicity or screening for disease 

resistance. 

675 

FUNGI ASSOCIATED WITH DAMPING-OFF OF SLASH PINE SEEDLINGS. 

INFECTION OF LIVINGSTON PARISH, LOUISIANA, LOBLOLLY PINE JUWG AngCAnEG KuH UNiv. OF GrA aNd SDAIFor. 

PROGENY BY SPORE COMPOSITES OF CRONARTIUM QUERCUUM F. SP. J.W. Huang and E.G. Kuhlman. Univ. of Georgia and USDA For. 

FUSIFORME. C. H. Walkinshaw, and R. L. Anderson. USDA, 

Forest Service, Southern Forest Experiment Station, Gulfport, Thirty-five isolates from 12 taxa of fungi were tested for 

MS 39505, and Resistance Screening Center, Asheville, NC. pathogenicity to slash pine seedlings. Isolates of Fusarium 

Loblolly pines (Pinus taeda L.) from Livingston Parish, LA, moniliforme var. moniliforme, F. oxysporum, F. fusarioides, 

grow well in the deep south and have valuable resistance to 

fusiform rust. We have observed tests in which 85% of bulk 

F. solani, Alternaria alternata, Rhizoctoniai solani (AG-4), 

Rhizoctonia-like binucleate fungi (RLBF) (CAG-3), Pythium 

loblolly pines are infected, but Livingston Parish progeny 

have only 40% infection. To evaluate the ability of field 

isolates to infect Livingston Parish progeny, we examined 

performance over many years. Over this time, using 30-gall 

aphanidermatum, Penicillium expansum and Cladosporium 

cladosporioides caused pre-emergence damping-off. Isolates 

of R. solani, RLBF and P. aphanidermatum also caused significant 

amounts of post-emergence damping-off. Three varieties 

spore composites from 20 locations form North Carolina to 

while Texas, susceptible Livingston Parish families progeny 

had 80 

averaged 

to 100% 

50% 

rust. 

infection 

The majority 

whil failis sucepibl hd 8 to100 rut. he ajoityto 

of Livingston Parish infected seedlings had galls that were 

of F. moniliforme caused cotyledonary infection; of these, 

F. m. subglutinans 

moniliforme 

showed the highest virulence. 

and F. 

F. m. 

m. intermedium needed higher temperatures 

cause cotyledonary infection. A baiting technique using 

thin with rough bark and resistance zones in the inner cortex, 

These symptoms are associated with resistance. 

slash pine stem segments provided a rapid, sensitive and 

accurate means of assessing inoculum potential of populations 

of R. solani and RLBF in forest nursery soils. 

679 

676 680 

OZONE CONCENTRATIONS IN REMOTE FORESTED AREAS OF NORTHCENTRAL 

PENNSYLVANIA. M. Simini, J. M. Skelly, and D. D. Davis, Dept. PHYSIOLOGICAL AND STRUCTURAL CHARACTERISTICS OF THE BROWN-ROT 

of Plant Pathology, The Pennsylvania State University, FUNGUS POSTIA PLACENTA. J. A. Micales, F. Green III, M. J. 

University Park, PA 16802. Larsen, and T. L. Highley. U.S. Forest Service, Forest Products 

Laboratory, Madison, WI, 53705. 

The atmospheric ozone (03) concentration was monitored at three 

forested areas on the Allegheny Plateau of northcentral Penn- An aberrant, monokaryotic strain of the brown-rot fungus Postia 

sylvania during the 1988 growing season using a TECO Model 49 placenta, ME20, was compared with other mono- and dikaryons. 

UV photometric analyzer. The test sites were located in Clear Strain ME20 produced insignificant weight losses in wood and 

Creek State Park, Jefferson Co. (lat. 41019'39", long. 79002' was thus used to elucidate the mechanisms of decay by brown-rot 

35"), Elliott State Park, Clearfield Co. (lat. 41007'02'', long. fungi. This strain was capable of producing H 2 0 2 , oxalic 

78 0 31'40') and Tiadaghton, Lycoming Co. (lat. 41020'05", long. acid and the carbohydrate-degrading enzymes normally associated 

77026'57"). A composite of the number of hours that the level with decay. It failed to form ethanol-precipitable 

of 03 exceeded 60, 80, 100, and 120 ppb for June, July, and carbohydrates in liquid culture and produced an atypical hyphal 

August demonstrated the lowest 03 concentrations were recorded sheath in wood as observed by scanning electron microscopy. 

at the Tiadaghton site. The NAAQS of 120 ppbh- 1 2x was exceeded This study provides additional evidence for the importance of 

for 14, 22, and 6h at the Clear Creek, Elliott, and Tiadaghton carbohydrate metabolism and the hyphal sheath in the woodsites, 

respectively. Peak 1-h 03 at the three sites was 152, decay process. 

136, and 142 ppb, respectively. 

677 681 

DAMAGE TO RESIDUAL TREES FROM A MECHANIZED HARVEST DURING WINTER ASSOCIATION OF ROOT FEEDING INSECTS WITH PROCERUM ROOT DISEASE 

AND SUMMER IN A NORTHERN HARDWOOD STAND. N. I. Hennessey, W. D. IN CHRISTMAS TREE PLANTATIONS. R. J. Nevill and S. A. 

Ostrofsky, and R. C. Lemin, Jr., Cooperative Forestry Research Alexander. Dept. Plant Path., Phys & Weed Sci. VPI & SD, 

Unit, College of Forest Resources, Univ. Maine, Orono 04469. Blacksburg 

A sugar maple-beech-yellow birch stand in northeastern Maine 

Procerum root 

is responsible 

disease 

for significant 

(PRD), caused 

economic 

by Leptographium 

|ossin easternf 

procerum, 

white 

was mechanically thinned in 1988 using a long-reach boom pine Christmas tree plantations. Insect split bolt traps were 

feller-buncher. Damage to residual trees from thinning during placed in 10 plantations, for 24 weeks starting ini May 1988. 

January and February was compared with damage caused during July Seven of the plantations had symptoms of PRO and three were 

and August. A total of 667 stems were evaluated along transect asymptomatic. In asymptomatic plantations, traps were placed 

lines representing each respective season. Results indicate randomly, and in PRD plantations, traps were placed in symptothat 

significantly more (P=0.09) stems were wounded during the matic and asymptomatic areas. Over 200 

summer bark beetles (34%) than of during various 

the winter (17%). Season of harvest species and 300 weevils, either Hylobius pales or Pissodes 

appears to be as critical in determining stand damage in nemorensis, were recovered. Bark beetles rarely carried the 

mechanical as in conventional harvests. In addition, 1720 stems pathogen. Root weevils carrying L. procerum were H. pales (65%) 

were evaluated in fixed plots established in areas harvested in andP. nemorensis (15%). The total number of weevils and weevils 

summer. Data from the fixed plots were used to develop a model carrying L. procerum were highest in the symptomatic areas of 

which relates species, crown class, diameter at breast height, the PRD plantations. These findings support the evidence that 

and distance from skid trail, to the probability of an insect vectors are the primary means of dissemination of L. 

individual stem being injured. procerum in eastern white pine Christmas tree plantations. 

1220 PHYTOPATHOLOGY

682 PHYTOPHTHORA CINNAMOMI. 

University, Clemson, SC 

J. C. Jang and F. H. Tainter, Clemson 

29634-1003. 

PATHOGENICITY AND HOST RANGE OF STRAINS OF Hypoxvlon mammatum. 

G. Bucher and D. W. French, Department of Plant Pathology, 

University of Minnesota, St. Paul, MN 55108. 

Hipoxylon mammatum (Wahl.) Mill. causes an economically 

important canker disease on trembling aspen (Populus tremuloides 

Michx) and also is found on other hosts as a parasite or a 

saprophyte. Isolates of the fungus from Salix spp. were compared 

with a pathogenic isolate from P. tremuloides. The isolates were 

grown on oat grain and used to inoculate Populus and Salix teP. 

One Salix isolate was found to be as pathogenic as the P. 

tremuloides isolate on the Populus spp. Other isolates from 

Salix have caused cankers on Populus spp., but these cankers 

were significantly smaller than the cankers caused by the P. 

tremuloides isolate. The P. tremuloides isolate was not able to 

cause cankers on the five Salix spp. tested. A saprophytic 

isolate from Salix was nonpathogenic on Populus spp. 

Calli derived from embryos of Pinus taeda, P. echinata, P. 

taeda x P. echinata, and P. virginiana were screened for their 

resistance to Phytophthora cinnamomi Rands, the lit~leleaf disease 

pathogen. The optimum culture medium with 10- M, 2,4-D 

at 22%C gave best growth of calli and caused the least synergis- 

tic reaction between fungus and callus tissue. Three methods 

were used for evaluating the in vitro resistance reaction: (1) 

diameter growth of P. cinnamomi on the callus surface; (2) 

amount of intracellular hyphae and cytological change of infec- 

ted callus cells; and (3) surface reaction of inoculated calli. 

Loblolly pine was the most resistant species and shortleaf pine 

the most susceptible. Resistance reactions in vivo were always 

correlated with a lesser growth rate of P. cinnamomi on callus, 

fewer intracellular hyphae, and necrosis-and accumulation of 

phenolic compounds in callus cells. 

687 

683 DEVELOPMENT OF IN VITRO AND IN VIVO SCREENING TECHNIQUES TO 

SEASONAL SPORE LIBERATION AND CANKER DEVELOPMENT BY DETECT LITTLELEAF DISEASE RESISTANCE. J. C. Jang and F. H. 

BOTRYOSPHAERIA STEVENSII ON JUNIPERUS SPECIES. N. A. Tisserat, 

A. Nus, and J. C. Pair. Depts. of Plant Pathology and 

Horticulture, Kansas State University, Manhattan, KS 66506. 

t Ktwo, 

Tainter, Clemson University, Clemson, SC 29634-1003. 

Two-month-old sand cultured pine seedlings were inoculated with 

five, and ten zoospores/chlamydospores of Phytophthora 

Liberation of Botryosphaeria stevensii macroconidia from 

cankers on Juniperus scopulorum was monitored from April 

through October in 1987 and 1988 with a Kramer-Collins spore 

trap. Peak spore release occurred during a 3-wk period 

between late May and early June. Few conidia were detected 

before or after this period. In December 1986, and May and 

August 1987, branches on one to three trees each of 31 

cultivars, representing four Juniperus sp., were inoculated 

with B. stevensii. Branches inoculated in December did not 

develop cankers, whereas 61% and 100% of inoculations made in 

May and August, respectively, resulted in girdling branch 

cankers within 4 mo. In laboratory experiments, macroconidia 

germinated in 4-6 hr at 25-30 C. These results suggest that 

spore liberation, germination, ' and ' infection occur in late 

spring 

cinnamomi Rands to determine in vivo susceptibility. Suscepti- 

bility increased with increasing dosage of both zoospores and 

chlamydospores for all the species tested. In order from most 

to least susceptible these were Pinus echinata, P. virginiana, 

P. taeda, and finally the P. taeda x P. echinata hybrid. Infec- 

tion rates from chlamydospore inoculation were consistently 

higher than for zoospore inoculation in all species, indicating 

that the former have greater inoculum potential. In vitro 

inoculation was done the same as above except that the plantlets 

were derived from embryonic cotyledons through organogenesis in 

a tissue culture system. In general, the in vitro inoculation 

results correlated well with the in vivo results, indicating 

that this screening method may be suitable for incorporation 

into a traditional breeding program for littleleaf disease 

resistance. 

or summer. 

684 MLD INFEICICN AND GROWIWH DECLINE OF WHITE ASH IN RELATION TO 

FUN ASSOCTED WIM PINE SE)LIM [ CN CCNVERM STAND CHARACIEPISTICS. T.C. Wigginton, P.J. Smallidge, Y. Han, 

ARICUL2URAL SE . G.B. Runion_ R.J. Mitchell and W.D. J.D. Castello AND D.J. Leopold, SUNY Coll. of Env. Sci. & 

Kelley. School of Fo61995, 'Auburn University, AL 36849. Forestry, Syracuse, NY 13210. 

To determine potential causes of pine mortality on converted White ash (Fraxinus americana L.) trees in 50 plots in the 

agricultural sites, roots of loblolly pine seedl 5 removed northeastern U.S. were indexed for ash yellows MID by DAPI to 

frcm five such sites on three sampling dates were rinsed in document the effects of MLD on ash grow!th. Two increment cores 

0.525 % NaOCl, sectioned, and incubated on moistened filter per tree were extracted from three or rore white ash per plt 

paper in petri dishes. Emerging fungi were established as measured and used to calculate basal area increment. Stand conpure 

Fungi cultures representing on potato 25 genera dextrose were agar isolated before from being 712 identified. seedlingspoionadsrcueweaalz.Arcntelnecurd 

position and structure were analyzed. A recent decline occr 

examined. represning 25gener were isolated frcmn 712 ofin 

basal area increment of infected trees, while the uninfecseedings. 

Fsarimsp. ted forr61%ofall fungi idenifihed ted trees did not decline. MID infection occurred in all ash 

Fusarium s n s accounted for 31% of all fungi isolated age, size, and crown classes. There is no relationship between 

a fran 55count60%fof seed ofral g fouri iofated MID infection and white ash basal area and density. Disease and 

test sites; it itoccurredon occurred on only 76% 7% of of seedlings seedlings from from thr the ofther other broom formation are positively associated with exposed stands. 

site. Macrophcmina Easlina was recovered from twice as 

many trees on Site 4 (42% survival) compared to the other 

sites (85 - 95% survival). Most of the remaining fungi were 

considered to be sapro~tytes. 

689 EFfO }IA EDE~ N O 

PINES. W.D. Kelley. G.B. Runion and D.H. Lard. School of 

685 Forestry, uburn University, AL 36849. 

PARTIAL PURIFCATION OF A METABOLITE OF ENDOTHIA PARASITICA WHICH Effect of thiram (Gustafson 42S) seed treatment on seedling 

INDUCES ETHYLENE PRODUCTION IN CHESTNUT AND SCARLET OAK. E..Y. emergenc of longleaf, loblolly, and slash pines was 

lkb[dand L. Shamn. University of Kentucky, Lexington, KY 40546. determined at four concentrations (0, 41.7, 83.4 and 166.8 

ml/kg seed) with and without a latex sticker (6.5 ml/kg seed). 

Filtrates from cultures of E. parasitica on chestnut bark broth stimulated ethylene production For each pine species, fifty seed/treatment were placed on 

by bark plugs of American and Chinese chestnut and scarlet oak, in comparison to fungus-free natural nursery soil in each of four plastic windowi boxes (33 

broth. There were no differences between species in degree of stimulation. The metabolite(s) x 13 x 11 cm) and covered with vermiculite. Boxes were 

passed thru ultrafilters with 5000 MW cutoff, and was partially retained by 1000 and 500 MW maintained in a glasshouse and energence counts were recorded 

filters. It was stable to lyophilization. The metabolite bound to QAE Sephadex Q-25 atp~s daily for 5 wk. The test was repeated twice. Significant 

above 5.0, but did not bind to DEAE Sephadex A-25 between pH 3 and 9. Three peaks of differene observed amrorg runs mast likely wer due to 

activity were eluted from a QAE Sephadex column by a gradient from pH 5 to 4. The three temperature differne in the glasshouse. The latex sticker 

peaks eluted at pHs 4.6, 4.4 and 4.2, indicating the presence of a carboxyl group. The had virtually no effect on en'ergence. The highest 

metabolite did not react with ferric chloride nor absorb strongly at 280 nm, indicating that it is concentration of thiran resulted in decreased emergence of 

not a phenol. Oxalate did not stimulate ethylene production in bark plugs. This metabolite, if lorqleaf and in increased energence of lcblolly, ocoupared to 

further purified, may be useful for eliciting blight resistance responses in Chinese chestnut. other concentrations. For slash, greatest seedling emergence 

Identification may lead to further understanding of the mechanism of pashogenesis of E. was recorded at the two icier rates of thiran. 

paras/t/ca. 

686 

688 

690 

WATER LOCALIZATION PATTERNS IN LOBLOLLY PINES AS 

A TISSUE CULTURE SYSTEM FOR STUDYING DISEASE RESISTANCE TOSTDE BYMGTI RSOAC MCOCP. 

Vol. 79, No. 10, 1989 1221

Macall and G. A. Johnson, School of Forestry and Env. 694 

Stud., Dept. of Radiol., Duke Univ., Durham, NC 27710. 

EFFECTS OF FIVE GLOMUS SPP. ON TISSUE-CULTURED ASPARAGUS. 

The technique of proton magnetic resonance microscopy C.T. Pedersen, G.R. Safir, *S. Parent and *M. Caron. Michigan 

was used to study water uptake patterns by roots of State Univ., East Lansing, MI 48824 and *Premier Peat Research 

loblolly pine (Pinus taeda L.) transplanted into sand. Center, Riviere-du-Loup, Quebec G5R 4C9, Canada. 

Signal intensity in the image of the sand increased 

with water content. Water depletion zones were Five species of VA mycorrhizae (VAM) were evaluated for their 

observed to first form around the woody taproot (within effects on growth of tissue-cultured asparagus. Plants were 

5 hrs of transplanting), followed by zones around grown in a peat-based inoculum provided by Premier Peat Moss 

mycorrhizal short roots and lateral roots. These Ltd., and then transferred either to the field or to the 

observations show the woody taproot is one of the sites greenhouse. Glomus clarum, G. intraradices, G. monos 

of water inflow into the plant. Bands of high signal and G. vesiculiferum increased total dry weight by approximateintensity 

could be seen within the root tissue, with ly 2-00-300% in the field. G. versiforme reduced the number 

distinct patterns in roots of different ages. of buds per plant, but had-no effect on plant dry weight 

Mycorrhizal short roots consistently appeared bright, in the field. Plant dry weight was correlated with the number 

suggesting the presence of tightly bound water. These of buds per crown both in the field and greenhouse, however, 

results show the potential usefulness of this technique fungal effects on plant growth in the greenhouse were not 

in the study of plant water relations, reliable indicators of field growth. Our results demonstrate 

that growth of tissue-cultured asparagus can be increased 

or reduced by VAM depending on the fungal species used for 

691 inoculation. 

INTEGRATED CONTROL OF WHEAT RUSTS IN NORTHWESTERN UNITED 

STATES. R.F. Line, Agricultural Research Service, U.S. 

Department of Agriculture, Pullman, WA 99164-6430 

Rusts frequently reduce annual wheat yields in the Pacific 

Northwest by more than 20%. Rust control guidelines for the 

region are based on predicting epidemics and potential losses 

and on utilizing resistance, fungicides, and crop management to 

reduce losses. Factors considered in developing the guidelines 

were type of rust (stripe, leaf, and/or stem rust); affect of 

environment and of regional and individual farm management 

practices on establishment, survival, and development of the 

rusts; prevalence and distribution of races and vulnerability 

(susceptibility) of cultivars to the races; kind and degree of 

resistance to the rusts, such as slow rusting, adult-plant, 

temperature sensitive, and race specific resistance; effectiveness 

of fungicides when applied at various rates and schedules; 

potential crop yield; and economic losses or benefits of using 

alternative control systems. 

692 

FIELD TESTS FOR EFFICACY OF HERBICIDES TO SUPPRESS ASCOCARP 

PRODUCTION BY PYRENOPHORA TRITICI-REPENTIS ON WHEAT STRAW. W. 696 

F. Pfender, U. Sharma, P. Bhatt, A. Nus, and E. Adee. Dept. of 

Plant Pathology, Kansas State University, Manhattan, KS 66506. DISTRIBUTION OF VESICULAR ARBUSCULAR MYCORRHIZAL FUNGI IN 

Glyphosate herbicide (Roundup), previously found to suppress SOILS OF PERNAMBUCO (BRASIL). L. C. Maia, and S. F. B. Trufem, 

ascocarp production by Pyrenophora tritici-repentis in Universidade Federal de Pernambuco, and Instituto de Botanica de Sao 

naturally-infested wheat straw under controlled conditions, was Paulo. 

ineffective in field tests. Further experiments suggest that 

inadequate coverage, and loss of the herbicide from treated The native VAM fungi present in six soil types grown to various crops in 

straw by leaching and/or microbial degradation, are responsible the State of Pernambuco were surveyed. The VAM spores were 

for the failure. A different herbicide, monocarbamide separated from soil and root samples and identified. The roots were 

dihydrogensulfate (Enquik), suppressed ascocarp production by examined for hyphae, vesicles and/or arbuscules, and percentage 

Pyrenophora on infested straw in laboratory and field tests. 

One or two applications to infested residue in the field colonization was assessed. Twenty five species of VAM belonging to the 

reduced ascocarp production by 32% and 62%, respectively, genera Acaulospora, Entrophospora, Gigaspora, Glomus, Sclerocystis and 

Either herbicide, but particularly Enquik, could delay Scutellospora were found present. In general, the different genera were 

saprophytic activity of Pyrenoohora in reduced-tillage residue, associated with a specific soil pH and P level. However, Acaulosoora 

and thus permit more effective biocontrol by an added was found in soils of widely different soil pH and P levels which indicates 

antagonist in an integrated control strategy. it is more widespread in nature. Species composition, total number of 

spores, and root colonization varied with soil type and with the host. 

693 

697 

SPORE DOT 

A REVISION 

ELISA FOR 

OF 

THE 

THE 

DETECTION GENUS 

OF 

SCLEROCYSTIS 

VENTURIA INAEQUALIS 

BERK. & 

T. Almeida BROOME. 

and N. Rogerio 

C. Schenck. Plant Pathology Dept., University of Florida, 

ASCOSPORES. J. A. Bergdahl, L. P. Berkett, and A. R. Gotlieb, 

Department GansilF321 

of Plant and Soil Science, University of Vermont, GievleF 21 

Burlington, VT 05405. 

Effective management of Venturia inae 

The 

.quais, 

genus Scierocystis 

causal agent 

is considered 

of 

by 

different 

some 

from 

authors 

the 

as 

genus 

not taxonomically 

apple scab, is based on multiple 

Glomus 

applications 

Tul. & Tul. 

of 

Since 

fungicide 

the establishment 

once Sclerocystis 

of 

mature ascospores by Berkeley 

are 

and 

found 

Broome 

in the orchard. 

in 1873 

Presently, 

as a result of their 

there 

description 

is no way to predict 

of S. 

initial 

coremioides, 

ascospore 

10 additional 

maturation 

species 

in 

have 

specific 

been 

spore 

described. 

ontogeny 

Based 

and 

on 

orchards without equipment sporocarp 

and information 

habit, we 

not 

concluded 

easily available 

that the 

Sclerocystis 

genus 

to individual growers. is 

The 

distinct 

benefit from 

of 

Glomus 

accurate 

and must 

ascospore 

be maintained 

species, S. 

with 

coremioides, 

one 

apcaonmaturity informationan thrWoUldderesbe 

with S. coccogena 

to aplctosbetter 

and S. dussll 

time 

synonymous 

fungicidewehv coremioides. 

to S. 

We consider S. pakistanica, S. microcarpus, S. indicus and S. 

developed an enzyme-linked immunosorbent assay (ELISA) using pachycaulis synonymous with previously described Sclerocystis species. 

nitrocellulose membrane strips as the solid phase to detect The species S. rubiformis, S. sinuosa and S. clavispora are treated as 

ascospores. This indirect ELISA modification was termed Spore Glomus species. The latter two species have a unique sporocarp 

Dot ELISA. The ascospores were applied to the nitrocellulose morphology with elongate chlamydospores arranged side by side in a single 

membrane in water suspension or collected directly onto the layer around a central plexus of tightly interwoven hyphae which differs from 

membrane from a spore tower. A dot of red-violet precipitate other Glomus species. With further study, these species may justify their 

indicates a positive test for individual ascospores. placement in a separate genus. 

1222 PHYTOPATHOLOGY

698 

SCREENING OF VESICULAR-ARBUSCULAR (VA) MYCORRHIZAL FUNGI FOR 

TOLERANCE IN A HIGH ALUMINUM ACID SOIL. H. T. Bartolome, Plant 

Pathology Department, University of Florida, Gainesville, FL 

32611. 

Selected species of VA mycorrhizal fungi were evaluated for 

tolerance in an extremely acid, Al-toxic Pacolet sandy clay 

loam (pH 4.2, 308 ppm Al). Gigaspora and Scutellospora species 

had the highest spore germination and the most extensive hyphal 

growth. These include Gigaspora gigantea, g. margarita, 

Scutellospora calospora, S. heterogama, and S. pellucida. Most 

Acaulospora and Entrophospora species were found sensitive. 

Of eight isolates from these genera tested, only Acaulospora 

scrobiculata showed some degree of tolerance. All six isolates 

of Glomus mosseae and G. etunicatum failed to germinate which 

supports previous hypothesis that Glomus species are not 

adapted to acid soils. Surprisingly, however, Glomus manihotis 

was found to be the only predominant VA mycorrhizal fungus in 

this soil. This suggests that species.of VA mycorrhizal fungi 

which are generally sensitive to extreme soil acidity and Al 

toxicity may have the ability to adapt to these conditions. 

699 

EFFECT OF AN ASPARAGUS ALLELOCHEMICAL ON GLOMUS FASCICULATUM 

AND GROWTH OF MYCORRHIZAL ASPARAGUS SEEDLING. T.L. Wacker, 

G.R. Safir and C.T. Stephens, Dept. of Botany and Plant 

Pathology Michigan State University, East Lansing, Mi 48824. 

o Mexperiment 

702 

EFFECT OF SOIL WATER MATRIC POTENTIAL ON INFECTION BY POLYMYXA 

BETAE AND BEET NECROTIC YELLOW VEIN VIRUS. J. S. Gerik and 

J. C. Hubbard. USDA-ARS, 1636 E. Alisal St., Salinas, CA 93905. 

The effect of soil water matric potential on infection of sugar 

beet by viruliferous Polymyxa betae was studied using a loam 

soil from the Salinas Valley of California which was infested 

with the pathogens. The soil was saturated with water and the 

matric potential was adjusted with a soil moisture extractor to 

potentials of -0.1, -0.2, -0.3, -0.4, -0.6, and -1.0 bar. 

Sugarbeet seedlings were transplanted into saturated soil and 

the adjusted soils in sealed glass beakers, and incubated for 

2 weeks in a growth chamber at 24 C. The plant roots were then 

assayed for infection by beet necrotic yellow vein virus 

(BNYVV) by sandwich ELISA. Plants incubated in the -0.3 bar 

and wetter soils were positive for BNYVV. No plants incubated 

in -0.4 bar or drier soil were infected with the virus. The 

indicates that P. betae, the vector of BNYVV, is 

The effect of ferulic acid, an allelochemical produced by unable to infect sugarbeet roots in this soil when the matric 

asparagus, on hyphal elongation and colonization of asparagus potential is -0.4 bar or less. 

by Glomus fasciculatum was studied. Spore germination in 

vitro was not affected, but hyphal elongation decreased significantly 

with increasing ferulic acid concentration (0 to 

400 vg/g). In the greenhouse, mycorrhizal colonization of 703 

roots and growth of mycorrhizal asparagus decreased significantly 

with increasing ferulic acid concentration, while FACTORS AFFECTING PRE-EMERGENCE FLOODING DAMAGE TO SOYBEAN. 

growth of non-mycorrhizal plants was not affected by ferulic R.S. FerissandJ.M. Baker. University of Kentucky, Lexington 40546. 

acid. Although plant tissue phosphorus levels were apparently 

unaffected by ferulic acid or mycorrhizal status, ferulic Aprocedurewasdevelopedwhichproducesresultssimilartothoseobtainfedwhenfseedsan 

acid inhibition of hyphal elongation in vitro and fungal planted in soil and then flooded, but with more efficient use of time, materials and facilities 

colonization in vivo suggest that ferulic acid production The procedure involves placing seeds in a 0.1% soil suspension in deionized water, 

by asparagus can alter the symbiotic effectiveness of the incubatingfor to5days, recovering the seeds, and then assessing viability by rating the 

fungus, and subsequently affect plant growth. seeds for germination status after 5 days incubation on cellulose germination medium. 

Viability was similar using autoclaved soil, baked sand, or no soil, but was reduced by use 

of pasteurized or untreated soils. Filtering pasteurized or untreated soil suspension through 

700 Whatman number 1 filter paper resulted in a minor increase in viability, whereas filtering 

THE EFFECT OF PERIODIC FLOODING ON INFECTION OF PEPPER BY through a 0.45 ptm millipore filter completely eliminated the soil effecL Viability was 

PHYOPHFTHORA CAPSICI. J.H. Bowers and D.J. Mitchell. Dept. significantly increased by seed treatment with carboxin-thiram, thiram, captan, penicillin, 

of Plant Pathology, Univ. of Florida, Gainesville, FL 32611. erythromycin, vancomycin or ampicillin, but was not affected by seed treatment with 

benomyl, carboxin or metalaxyl. Viability increased with decreasing soil suspension pH. 

Periodic flooding increased the mortality of pepper plants Overall, these results support the involvement of soil bacteria in flooding damage. 

grown in soil infested with 25 oospores of Phytohthora 

capici per gram of soil. Plant mortality increased as the 

numiber of 24-hr flooding periods at 10-day intervals increased. 

Plants grown in infested soil at a constant 

soil-water matric potential of -125 mbar were not infected 704 

after 37 days when plated on a selective medium. However, THE INFLUENCE OF FUSARIUN SOLANi ON POPULATIONS OF PHYTOPHIH 

when infested soil at -125 mbar was periodically flooded, 20, CITROPHTHORA ANDC P ARASITICA IN RHIZOSPHERE SOIL OF CITRUS. 

53, and 100% of the plants died after 1, 2, and 3 flooding 

periods, respectively. At -25 mbar, 0, 80, and 100% of the L.M. Dandurand and J.A. Menge, Dept. of Plant Pathology, 

plants died after 1, 2, arnd 3 flooding periods, respectively. University of California, Riverside, CA 92521. 

Only one of 15 plants was infected in soil at a constant 

soil-water miatric potential of -25 msbar. These results The influence of F. solani (Fs) on populations of P. citrophreinforce 

field observations in which heavy rainfall with thora (Pc) and P. parasitica (Pp) was studied by sequentially 

subsequent flooding of the soil was associated with incrase sampling the rhizosphere and roots of citrus which were either 

in dseas ogrss.pre-coionized or not with Fs prior to inoculating with 

in dieaseprogesszoospores of Pc or Pp. Initial rhizosphere populations of Pc 

were significantly higher from roots pre-colonized with Fs 

than from non-colonized roots. Six weeks after inoculating 

with zoospores, Pc populations in the rhizosphere of noncolonized 

roots were significantly higher than from roots precolonized 

with Fs. In a second experiment, when citrus was 

heat-stressed, populations of Pp from roots pre-colonized 

with Fs were significantly lower than from non-colonized roots. 

Root weights and root lengths were significantly lower in 

plants pre-colonized with Fs than non-colonized plants. 

705 

Gl31HOF GENETIcALLY-AlEE PSEXDCZ4DNAS SOLANACEAINJM 

IN RHIZOSPEERE AND NONRHIZOSRHERE SOILS. J .W. WilliamsnonI, 

P.G. H~artll and M.A. Schell 2 . Depts. of IIocmy an 

Microbiologyz, Univ. of Georgia, Athens, GA 30602 

The effect of genetic alterations of the IIA gene encoding for 

an a-l, 4-endcpolygalacturonase cm the growth of Pseixiamonas 

Vol. 79, No. 10,} 1989~ 1223"

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