SYNERGISTIC EFFECTS OF MICROBIAL ANTAGONISTS AND ...

SYNERGISTIC EFFECTS OF MICROBIAL ANTAGONISTSAND PHYTOEXTRACT ON RHlZOCTONlA SOLANI K ~~HNINFECTING GLYCINE MAX (L.) MERR.THESIS SUBMITTED TOPONDICHERRY UNIVERSITYFOR THE AWARD OF THE DEGREE OFDOCTOR OF PHILOSOPHYIN LIFE SCIENCES (BOTANY)DEPARTMENT OF BIOCHEMISTRY & MOLECULAR BIOLOGYSCHOOL OF LIFE SCIENCESPONDICHERRY UNIVERSI7YPONDICHERRY 602 014INDIAMARCH 2005

PONDICHERRY UNIVERSITYDEPARTMENT OF BIOCHEMISTRYMOLECULAR BIOLOGYSCHOOL OF LlFE SCIENCE5PONDICHERRY UNlVERIlTYPONDICHERRY - 605 014, INDIAI.. B. KannabiranaerE-mail: ushakannabiran@yahoo.comCERTIFICATEI hereby certify that this thesis entitled "SYNERGISTIC EFFECTS OFMICROBIAL ANTAGONISTS AND PHYTOWTRACT ON RHlZOCTONlA SOLANlKUHN INFECTING GLYCINE MAX (L.) MERR " submitted for the award of degreeof DOCTOR OF PHILOSOPHY in LlFE SCIENCES (BOTANY) is a bonafideresearch work done by the candidate Ms. ASHA. A. N., during the period of herstudy under my guidance in the Department of Biochemistry and Molecular Biology,School of Life Sciences, Pondicheny University, Pondicheny and the thesis has notpreviously fonned the basis for the award of any Degree or Diploma or other similartitles.candidate.I further certify that the thesis represents the independent work of theDate: 15,3,2 rc5,- I 1,:,, LC. Ci. i. cci- .L--hDR. 8. KANNABIRANRESEARCH SUPERVISOR& w m316PRO R VllA NPROFESIOR P. P. MATHUR- 1DEANHEAD OF THE DEPARTMENTPROFESSOR $ HEAD,ot?~ fir Ilo(utYiilr' 4 I,.:,, ri D ?,USPONDICHEREb UNI'IFRSITYP0NDICHtRF.Y .605 014.

MS. ASHA. A. N.DEPT. OF BIOCHEMISTRY & MOLECULAR BIOLOGYSCHOOL OF LIFE SCIENCESPONDICHERRY UNIVERSITYPONDICHERRY - 605 014INDIADECLARATIONI hereby declare that this thesis entitled "SYNERGISTIC EFFECTS OFMICROBIAL ANTAGONISTS AND PHYTOEXTRACT ON RHIZOCTONUSOLANI K~HN INFECTING GLYCINE MAX (L.) MERR" is a record ofresearch work done by me under the guidance of Dr. B. Kannabirm,Department of Biochemistry and Molecular Biology, School of Life Sciences,Pondicherry University, Pondicherry, submitted for the award of degree ofDoctor of Philosophy in Life Sciences (Botany) and has not previouslyformed the basis for the award of any Degree or Diploma or other similartitles.Place: PondicherryDate: IsMarch 2005

CONTENTSPAGE NOACKNOWLEDGEMENTSABBREVIATIONS1. INTRODUCTIOI\I2. MATERIALS &METHODS3. RESULTS4. DISCUSSION5. SUMMARY6. REFERENCES7. LIST OF PUBLICATIONSIllv1- 2728-585 9-7474-113114-118119-152153

1 am e~retm-4 gratefill to my supervisor 0. mnna6uan, @ader,Department of (Blochernutry andSMo&ctrlbr (Bwlbgy, .Srhoof of Lfle ,Sctewes, PondtchenyUntverstty, for hrc mntwatlon, valu~tibgulriance &tylvlth constant arnurqement andmgqestmm 7fu bucusstoru vlth h~in dunrlg tfu coirrse of my research prdda put&afrfencouragemenr towar& the complktum of thu wort,I wirh to express my hearty tlia* to @?of: B @ayaq Dean School of Lge.Srhes, cPondichmy Vnfuersity, for ahqg me to maif the h6oratory andinzastmrture fmfitiiv at af7 o&itimes dufjnB the pmnd of my research. I am high4than@lto Bmofessor R 8. Mafiw, !Mead Department of @wchemutry andMoli?culhriBwCoay, Schoolof Lfe SScimcs andOjfjker-In-cfiargc, c8wifirmatirs center, IPoduheny'Lhrrversity, fir hic mur(~~ement by p r d 4 wrnputatio~l and intrmet fwtlitiesduring the course of this wort; I ia& thic opportunity to eqess my tlionk to the staflof the @ioinj~nnatus Cenw, for &wingmc to use the Ll;lt(lhase fdties. lliumc~ruragcment andfie@ of 4k. 3.4 anda K, -, fdty mem6ns of thicDepartment, are fat@&act&w&~edMY Mywere r f i ~ are s due m Hkfandfaculiy mem6ns, Department of rBotany,. 7 m Laage, Port&ir Qx. ?? srrvb ad Qk. 3-, 4k.T~WIAS, 4k. Jaymdy dm.thrs Lrrl~fsrrrdy.Hahmi, for their nrcourqmat andsupport to reach1 am e@mlygratefuf m ~Doctoml(,i)mmtt&e mm6ns. Qx. Dr. w,S.G&ctum, 'h.,%. mtrc for cP. G. Stubis and0. LX 8&mathy,Y7cakrDqrtmnt of3~Kc0lnay ardBnwonmmtafS, .Scfux,~lof L#e .Scietu8s, &tdufq'Lhtiversity fortherr suggrstic~i~s andvaluablk &as.My hhcnrtfeit grantudi must aGo reach the o&1Birrfiemutry andMelkulhr rBwCngy, %c appnntineons Ms.and ~ r ya&- . -for diir help.personnelof the Wpam~UofMK Veerma@

I atn hhhly iiA6ted to Dr. ?d V:a&hivel Departmat of cBwtechmhgy forjmwufzng rnc the neededculture for thts prolect. I am thaic&5dto m. 5. &&zrhnana,flf4 Department of Earth Sciences, for prnoiding the necessary Ih6oratoty fmfities. Iam h@h(y gra~elfirf to @of: ,4.S;. R#muchfidran Naic :Department ofChLtty,R~ndiCfi"y 'LJniversity, Rmdufi fir hrcgenerosity andcnticalcomments inadyzitrg the spectralstudus.I k d y that&$ to m. ~ufu Das andm. 'tfmala, (wartmen1 of@%atlt Patfubgy,CollEgc dpgncdure, Tkandrum andwearch Scholhrs i fTWV, Coimbotme for theuvdua6h rounseGg andprod? the nee&dinftmtion with esteemedcnnmn.I qrcss my than.f,v to my seniors Qk.Q gomathi, cDr. R,So#cheandDrACka fm m 6 f i me to ham the 6nsic techniques andtfiw support during the wwsc ofmy w& I uwulii fikt to thank my cofiagues a n d w , W. W'athrlbgmn,$cgnwri, fMr. Girirh andothn cofiagues of this c~epartemtn for thek time5 he& andsupport at some pod of thls research 'WM~ I am aLro t/in&dto myfnendF m. Dr.-*. @&+ W. @&a and Dr. R@ for thr mggestwm, fimithss sup tliat6acW me at all stages and &pt me going atiead m spr'te of a.a trou6h. Ny fuicrvfebto Ms. *a&hz, W. Haritk Mr. (DCY Mt SLfjini Ms. Qlkljmn andothertiostclmatesfir thca momlsylpm andma6ng my stuy in the hstcCa memora6b ow.Mr.I rlurr& myfind NK BmMn fir liir ~ppirt, cooperation, encouragement andpnniding tfi necdidsourcc of rmprmtwn ddng my w&.rlwTa'mosi cf&&MI it1 wrd( tfit easi[Y.I o w tmmely to my parents for cwrythi~ that cannot &put

APSUSACBBEDTAHEPES0-MEI ~ MI'C APMSI;POPPOSAllSDS-PAGETBATCATEMEDTLCTr~sAmmonium persulphateBovine serum album~nCoomassie brilliant blue diarnineEthylene diamine tetra acetateN-2-hydroxyethyl p1perazine-N'-2-ethanesulphonrc ac~dP-rnercaptoethanolMill1 molarPerchioric acldPhenylrnethylsulfon).I fluoridePeroxidasePolyphenol oxidaseSpecific activ~l). unitsSod~urn dodecylsulphate -polyacrylarn~de gel electrophores~sThiobarbituric acidTrichloro acetic acidN N N' N'- tetrarnethyl ethyleneThin layer chromatographyTris (hydroxymethyl) arninornethane

GenernlThe world's population is currently estimated to he 5.4 billion and is expectedto stabilize at 11.5 billion after 2100 The global area under crop production isapprox~matcly 1.5 billion ha. An increasing population demands a corresponding risein food production, but despite dramatic increases in production during the last 50years the world population is still affected by food insecurity. No matter how cropproductivity is affected by the environment or by the farmer's practice, theimplicat~ons of the growing imbalance between global population growth and foodsupply are contcnual source of debate (Weber. 1994)The rise in number of people creates enormous pressure and demand on foodsupply resources To meet these demands, man has developed a range of crop plants.of wh~ch 10 to 20 species are highly evolved and domesticated which provide 80 to90% food rcqulrement of the world. Enormously two cereal grains i.e. wheat and rice,predominate the large proponion of cultivated area of the world. Wheat accountsabout 508 x 10" ( 16 3%) and rice 485 s 10" ( 15.6%) in terms of world production(FA0 production yearhook). At the same time leguminous crops occupy nearly onequarterof the world's dietary protein requirement. World production of leguminouspulses accounts ahout 55 s 10" (1 .R%) and soyhean alone 92 x 10" (3.0%) (Langerand Hill. 1994)Sufficient micronutrient level in the dally diet is one of the prerequisites forhuman health. Est~matesuggest that some 81 5 million people worldwide suffer frommicronutrient deficiency. Though a number of leguminous species are known to meet

- --the dietary requirement of man, the inevitable importance of Glvcine max (L.) Merr.(soybean) in the farming system and dietary needs of the growing population has beenwell recognized in the recent past. Ind~a is the fifth and second in world and Asla inaccordance with the area and production of soybean (Bhatnagar 1994).The seeds of soybean have been used for centuries as food in Ch~na. Japan andKorea and constitute a valuable protein supplement to the rice diet. Other Asiancountries in whlch soybean is grown to some extent are Ph~lippines. Indonesia,Thailand and India. It IS also cultivated in America, Canada, Brazil, Argentina, USSR,Germany, Romania. Bulgaria, C;rxchoslovakia and Yugoslavia. In Ind~a, it wasintroduced in as early as 1880s and is now being cultivated mainly in the western andsouthern states such as Raiasthan, Madhya Pradesh, Maharashtra, Karnataka, AndhraPradesh and 'Tam11 Nadu. It is now cult~vated in other states also like Assam, Orissa,West Bengal, Man~pur, the Khasi and Naga I-lills, Kumaon hills of U1' and in Punjab,Himachal Pradesh and Kashmir upto an elevation of 1830 m.GIydne marG/ycme mar (L.) Merr (soybean) is a subtropical economically importantcrop plant wh~ch 1s cultivated both In temperate and tropical reglons of the world.Though G, mar is essent~ally a sub-tropical plant, ~ts cultivation extends to temperateregions, upto 52 ON lat~tude Numerous forms differing in cultural and botanicalcharacteristics exis1 in this species. They have been broadly classified under twogroups, the northern or erect types bearing rounded. l~ght coloured seeds and thetropical types with trailing or semi-trailing habit bearing flattened, dark colouredseeds. The erect types are probably evolved from the trailing or semi-trailing ones. Itdoes well under high temperature

TaxonomvG. mm belongs to the family Leguminosae and sub family Papilionoidae. Itis an annual herb. which is usually sub-erect or twining. bushy and rather leafy1,eaves are alternate and trifoliate except at the first two nodes. Petiole long: leafletgenerally ovate and lanceolate in shape; flowers generally small, whtte or purple incolour borne on short ax~llan racemes: sepals five in number, connate: petals littleexserted. stamens ten, monadelphous or at length diadelphous; ovary sub-sessile;styles short and lncurved, sttgma terminal and capitate; pods often small, flattened orcylindrical. in clusters of 3-5, densely haity: seeds 1-3 in a pod, or more sometimes;seeds elliptical, compressed, yellow, chocolate or black-coloured. Soybean isprimarily a short-day plant and sensitive to photoperlod.Climate nnd soil conditionIt is generally raised as a kbariflcrop and seeds are sown during the period ofmonsoon from June or July and harvested in December or January, three months crop.It thrcves well in the moist cl~mates and shady sttuatlons; it can be grown also in thedrier parts under irrigation. Soybean is raised either pure or in mixture with maize. Insome pans of Assam it is grow along with aus paddy. Being a legume, soybean canhe cultivated with advantage as a rotation crop with potato, as in Assam, or withsugarcane as In Rihar. It IS grown as a green manure or cover crop in tea estates It isalso ven effecttve In keep~ng down weeds and in pre\,enting so11 erosion.The plant thr~ves best In rich sandy or clayey loams or well-drained alluvialsoil. It can grow In acid as well as in neutral or alkaline sotls. In acid soils, rt respondsto the applicat~on of lime. When grown on product~ve soils, the application of thenitrogenous fertilizers is not necessary, as it IS capable of utilizing atmospheric

nitrogen through the symb~ot~c bacter~a present In 11s root nodules In USA and fewother countries, artificial inoculation of nodular bacteria has been adopted withbeneficial results. In India, however it is not required It grows well on all types ofsoils.OccurrenceC. nrax IS bel~eved to he der~ved from G soja Sieb. & Zucc. (syn(;. ussrrriensis Regel & Maack), a slender prostrate, twining legume which IS foundwild throughout eastern Asia, possibly in hybr~dization with G, romenfella Hayata(syn. G. romenrosa sensu Benth., non-L.), which grow wild in southern China.Because of its long history of cultcvation and more recently because of an intensivebreeding programme, a great many cultivan exist. direring in their growth habits,maturation period, oil content and composition.Soybeans are thought to be native to northeastern China, where thedomestication is hypothesized to have occurred about over 7,000 years ago. Thesoybean plant was supposed to have given to human beings by Shen Nung when heintroduced people to the art of c~vilization. According to the Ch~nese writing datingback to 2800 B.C. it was known as one of the five principal and sacred crops. Broughtfrom the orient to France by missionanes, soybeans never became popular outsideChina and Japan until 1890Soybean ranks high in various aspects among the leguminous crops of theworld. In China. it is considered as poor man's meat, indicating their importance inthis populous country. While all the legumes can be considered as partial substitutesfor animal protein, the soybean has more protein. greater versatility, than any other.As soybean contains about 40% of high quality protein (richest source of vegetable

protein) and about 20% of oil, it occupies an intermediate position between legumesand oilseeds. Soybeans have less carbohydrate per unit weight than any other pulsesexcept peanuts. The flour obtained from the seed is very good for dtahetes stnce it islow in carbohydrates and hlgh in proteins. All the essential amino acids are present.These factors combine to make soybeans the most important legume crop in theworld. In western countries, soybeans are used primarily for oil extraction and animalfoodBiochemicnl constituents and chemical nnturrGreen seeds are occasionally used as vegetable. Black seeded varieties arerichest in protein and have a low percentage of 011. On the other hand yellow orchocolate seeded forms have htgher 011content but low in proteins. Soybean seedcontains moisture (5.02-9.42%); protein (29.6-50.3%); fat (13 5-24.2%); fiber(2.84-6.27%). carbohydrates (14.07-23.88%) and ash (3.03-6.35%). The averagevalues reported for the Indian seed are as follows, moisture-8.1%; protein-43.2%;fat-19 5%; fibre-3.7%; carbohydrates-20.9%; ash-4.6%; phosphorus-0.69%. calcium-0.24% and iron-] 1.5% The composition varies according to the type of seed beingculttvated, soil and cltmatic conditions. As a ~ lwhen e the protein content is high, theoil content is poor. Thc fatty acld constituents of soybean oil are- oleic acid- 23-34%ltnoleic actd- 52-60%. palmitic acid- 7-14%. steanc acid- 2-6%, linolenic acid- 3%and higher saturated fatty acids upto 2%. Also it contains the glycosides genistin anddiadzin (daldzin) and four saponins. The chief form of proteins are globulin(glycinine), accounting for nearly 80-90% of total seed protein. The amino acidcomposition is as follows: cystine I. I%, methionine 1 .8%, lysine 5.4%, tryptophane1.7%. threonine 2 1%. leucine 9.2% isolucine 2.4%. phenylalanine 4.3%. tyrosine3.9%. histidine 2.2%- valine 1.6%. arginine 8.3%. glycine 0.7%, alanine 1.7%,

- - -aspartlc acid 5 7%. glutamrc acrd 190% and prol~ne 4 3% Bes~des thrs, anotherglobulin (phaseolin) and an albumin (legumelin) are also present. The decorticaredbean contains about 12% polysaccharides (dextr~ns, galactans, pentosans and 1%starch) and 12.5% sugars (sucrose 6%; stachyose 5% and ramnose 1.5%). Besrdes thetrue proteins, soybean seed contains the following nitrogenous substances in the freestates: adenine, arginine, choline. glvc~ne. betaine. trigonelline, guanidine andtryptophane. The total non-protein nitrogen varies from 2.8 to 7.8% of the totalnitrogen. Mature soybean seed, like other pulses, contains small quantities of carotene(1 10 IUIIOO g). Soybean contains several vitamins, especially of the B-complex typeand some important minerals. It has more of calcium and phosphorous, more thaneven peas and beans. It is also rich In iron. potassium and magnesium. Chinese wereso dependent on soybeans that they managed to overcome the deficiency of vitamin Cin their diet by using the sprouted beans while making salads. Soybean containsamylase, urease. lipoxidase. lipase, peroxidase, protease, glucosidase, carboxylase,catalase. ascorbrcase, allantoniase, phytase and uricase. It is a good source ofP-amylase. Soybean urease is employed as an analytical reagent for the estimation ofurea in physiological fluids. Lipoxidase in the form of soyflour exhact finds use as ableaching agent for bread flour. Soybean contains a variety of pigments-carotenoids,isoilavonc glycosrdes, anthocyanins and chlorophyll The glycosides present includegenist~n [C2,H2,,0~, mp 251-256' (decomp )] whrch yrelds on hydrolysrs glucose andgentstern(5:7.4'-trrl~ydroxyisofla\~one; mp 296-29S0), daidzin (C~IHIOOV;mp 234-236") \r.hich yield on hydrolysis glucose and daidzein(7.4'-dihydroxyisoflavone; mp: 323") and 4 saponins Three crystalline isoflavoncshave been separdted from the germs, one of which is identified \\.ith biochanin C[CaHl+04N1; mp: 310" (decomp.)] isolated from gram (Clcer arietinunt Linn ), the

second substance is spar~ngly soluble in alcohol and crystall~ms in colourlessprismatic rods, mp: 322-323" (decomp.), the third substance (CI~HIZOJ ) which ismore soluble. cvstallizes also in colourless prismatic mds, mp: 31 6-31 7" (decomp.)and is identical to tatoin, which is now regarded to be daidzein contaming genlstein asimpurity. Soybean lecithln is the term appl~ed to the total phosphatides (I.5-2.5%) ofsoybean. It is ohtamed as a by-product of the soybean oil industry. It is yellow, wax-like material consisting of lecithin (29%), cephalin (31%) and inositol phosphatides(40%) (Anonymous, 1948).Medicinal valueNowadays, soybeans are widely employed for the industrial production ofantlbiot~cs. l'odav, most steroids which are w~dely used to treat arthritis and toachleve contraception are made from natural compounds called sitosterol andstlgmasterol, a product of soybean processing. Stigmasterol and sitosterol arepromising materials for the synthesis of hormones. Soybean tocopherols have beenused as antioxidants for vegetable oils. Recent research studies indicate that womenwho eat soybean r~ch dlets are upto eight times less likely to develop breast cancerthan other women, probably because of the presence of antiestrogen compound suchas phytoestrogens (Hill and Sharma, 1996).Economic lmportmce and usesSeeds are used In the preparation of certain confectionary products.Foodstuffs made from soybeans are curd, cheese, drinks, sauces (soy), "greens"(sprouts) for salad and oil. The dried or fresh seeds of the soybean are also eatendirectly, but In the orient they are used in the more processed form to provide varietyof nutritive products. Of these, tofu, okara, soya milk and soya sauce are the most

important ones. In maklng tofu, drled soybeans are soaked, rinsed and then crushed inwater. The slurry is heated and the liquid decanted. This liquid is called as soy milkwhich is used as drink for the nondalry infant formulas used in Unlted States. Thesolid portion left after decanting the mllk is called as okara, a spongy mass that isused as cheese. To make tofu, the soy milk is used. Curd is scooped, dralned andallowed to settle into firm. smooth-textured tofu cakes. Tofu is extremely nutritious,very digestive and quite bland. Soy sauce IS another product made from these beans.Fermented cakes. after they are soaked and rinsed are used as cheese in China.American soya sauce is often the product made by flavouring salt water rather thansoaking fermented soy cakes. In India, soybean has not attained much importance as afood crop. The seeds are consumed locally after splitting as dal. They are also parchedand used as bhuqa or ground into a meal (satfu) and used in food preparations; afermented product is prepared from soybean in Manipur. As it possesses acharacteristic nutty or bean flavour which is not much flavored in India, efforts havebeen made to select types devoid of the flavour and to popular~ze the use of soybeanin non-cereal catering organization for such preparation as porridge and biscuits.Considerable work has also ken done to popularize the use of soybean 'milk' (Beryland Molly, 1986)The oil obtained from soybean is semi-dry in nature and IS used in cooking. Anotable feature of the percentage distribution of fatty aclds in the oil is the constancyIn the ratio of saturated to unsaturated acids, irrespective of the total amount of oilpresent in the seed and the iodine number of the extracted oil. The percentage oflinoleic and linolenic acids increases more or Iccs regularly with increasing iodinevalue of the oil, while the reverse is the case with the oleic acid. A large proportion ofthe soybean oil is used for edible purposes in the United States. particularly as a salad

oil and for the manufacture of margarine. It is also employed for packing sardines,tuna and other kinds of fish. It also finds use in industry for the manufacture of soaps,glycerine, grease, lubricants, waterproofing material, oilcloth, putty, palnts, varnishes,candles. printing ink, resins, ~nsecticides, disinfectant, leather dressing and linoleum.Soy-lec~then, an Important by-product of the oil industry, is also used in the foodindustries as an emulsifier and in the cosmetics, pharmaceutical, plastic industry andin detergents. Soybean meal is suitable for live stock and poultry feed and is used inmaking plastics (Sen. 2000).Before 20th century, it was not used as livestock feed because the rawvegetable contains a hypsin inhibitor Trypsin is an enzyme that is necessary foranimal digestion of protein. In the early part of this century, it was discovered thatheating destroyed the inhibitor. As a result of this finding, soybeans becamecomponent of animal feeds and worldwide demand for the crop started It is oftengrown as a pasture, forage or fodder crop and used either as hay or as silage and alsoas green manure. Soya meal, the residue left after the extraction of the oil, is a richprotein feed (40-48%) for cattle, pigs and poultry. The meal is also used in themanufacture of adhesives. sizings. waterproofing, plastics, synthetic textile fibre.foaming solutions and as fertilizersSoybeans were not commercially planted in the United States until 1920.S~nce then there was a rise in the production In the soybean crop that they became theworld's largest supplier of soybeans together with China and Brazil and the beanbeing called a "C~nderalla crop". Other important producers are China and Argentina

with some supplies coming from India, Canada, Indonesia and Eucadir, Italy,Thailand, Russia Federation, Korea Republ~c and Colombia.DiseasesLosses due to various d~seases is one of the important causes for holding thenational productivity of soybean in India at low level of Itonlha which is half theworld average. In lnd~a major diseases of soybean are bacterial pustule, pod blight,yellow mosaic virus, Rhizoctonia aerial blight, Myrithecium and bud blight and majorinsect-pests are green semilooper, girdle beetle, stem fly and blue beetle (Bhatnagarand Tiwari 1993). So far 20 pathogens belonging to fungi, bacteria, viruses andnematodes have been recorded in soybeanSome of the major sovbean diseases are listed below:ILeaf and stem diseases of soybean.+ Bacter~al blight: Pse~tdomonasyringae p1 gl,vcineua Aeria!/fol~ar blight. Rhizoctonia solani* Frogeye leaf spot: Cercospora soiino+ Cercospora leaf bl~ghl Cercospora krkzrchrra Broun spot. Septoria glvcmes+ Pod and stem blight: Diaporthe phaseoloruni var, sojae and Phomopsisrongrcolu+ Downey m~ldew, Peronospora ntanshurrca+ Powdery mlldew: Microsphaera manshurica+ Soybean mosaic Soybean mosaic virus2. Root and stem diseases:* Phytopthora rot: Phytopthora sojae4 Rhizoctonia stem rot: Rhizoctonia solani

3. Root and lower stem diseases:& Sclerotinia stem rot: Sclerotinia sclerotiorum& Brown stem rot: Phialophora gregata& Sudden death syndrome. Fitsariirm solani4 Charcoal root rot: Macrophomina phaseolina4. Soybean cyst nematode: Heterodera glycinesAmong the soybean diseases, foliar blight caused by Rhizocfonia solani KUhnis considered to be one of the most menacing diseases which have been reported tocause yield loss of about 40-50% (Anonymous, 1975). R. solani has been reportedfrom most of the soybean growing regions of the world (Sinclair and Beckman, 1989)and causes substantial yield losses (Tachihana, 1968; O'Neill et al. 1977). Especially,foliar bl~ght causes comparatively heavy loss in the yield and standing crop in thewarm and humld parts of lnd~a (Dubey, 1980). Soil of Punjab. Haryana and WesternUttar Pradesh are totally infested with R. solaniHardly any full term potatoconsignment from these regions is free from black surf disease. R. solani is foundhoth on the seed and soil (Sen. 2000)PnthogenR. solani occurs globally and causes various maladies starting from seeddecay. damping oil', root and stem rot, canker, sheath bllght and ear rot in moncotsand dicots. It has a remarkable capacity to remain in soil saprophytically in theabsence of hos~ plants (Parmeter, 1970). R. solani can survive in soil and in planttissue as dark, thlck-walled hyphae as a result of parasitic or saprophytic colonuationof the plant tissue (Boosalis and Scharen, 1959) and also as dormant sclerotia (Naiki,1985). Typically, these propagules are concentrated in the upper 10 cm of soil and can

emain for several years in soil and plant debris and serves as primary ~noculum fordiseases caused by R. solani on subsequent crops (Herr, 1976; Mac Nish andDodman, 1987).Twonomy, structure, occurrence nnd di.~lrlbutlonR, solani is a soil borne pathogen belonging to Fungi Imperfecti and has awide host range and extensive adaptability to various environmental conditions. Theform genus Rhizocfonia contains 15 species, Since conidia are not produced, thetaxonomy of the genus is very dltficult. According to Parmeter (1970). the genusRhizoctonro is a heterogenous assemblage of mycelia of Bas~d~omycetes,Ascomycetes and Deuteromycetes R solani frequently exists as thread-like growthon plants or in culture Asexual spores are not produced and thus the fungus wasplaced in a taxonomic group with sterile mycelia only (fungal thread). Charateristicsof sexual spore formation and the structures which produce them belong toThanafephorus cucumeris (Frank) Donk. (perfect state), a basidiomycetes.Infrequently. sexual spores are observed on diseased plants. Species of Rhuoctoniaare facultative necrotrophs. Speclalised structures-basidia are produced for theformation of the sexual spores. Four spores are produced on each basidium. Basidiaare formed when the environment is moist and sufficient growth of the fungus hasoccurred. Basiodiospores are wind dispersed and germinate in the presence ofmoisture. Each basiod~ospore has a single nucleus. The hyphae produced bygerminating spores will fuse (anastarnose), forming new hyphae with a combinationof nuclei. It has been observed that basidiospores are not formed in the host tissue.Several methods have been developed to produce basiodiospores in pure culture butstill remain difficult. R. solani produces thread-like structure called hypha. Thehyphae ofR, soloni have the following characteristics: some shades of brown; a

special type of cross wall within the hypha, called dolipore septum; each cell ismultinucleate rather than binucleate; branches are produced at right angles; no asexualspores are produced. In general, the growth rate of R. solani is very rapid and a typicalisolate can grow across a YO mm petri plate in three days. Small, oval cells producedin branched chains or clusters are formed. These are called moniliod cells and haveslightly thicker walls than the mycelium. Large aggregates of these cells are calledsclerotia which are black to brown and 3-5 mm long. Parmeter (1970) has broughttogether and integrated all of the available information on R. solani in all it variousaspects, especially ecology and physiology of R. solani that directly or indirectlyprovides access to all the important work on this fungus.The hyphal cells of R. solani have many nuclei (commonly 4-8). Isolatesbelonging to R. solanr are classified into different anastomosis groups (AG) based onhyphal anastomosis, cultural morphology and pathogenicity (Sneh er a/., 1991).Currently, there are 12 AGs to which isolates of R. solani are assigned, Isolates ofR, solani from soybean belong to Ati-I, Ati-2-2, AG-4 or AG-5, although isolatesbelonging to AG-3 have also been reported to infect soybean (Jones and Belmar,1989; Nelson et 01.. 19%). AG-I and AG-2 are further subdivided into AG-I IA,AG-I IB and AG-I 1C and Ati-2-1 and AG-2-2. Subgroups AG-1 1A and 19 areprimarily foliar pathogens and cause aerial blight and web blight, respectively,whereas isolates in the remaining groups cause seed, root and stem rots. Thisdlstlnguishes it from similar fungi that have only 2 nuclei per cell. Those fungi withhyphal characteristic s~m~lar to R. solani, but with onl! 2 nuclei per cell, are calledbinucleate types and are generally non-pathogmic. Certain fungi closely resembleR. solani in mycel~al characteristics but possess predominantly binucleate hyphalcells. The perfect states of some of these binucleate Rhiroctonia-like fungi (RLF)

have been identified as species of Ceratobasidium Rogers (Parmeter, 1970). Th~sgroup of binucleate RLF has been shown to possess discrete anastomosis groupsimilar to those within R. solani (Burpee eta/., 1980).Shenvood (1969) and Ogoshi (1987) divlded follar blight ol'soybean into twotypes on the basis of symptoms: aerial blight caused by R, solanr AG-IIA(=Shewood's types and web blight caused by R. solani AG-I 1B and AG-I IC(=Shewood's types 1and 3) Aerial blight is characterized by the production ofsasakii type sclerotia on diseased tissue. The pathogen spreads in the canopy bymeans of mycelial bridge between leaves. Ogoshi ( 1 987) subdivided R. solani AG-2-2into two intraspecific groups (ISG) based on pathogenic specialization as reported byWatanabe and Matsuda (1966). In Japan, isolates of AG-2-2 1116 (rush type) causessheath blight of mat rush (Juncus effuses L.var, deciprens) and false sheath blight ofrlce (0. sariva L ). isolates of AG-2-21V (root rot type) cause root and crown rot ofsugar beet. Isolates of AG-2-2 1lIB represent a high temperature group capable ofgrowing at 35 "C. \+hereas isctlates of AG-2-2 IV do not grow at 35 'C Recently,researchers have identified pathogenic cultures of R. solani AG-2-2 from soybean as1118. Engelkes and Windels (1994) reported that an isolate of R. solani AG-2-2 frompinto bean showed h~gheroot rot rating on sugar beet than did an isolate from sugarbeet. but the culture d ~d not identify to ISG. Schuster and Harris (1960) reported thatisolates of R, solani from sugar beet were nonpathogenic to field beans, butanastomosis grouplng of their isolate of R. solani AG-2 that was pathogenic to sugarbeet was also pathogenic to bean. Kuninaga et at. (1979) detected a high incidence ofisolates of R, sobni (AG-6) in non-cultivated soils in Japan. Isolates of this groupwere only marginally virulent, whereas isolates of AG-I, AG-2 and AG-5 exhibited arange from weak to highly vtrulent. AG-3 isolates were reported to infect potatoes

---- --primarily (Chand and Logan, 1983) and AG-4 was common in the field and wash~ghly virulent on a vanety of hosts Recently, Christopher et a1 (2004) havedeveloped a technique called rapld ~mmunochromatographic lateral flow devlce(LFD) uslng hybrrdoma technique which allowed the product~on of specrticmonoclonal antlbodies (MAbs) and enabled the development of immunodiagnosticassay that detect the activity of the growing mycelium of R. solani alone. Accordingto pathogenicity, cultural morphology and hyphal growth rate it is possible todistinguish three different cultural types within R, solani. Salazar etal. (2000) showedthe complete DNA sequence of the ribosomal internal transcribed spacer (rlTS)region of 28 isolates of R, solani AG 2-2 from the previously identified cultural types.R. solani differ in their rDNA ITS region which correlates with the difference in theirbiolog~cal properties (Boysen er a/.. 1996: Kuninaga et al., 1996; Salazar et a/., 1999).All the data available show that there is a completely conserved 5.8s region, but showvariation in both ITS regions, and this variation in size and nucleotide sequence isgreater in the isolates which are different by anastomosis, host range and otherbiological criteriaEpldemlolo~~Epidemiology of the pathogen is that it can survive in soil, within the diseasedhost material as sclerotia It can persist in soil for years, since so many plants are hostsfor this pathogen. Symptoms on the plants vary from reddish-brown to bmwn spots,which can be sunken and can expand to kill the host plant These rots inhibit growthand cause stunting and poor vigour. Callus formation and thickening of the cell alsooccur. Leaves are covered with fungal threads. Being a pathogen of great diversitywith wide host range and lack of sharp differentiation among its specialized strains,R. solani finds great difficulty to develop resistance in crop varieties. Some of the

--funglc~des. vlz , thirem, pentachloron~trohenrene. captan, benomvl. carboxln, etc ,known to control H solonr have adversely affected the soil and human health maklngthew use counter prnduct~ve (Parmeter. 1970)('hemirnl ControlChcmtcal control IS one of the classtcal methnds of disease management and aWI& spcctrum of diseases IS controlled by \.annus modes of applical~on of fungicidessuch as sccd drcsslng. fol~ar spra? and sot1 appl~cat~on (De\ and hlar).. 1986. Roy andSatkta. 1976) S~nce the dlscoven of Hordeaux mlxture in 1846 and 1882, the rolepla\ed h the fung~c~des In the agr~cultural sector has heen diversified and changeddramat~call\ (irentcr demands for chem~cals to control agricultural pests and diseasevectors resulted In suhrtanf~al gro~th In the production of chem~cals and also thequantltla relwxd Into thc en\ lronment The estent to \+ hlch the mb lronment food.fd. mt ctc hud become conwmlnatcul \\as revealed b!major Improvement Inanalytical ckm~stn In tk late lv5(k and earl) I')hOsAner the cnlrodw-twn of organlr p~st~cdcv on a large scale In the decadefc)llo\rlng thc Scrond World War, fklr sp~~culur knefits \\ere qulckl! rrcognuedand tk truhnolog? HIIS rap~dlh wk~ptrd on a \\orld\c tde has~s 'The lntrnductron of thes~stcm~ 1Lnp~lJr.s brought re\olul~on In tt~c I'unp~ode dlsco\cn and Inipro\cmnt Inl'anncr's pmclre I;ungrc~&r arc Icirlc subsmnc~~ USL?? to Llll or tnhlh~the gro~th oflung1 that etthcr cause cxx)nomtc damugc to thc crop or ornawntalplants orc%hingcral tk health of hmatrc animals or humans Ober a umc. ekiessl\e ux ofsynthcltc compounds has multcd In srr1ous problc'ms. IlLe. &\.elopment of Insectrcststancc, pestwlde ~nducrd nsurgcncc of path>gens, adverse enk-ts on non-large1orgunurns such as hc na~ural mles ol' ~nscut-parasltrs, predators. honeybees.

pollinat~on. fish, blrds. cattle and human be~ngs. 01' late, the alarming rlse In thepesticide cost adds to the need of not relying on pesttcides alone for agricultural pestcontrolIncrwslng en! ~ronmcntalin/;lrds due to the lncrzasltig usr ol chc~n~cals haizbrought changes In the managcmt'nt practlce lor thr control of pest. pathogens andwwQ ('hemical Ikrt~ll~ers. pc~tlcldes and cropplng practices hate brought togetherundes~rahle changes In the nature ofthe so11 Regular and continuous use ofthese hasbrought m ne& prohlems Most alarm~ng among the drawbacks are pollut~on of theen\ tronwnt. restdual tox~c~t! ol' prsttcrdes, developrnenl of resistance In pathogensand ~mect pests and alw~ &veloprnent of harmful envtronment Ibr human bangsChem~cals ha\e plabed an important role tn protecting agriculture crops fromthe dreadful dtscasn At the utrne tlme the\ have hrought In harmful effectsResponu of pthogem to \arms chernlcal fungicides dtffen cons~derahl\ Vanouschemical funpt~l&\ llLe organom~.rtur~als kn~~mldamle< p\ nmldlnes$en+ Iamldc~ d~carho\lrnld~> ~arho\lrn~d's and rnorphol~ws shot4 \ ~r1ou5 degreesol 1nhlh111)n mtton agatnst \ arlous pathogens (I let\ In 1994)In general, pcst~clde seed tratrnents are done In almost W)OO/O of all food cropsbul st~ll h\e la oun dra~bacls i:unglcldes d~d not succeed 111 all crops. Forcmplc. Soybccln crop m On~t~rl Sliltc~ mid crrcals In burope are gmwrall! aKectedbj, thc latc-season fungal d~seau and poudrn rn~ldeu dl%respa.t~\el! before theux of systcmr funp~c~dcs An earl) trualrnent \\~th knomyl dela!s senescence andma) k able to Increase )#Id uplo 1090 through the comhlnat~on of fung~ctdes andplant growth hormone3 Selerel studles were undertaken to find out the tnh~h~toneffws of chcrnicelfunglcdrs ~ncluding cahutn. penbchlomn~trokmne.

din~troanlline and tnfluralin on R. soluni (Bauske and K~rby, 1992) Response of R.sol an^ was found to differ cons~derably in terms of lnh~b~tory effects to these chemicalfungic~des.In vleu of the rncreilslng toxic elkts such as non-degrdable and persistentresrdues causedthe continuous and constant use of chem~cal fungic~des, researcheswere lnltlat~d on Ihe b~ocontrol of' varlous diseases 'The hlopestic~des have seberaladbantages o\er chemical fungicides (Suseela Bha~. 2000) Hiopestlc~des are usuallyIcss IOKIC than conventional fungicida Thc? gmerall? affect onl) the targetpathngcn~c organlstns. In contrat to hrnad spectrum. con\ent~onal fungicides thatma\ affc~t orpanlsms as d~ffer~nt as hlrds. In%Tb and mammals Also the!areclliirl\e In 1-small quantities and oRcn decompose qu~ckl!. thereby resultinglourr elpmurcs and largel\ etoldlng the pnllutlon prohlems caused h> the chemicalfung~cldesllse of htolog~cal control to varlous fungal diseases is nnt a new concept Ofthe var~ous hiolog~cal pest control methods. cultural contml. gmwing resistantvandies. mrchan~cal rnethnds. phvs~cal contml. h~oloplcal control. genetic methods.cmaol h uslng plant products form a sort of hrtter control approach Micmhes.mirmhial mrvrholrtcs and plant c.itracts are supposed to induce resistance in cmpplan& agalnst s \vlb ranFc of parhogens Thc efic~ent use of indigenous soilmlcrohlal antapnlzts and ph~lw~tracts In a consonlum for greater stah~lip. andrcl~ahilln rather than trirh single spec~cs or strains for acrompl~shmnr of ~mpmvedhinlogical control In cmp poductlon can he annrhcr optlon

Recent reports In the field of blocontrol in controlling crop diseases includethe use ofantagonrstlc m~crobes e~ther slngly or in comblnat~on w~th a low dose of thechemical pestlcldes or botanicals Hlolog~cal control by means of bacter~a holds greatprornlws for wntrolllng fungal diseases Fluorescent pseudomonads are non-sporeforming bacter~a and thus are sensltlve to des~ccation and other adverse environmentalconditions The challenge IS to Improve the b~ocontrol systems to make them morerellableSc\eral studies havc ~dent~fied agents ~hich are used as d~sease antagonlstsAntagon~sm that trcurs hetween fungal pathogens and fungi. hacter~a or plants offersoppoflunltl~? lo exploit new rncxk.s of' actlon Betng a partner In the Integratedmanegemnt s)i?slrm huse 01' 10% cost of development, small scale marketproduct~on and pafllcularl) l~rs commsrc~al competltton. th~s 1s complemented for theW~t~onal approach for the protcvtron of cereals. rice, grape\ines and top fruitsKwcnt ach~cvernenls In the lii?ld ol' hloconuol of crop dlseases are encouragmg.l.!nch (1988) rrponcd tk swccsst'ul wc of hlocontrol agents One bacterial spxtesand fivc fungal y s l t s utre avarlablr on small scale to grown tor management offungal disease S~ncv then lot oi antagonlsts wre commrclal~zed for the control ofyb~hrunr. Hh:wtanro and ~~u.wrrum spr?c~es (Man~n. I W4) Accord~ng to Powell(IW3) the n~ahl~shcd h~olog~cal control agent 1s less ellicunt and less rel~able butsafer than chem~cal control It IULS P narro\+rr range of actl\ IF and can he I~m~ted h)the t).~ of crop or the en\ IronmwntSo h: rmllsilc one should not exwt a venhcMd mnpc of pcs~ or dlwax control from the hiologlcal agents In terms ofcnntmllinp m)or PISor pest complexes In rnqor crop In a wide range ofenvironments SIW bmlogtcal c-ontrol agents are. h!their nature. more l~m~ted thanthe equivalent chemtcals. the~r uw necds to he laqelod carefully. hased upon an

appropriate charactertsatlon of the different stratns to enable the~r dtscrlrntnateselect mnHlologlcal control agents could not perform well In the cereal market whencompared to other crop The largest use of fungtclde as seed treatment of(;aeumannomvces wamrnrs f sp rrrrrcr has been reported by Thornasho~ and Weller(1988) The expottatlon ol antagontsts to control specific unrnet needs In cereal croprnav be commerctall\ vtahle The use of Pseudomms (Cook. 1988) and Rarrllus spp(Capper and Camphell. 19th) lo control ti prmrrnrs var nrricr ISan example of apntentlal agent Agrtcultural and forest ecosvstern's possess vast mlcrobtalcommunlttes that are respons~hle for the natural suppresston of plant pests~cudommer m a pfen fid Moronhol a#ntFluorrrcent pseudomonads emplo! a nurnher of d~fferent rnechanlsrns in thesuppresston of plant pathogens and the toptc has been revtrwed eutensi\'el?(O'Sulltvan and tiara.1992) The mechantsms of hlolop~cal control lncludecompctltton. tndwed reststarwe and the prnductlon of ant~mtcrob~al substances suchas antth~otlcs, sldernphnrcs and h\drogen c\anrde Antagonlsttc Pset~&mnna.t speclesprnducc WI&varlet\ of sruondan mctahol~tes that ad\.erxl! aTiect the grouzh andrnetahnltc XII\ ICS(of the plant pathogenic fung~ The prrsence of four most cornrnnnantthlotcs found In fluorcscmt I'.ter~dornt~nn.t stratns \\ith hicxontrol actn lt? are2.4-&cc(\l phlorngluctml (I):\Ni). Phcnaltnc (Ph;.). P\rrolnttrln (Pml andRolutmrtn (PI!)I'he eficac). of nlnc lsolates of tlucorcsccnt PsruJomoms was tested agatnstXdm. F~~arrum aqspnm. Sclemrum rd/srrand Altemarra bmrrtme byShd;har uwl Cbubc ( IW) They found that tnhtbcon potenttal of all nlne hacteml

solates d~ffered s~gn~ficantlv from each other Mutha~yan (2000) reported that the rtceblast caused by Pvrtc~tIar~a omwe was effect~vely controlled by P fluorescemlomulat~on Manoranl~tham er a1 (2001) reported that damp~ng-off of tomato causedtn Pvthrum aporndennarrtm could eas~l) be reduced b\ so11 appllcatton ol talc-basedfonnulatron of I vrrrde and P fluorescem e~ther lnd~vrdually or In wmbrnatton Itreduced the pre- and postemergence damptng-ofl and also Increased the helght of theplant as well as the b~omass product~on Kav~kumar (2002) recommended the use ofPseudomorn sprav ( I (W) g rn 10 1 ol water) to control the leaf spot drsease and use ofInchoderma spp for rhl/nmc treatment to get h~gher k~eldKalathllagam (IWI and Puguknlhr (2001) studted thern~crob~alantaponlslrc effect on ( ' capsrcr Sourechc (202) reported the control of C capstcr Intunncrrc h\ uslng rn~croh~al antagontas such as herrdomonas and (;ltocladtrtmMan) mrcroh~al anlagonlsts Titchoderma spp . Pseudomonas spp and Baollus spparr: commrctalrrrd and usrd In plant pest managementTdrhoderm as a Moronrrol agar7nchuJermu spp arc common fung~, lbund In almost all t).ps of soilMemhen of this genus arc antaponlstrc to other fung~. rncludtng plant-pathogen~cspxlc!,Possrblc tw~hanrsm rniol\~d in Irrc~hcdrnw antagonism arc. Antib~os~s.where II produ~x-s s olnttlc or non-\olat~lc ant~h~ottcs (Lknn~s and Webster. 1971a. b),compcrltbon. %hers spacr or nutr~enls I c, carbon. nltrogrn, mtcroelements are thel~m~ttng factors (Sch1pp.n er 01. I9117), m)coprasttlsrn. where 11 attacks anothertLngus by excreung I5tr -me5such as, proteasss. glucanaws and chrtlnases thatenable 11 to &grade the cell wall ofthr host (Ckt, 1%).Cool. and Baker. 1983) andpnsltism. u b 7hchodermu spp raw UK death of thc host fungus (Barnett and

B~nder. 1973). Trrchaderma and C;liocladium are closely related fungal blocontrolagents Each produces antlmicrob~al compounds and suppresses disease by dlversemechan~sms. Including the pmduct~on of the structurally complex antiblotlcs gl~ovirinand gliotoxln (Howell et 01. 1993) The Importance of the lyt~c enzymes produced bylitchodermo spp In the b~ologlcal control of plant pathogens has been brought out byseveral workers ('f'ok~rnoto. 1982. Schlrmbock er a/. 1994. Haran er 01. 1996. Lew~sand Papavlzas. 19Y1) They have confirmed the antib~ot~c efiicacy of Trrchodermr,spp and (;lrr~ladtum vrrens fonnulat~on agalnst conon damping-off caused by R.snloni tiouell er ul (2000) reported that the defense response. panlcularly terpenoidsynthesis cn the seeds treated \v~th Trrchodermo spp may be cons~dered as anlmpnanl mhan~sm In the h~olog~cal control of R solanr lnclted conon seedllngdlsasc Shanna and Trlpalh~ (2001 J found oul that sevd treatment and fol~ar spray ofT hucronum and (; vrrerrt Sormula~ton uas rnixt~\e In the control of web bllghtdlseasr of urd hean caused h R solonrAccording to Howell (2002) Trichalemo treatments In other plants tndlcaethat Ihe mhiinlsm and nature ol thls h ~wntml agent IS strll ver) elus~ve and needlot more lnterprrtatlon In older I~tera~ure. the prlmaty rnechanlsms In the plant-psthogmr fungal tntersctlon were cons~dered to he rntcoparasltlsm. ant~blos~s andcompct~t~on More racntl\ man) other mechan~srn such as competltlon for fungalgcnnlnatwn el~c~tors (Ho~ell 2003) and lnhlh~tion of ermine rqulred for lnfect~on(/.~mand. 1%)d~ffmt fmulattons of

were very effecuve in reducing the disease tnc~dence as well as In produc~ng highery~eld. Harman el 01. (2004) reponed that seed treatment with T. horrianum strain 22shows substant~al elTect on the prowh of mawe inbred line Mo17 Seedl~ngs treatedwtth '1'22 were larger than untreated ones lnfestat~on of so11 with P. utimrrm had veryllnle en'ect on the growth of Mo17 It also Increased the level of proteln. &I, I3glucanase. exochlt~nase and endochltlnase In the roots and shoo&. It also Induced SRIn rnalzr agatnst ( ' pmmrcoluPhytoa?racl as a potential biocontrol agentl'h~toestracts ot' several hlgher plants have been reported to control varlousf'ungl dlsases (Srnastava and R~har~. 1997. Prarntla and Duky. 2003) Jasrnlne(lW7). Senth~l Kumaran. (1948). Asha (1999). Gomath1 (2001) and Chttra (2002)have s~ud~cd the ~nhlh~ton eltkts of (armus ph~torrctmcts on Colletorrrch~rm capsrcr(jornaiht (2001) brought out the whlbltog enects of the aqueous leaf emacts ofI-rumere/, Prroprs juirjloru and L%)lonum ronvm on (' rapsrcr lnfecttng( 'aprrorm annrrm Sr~vastava and B~han ( 1997) r e p 4 that out of ten plants tested.fresh leaf of Atadrmchta mndrcu. tblonaprs procera. Ocrmum basrircrrm stoppedmyccl~al growzh of ('un.ulono rubermlata and Altemrra alter~la Grhlot andBohra (1%)rrponed anttfungal actlvlty of certatn halophytes apalnst A solunr Thebarl and leaf extracts ol' 7bmarrr a&.Na.leaf and stern estracts of Solaola lu7nnsmu.stem and root c~tracts of Alrrplex Ient~forn,rs and onl! stern esuact of ~lulon~lonrecunrm shod tolal ~nh~b~t~on of the potato blight pathogen 'Thopill1 (2002)reponad anttmmohlal actlv~y of essential o~l of Arfemrsra nr1,qrrim. a strong11scent4 we& herb of Astcraccac The o ~l was tour to SIX hacterta and seven fung~, In vrrro mttfungal actlvtty of Ttmupra cwd~fdru Has stdtrd by Brino cr a1(2002) ustq chloroform and ethnol extracts They ohserved that mgcel~al growth ol'

oth the test lung1 was affected and the inhibitory rate increased with increasingwncenuauon Among the two solvents chlorolorrn was Sound to be superior overethanol. Cohen el a/. (2002) provided w~dence Ibr the antifungal actlvlty In plantextracts made with organlc solvents. including methanol. ethanol, ethylacetate.acetone. chlomlbrm and n-hrxane Llslng thln layer chromatography overlay assays,seven cnhthltoty zones against ('ludosporrrcm nrcltmerrnrtm were observed In theextracts Laves of Imlu vrscosa Here extracted ulth a mlxturr of acetone andn-hcxanc I hc oil\ \rater lnsoluhle paste c~ther d~ssolved In acetone or emulsified Inuatcr ckt~\el\controlled doum mlldeu of cucumber. late bl~ght of potatohomato.poudn mlldeu of \\hat and rust of sunflo~er caused b) Pseirdoperonospomc-ubrmrs. P~fopfhoru mnfesrum. Hlirmer~u grumrnrs and Helranrhirs unnirs (Wangcr a1 ?(XU )Infrprorrd approach to fhc biuconfrol sfrafegies'Ihe intcgratcd ux of h~ocontrol agents In conlunctlon wth other diseasecontrol measures holds grcatcr promises for promoting the \\idesprc.nd adopt~on ofh~ological dlsea~c: control strateglr3 In the agricultural 5 stem (Mark. 1998) Severalresearchers ha! e ~n\cstigatcd thc wr of co-application of fluorescent psrudomonasulth spxla of' 7rrckdermo or other fungi I\ ith disease control capahlllh In \leu ofthc tact that the mctaholiles prcducr.d h!I'srluforno~s spp that lnhlhit plantpthogcnic rung1 rn~ght lnhlh~t thr hlcxontrol hng~ compauh~llt\ ht\wen thesehtocontrol agents arc to hc e\aluatcd According to Dnndurand and Knudsen (1993)there uas a slight hut trnnsient Increase In the rad~al gro\!th of T horrronum from pea&swhen co-cnoculated ullh P fiort*srcn.r 3-79 strnln None ofthe file strams offlurimcent Psrt&mmxcsspp rcduc-4 the actn it\ of T h ~ n g uhen ~ r applled towhclll simulbncousl\ (Ihff\ er 01. 1995) B~olog~cal contrnl of Fusar~urn u~lt uas

hrought out bv the use of e~ther non pathogen~c I.'oxvsporum or fluorescentPsessulomonas spp I he combination of these two agents has been ver) effic~ent andconslstenl in controlling the Fusarium wilt dlsease (Lernanceau rt a/, 1992.lxrnanceau and Aiabouvette, 1W1) Appllcat~on of combination ol strains oftluorescent Pseudomonos spp on C;aertmannom.vces gramrnrs \ ar grumrnrss~gn~ticantl\ wpprewd the hhc-all dlcease than the e~ther treatment gl\en alone( L)ull\ and Weller. 1W5)Wqciech Janlsic\r lcz ( I W(l) scr~~ned 2 I !east antagonists trh~ch \$.ere testedtbr the control of hluc mold d~scase of apple In 13 different mlvtures Out of thls, 3mlxturcs werc dlstlnct~vel! Sen cff'c~tl\e and bctter than an!of the indi\,~dualtreatments BILK' mold dtwclsc. of apple caustd h!Penrcrllrtrni e.rpanstinr \\.asc~TK.~I\cI\ controlled h the comblnat~on of two yeast strains. T5-D3 and 7'5-E2brhen compared to the indlv~dual antagonist lreatmenr Enhancing b~ologlcal controlh\ uslng mlxture of hlocontrol agents has the following advantages* 11 ma! hroadcn the sprtrum of act^\ IE4 It nan? enhancc the ellicac! and reliablllt) ol'the b~olog~cal control4 It allow the comhlnntlon of varcous tralts nithout the employment of geneticenglnccrlng (Wo~clwh Janisr\rlc~ 1%)'l'he comhtnatlon of P .nrr%w and pink \east. Spombolom~res mscus. eachen'cc11ve agatnst hoth M u nmld and green mold dlxases of apple, enhanced controlof thac drscavs an mturc apple hilts (Jan~s~e\vrr and Hon. 1W5) Lernanceauel d (1992) ~m aMe io control Pusanurn n11t diseax by combining a non-palhogm~ antagon~st~~ straln ol b' c ~ s p w m wlth f'prrrtdu. whh were notefk~wc when either of thc antagonla was utad alone

The integrated use of b~ological control agents In conlunctlon wrth chemlcaltreatment can be used to Increase the level of disease control actlvity or extend theprotective act~vity durlng the course of plant development Although it is a goodalternative, this could sometimes lead to negative Impact on the non targetmicroorganisms and activ 11) of the b~olog~cel control agentsD~flerent combinations of h~o and chemical agents, like. G virens, Pongamiaglahru cakc. chemical fungicide-carhnrin and Rhcohrttm \\.ere used by Dubey (2003)for controllinp R solanr causing \rch hl~ght of urd (I'rgna mttngo) and mung bean(I' rodrura)Jessica er a1 (2004) reported that choline acetate and glycine betaineextracted from wheat when treated alone has ken repned to stimulate the growth ofI.' gumrneurunt causing Fuw~um head hl~ght of \\heat. \shereas the equlmolarmlxturc of tuo compnunds sho\\ed sl~ght reduction in gronth rate at higherconcentration &$hen compared to control Rlanca er o! (2004) hrought out the effect of~ntegratcd approach In the control of Fusar~um wilt dlseasc In chickpea caused b> Ftmspwum lntegratcd approach cnns~stcd of chiice of sowing date. use of pan~allyresistant chickpea genotype and seed and soil treatment with hlocontrol agents (Bmemcrrrrm RCiAI: 5 1. H srthsrr1r.c (;U03, non-pthogen~c F a\l:cpnntm Fo 90105and P ,fluorrscenr RG 26) 'Ik\ dcmonstmted thc Imponancr. of tntcgrated controlpractices along u.ith othcr hio contml mtmurcs to manage the Fuwrium tr.111 andincrwc the \ ~eld of chickp P ,/ltrcuesrcn~ UZ and H srrhtrlts MR 1600 weretrca~d e~thcr singly or In combination agatnsi f?,rhtum drtntum H ~ I C fauses ~damping-ofT on sugar beet xadllngs Combined treatment H ~ S found to he moreelf~t~ve than s~ngk treatment B swbrilis MB 1600 alone d~splayed no sign~ficantrntagonutlc etfect rgr~nst Avhtam ~Itamum wtm c~nparrd to P /luorurcn~ BS. but

d~dn't lnlluencr the perionnance of P ,/lrrorescens B5 in the comhlned lnocula(Schmidt el a/. 2004)Kevtc~ of Itterature reveals that several research studies ha\e ken conductedto stud\ the nnrrpisttc ellixts of dttTerent antagonists. antagonists and chemicallunglcldes. anlagonlst and phytoproducts on the control of pathogens Ilot\e\,er. therescvrns to hL' nn repon on the s)ncrglstlc lnh~h~ton cfTccects ofactl\c prlnople Isolatedfrom plant and chemical iunglcldes and products of pht?oextracts. and antagontstsand ph) tncvtrartsllrws the prcent stud! \\as undertaken to find out the synerplstlc effects ofsclrrtrd rnrrohial antapontsts and ph)tncrtract In companson br.tth the lndtvidualelrcrls of antagonistic m~croln-s and ph\ t~xxtract on X solcmr Infecttrig (; mmY@LFCTI\'ES OF THE PRESENT S'1'1'DI':81 1'0 screen the Isba of live spclcs which uvre reponed arllrr (Kuruche\e era1 . I W7) to have ~nhthlton oct~\ th and themtahlc pmprm grontng in andaround Ihe Pdrhern I Inn erstt? campus81 1.0 find out the k tplant leaf extract sho~l~gmaxtmum lnhlhttlon on rad~alm\ccltal gro~lh or H .soktnr tnl'.c.ting (i may -1.0 find out the hr~t cnmhinationof mtc-mh~ol anlnponlst a d ph\tt~\tml (m \,rro and rn vn.c)l shotrtnprnaslmum stncrgcstlc ~nhthlton clliil on N sf~kvtl At the sanh! Ilme 11 u.ascnsurrd that hnth the biological agents arc ncllhcr 11ntc 10 each other nor ha\ea stimulatun cllix-l@I To n*Ihc hast-palhogcn ~ntaxtlon by analyzrnp the bmhemtcal changestn the host piant undrr health?. tnfatd and treated CO~~~KXISEl To ~robtc and chmcicnn the #-tt\c pnnclplc pnsent m the ph?tcxsuacl

MATERIALS AND METHOD5In the present studv exprrrnents were conducted to find out the effects ofthe each oftwo mtcrohtal antagontsts and ph\toextract etther ~nd~vtdually or In cornblnatlon on R.rolanr lnfealng tilvcrne mm I.1. SO1 R('E OF 'I'll): PA'I'I1

vtz.. ptroleum ether. chloroform, acetone and ethanol were obta~ned using soxhletapparatus Three concentrattons of the above extracts (2 5. 5 & 10 %) of each of fivespecles were tested for the~r anttfungal actl\~t\ agatnst X solonr in vrtro2.3. Preparation of extracts:2.3. I. Preparation of extra& with solvents: For the screening of plants. extracts werepepred ~tth d~llcrenr orgunlr. sol\ents lh~s IS kuusct ofthe fact that \ar~ed typesol' suhslancr% extracted In d~llkrcnt solrents m~ght have potenttal ~nh~b~to~suhslanc~~ against palhogcns (Coop and Ciunns. 19'45)SoI\cnls uscd tor the cttrarllon rrcrc4 I'etrol~wm ether I o xparatc rvates. sterol, strro~ds etc (60-80 Y).' C'hlorotormAc~lonelo x-parete aihalotds 160-h2 OC')I o sc-parate reslns ( 55 i-56 5 -i')4 1:thanol 1 o separate rcwns and lo\+ plar gl! cosldcrs ( 70 "C)2.3.2. Edtadim Prorrs5: Ihr. Ica\cs rrcrc rtashed In d~sttllal Haler and dr~ed at.37 "C' I! mar then pr)urk.red and taken tbr sttractton Ihe r\tractlon uns done Insoxhlet applratus Afkcr pch~ng the r ~al tr~th cotton. 15 g of the test matertal waslill~d lthc rr-spn.tt\c wlren! rrhlch &\a\ und Ibr the extractton uas 1*1llcul In thehottom 11mh Ihe apparatus has a condmscr In rrhlch the lapour artslng rn the tlashpas- h\ Ihc. SI& ~ u Into k the condcn.rcr I krc tt conden.ses and drlps into the ho4\01' thc cktrwior, n prtn~on pisslng Into thc s\ptwn luk s~phons orer and runs tnto theIlasA, thus cmptr mg hc hhJ\ t k e\twtor I'h~s alternnt~on of lill~ng and ernpt? in$the bud) ofthe cxuac-tor \rmt on wn~tnuousl\ h\ rrh~ch ItK. %~lublc mantr Jtswl\.edfrom thr lrsl malcttal In he cxmmr rernn~nd In the Ilash. uh~le the .wl\snt *asrcpealnll) rola~~ltrrd I'h~s prxc'u; \\as repatrcf 14 to I5 trmes unt~l~qu~d In ther~phm tubc hecame ~wlourlrss (t8skc and Vuon. 19971

Unrversrty. Coimbatore and Department of Biotechnology, Pondrchem Untversitv,Pondrcherry respcttvely .3.2. In dtro screenin# of the nntogonisls nnd phytwwtrnm against R. solani: Themtcrob~al antagontsts 7' hacranum and Pseudomonas strarns were tested for therrantagonlstrc elt'ect agatnst R solanr by the followtng methods3.21. Development of ontlbiorlr-resistmt strain: Antib~otlc resistant stralns ofPscudomonas wsre developed h plating O I mi of a turbtd bacter~al suspension onKtng's d tum 13 agar (KMB) containtng nfarnplctn (195 pgirnl) Krstsrant colonteswwc wlecled and streaked on KMB \\~thout antthtottc From thls. single cnlonteswnr xlrctcd and restreaked on to rtlhmptcrn supplemented medlum Marker stratnHas oharned h repattng the process three ttmes The resistant strain thus ohtarned*a5 drrtgnated as P,flwe~cer (KiIW) and used for sun r\al wud~ru The tnhlhttoncapctt\ of the stratn Has also tested agalnst the pethogen3.22. Ikd (itlhtrr Twhniqur: I hc. rn\copras~uc action or Psrorlr~ntom~.~ strains-I'f?:. C'CiN,. I'VM?. I'\'Mt. 1 ula & Lollam and 1 kcrancrrn Has r\ a1uatr.d agatnstR SC~UJII In ItK laboralon h hc dual culture lrchntque D~rs (V mrn dia ) ot'~hern~crhtal antagonists a5 \\ell as the pothopn were cur ~ith the help at'stertl~/~d corkborcr from thc cdgc ol 5 da! s old culture and then plad apn on soltdili~ul PDArncdtumIn the caw ol' /'SFI~/((MIOM.T Stratns. tk PI)A plates \\rrc' lint lnc~ulalrdwt~h the pinhogen dtx on the first Ja!. l'nllo\rd h thr suraktng 01' the prcparcdcultun: tiloratti of Ihr PseurkwrYwurs strnlm at one c d ol' the peutplates on xwndda)lr~pltc~tes HC~C rnatntarwd li~r mch tratmnt Ik plates \\ere tncuhted?U+O 2 "C' iic 3 Ja?s lnhthrt~on of m~celtal gnn\th of H solonc h\ rach mtagontstas lbund out on 11.1~ harts 01of lhe conuol (R srdanr alone) plrvmw ol'radu~l yro~ih oi X solunr tn dual culturn to that

3.2.3. PoLFoned Plate Technlquc The mycellal growth of R soianr was measured bypotsoned plate techntque. Afkr the ster~lizat~on of' peutplates (9 cm dta ). PDArnedtum. cork borer and other glass wares in an autoclave at 121.5 "C for 15 mtn wtth15 IWsq tnch pressure, the prepared culture filterates of' the b~ocontrol agents andphgioextracts three dtli'crent concentrattons vtz. 2 5%. 5% and 10% were added tothe warm I'iIA In three Petrt plates Then the plates were tnoculated by plac~ng 9 mm(d~a) dtsc cut from the gro\rtng ttp 01- 5 days old culture of' R soian~ All thts wascaned out under the stertle condttton of' lamtnar flow chamber PDA wtthout theextract mrxl as controlI he control and treated cultures were rnalnta~ned tntr~pl~ates I'hr ~noculatrd plates were waled wtth parafilm and tncubated in the~ncuhator at ?Us0 ? "C'urAm along thc radtal IIWOn the 5 "' di~! ol' tncuhat~on. the measurement (em) brascil thc m\cel~al gro\rIh tn thc ptrrplates I he percentage ofm\crlwl groulh tnhrhlt~on Has calculated h mlng thc tbrmula of Vtncent ( 1927)C'- II , ----I. * 100c'H'krc.I -: Inhth~tlon of m\ecI~aI gro~thc' -.. 1)lamrtcr gro\\rh In control4. ('E1.1. \I Ai.1. DE(;HADISti ESX1'3IE ASSAI S:4. I. EuMcilm Procedwrc l.or p~~~nol)ttc sw! mc prndwtlon the pathogen wisgrown tn crapck's broth. supplemnt~d ~tth Frttn a.r carh~n source replac~ngsuurosc S~m~larf\. for ccilulol~tr enq mts mtcrocp stalltne ccllulos+. and carhou!mh) 1 ccllulosc w e usrd

To 50 ml stertllzed czapek's l~quid medium In a 250 ml Erlenmeyer contcaltlask. the culture filterate of' 7: harz~anum and Psuedomonos strains In thew MIC wereamended to the med~a separately 'i'ufo discs of 9 mm d~ameter were cut from thegrowtng t ~p of the 5 days old culture ot' R sokun~ w~th the help of a cork borer I'hqwcrc ~mculated In each tlask and Incubated In the BOD incubator ZSK, 2 "C for5 da\s The control and treated llasks were rnaintalnrd In trlpllcates Afier ~ncubatton.the fungal mat and 11qu1d medta were scwrated b!double la!.ered Whatrnan No Ifilvr paper placcd on 13uchncr funnel under suctlon h\ \accum pump The filterateswre further centrifuged In a htgh speed. ccwllng cenrr~tuge ar 5WO rpm for IU mlnand thc supmatant uas ud as the c w me source4.L ESI'lhlA'I'1ON OF PE

through the large arm of the vlscometer to mix the contents and the suctlon was alsoapplied to the small arm to determlne the viscosity of the mixture (I e 7xro tlme) Theclllux time of the reaction mlxture at e\.er) 30 mln Intervals for 3 h uas measured andthe percentage loss In v~scoslty was calculated by the formula\' - ---*---*'1'1, - -1.1'1.1, - 'Is 1 (K)H'kre. V = Pcrccn~ loss In \ ~woslt\'I,,- Florr 11m ol'rcxu3lon rni\ture at 0 rnln'I I --l.10~ tlm. ot'rarlton rnr'iturc a! a particular1nrr.n al1 . l.lo\r rlm oi Jtsr~llrd {rater4.2.2. .4rcqv of E*(FPMG: 'Ihc arllt II\ oi c\c*l'hlCi was assa!ed h! rncaurlng themonomeric gala-ruronw aclds rcleits~d h\ the enj?mr h! calal!rlng the pttndcgrodalron I-hc rtsulr> &\err e\prc~wd as spc~fic actn I!! unlts (SACI)Wn(lats:I I)inltrnsaI~~~latc reagentIF oi 3.5 I)ln~troultc~latc. 30 g of dlurnpnusslurn Lnrtnmte and 1 h g ~~I'wdlurn h\dro\~dc rrcre dlssnl\cul In 80 ml ofd1st11lrJ \rarer m J mdc upio I(W rnl2 Scxllurn a~wa~c-acctt~~ w~d hutlbr pll 5 23 Standard mallow Irng*rnl solullonMrtM: From t k 3 hour tnzuhtcd rearrtnn ml\turc. 2 0 rnl alyuots \\ere plpsnrdout To this. ? ml of' ONS reagent rras adhi and heated In ho~llng r\otcr hath for 10mrn lhcn 11 wasand Jllut~d HII~ I0 ml of dlsltllcrl water I'hc orange rrrlcdwr w rad 81 575 nm Ccmwol ma3 mcrintatncd uilh hiled cnntne reactton

ntxture. The enzyme actlvlty was expressed as specific actlvlty unlts One unltrepresents vg of maltose release&4.3. Ptctln Trnns Eliminnsr (PTE): (E('.4.2.2.10): The enTme PTI: cleaves pectinclthcr randomlv (endo) or termlnallv (cvo) thereh\ reducing viscos~t?. of substrateand produces 'TRA reacttng substances I-ndn-PTE actlvln. was determined b!,mcasurlng the loss of vlscastt? In the rnlsture and esn-PTE h> determtning theproductton of THA reactlng suhstances (Mahadevan and Sr~dhar. 1986)Reagent:I I3or1c iic~d-horax butter. pll 11 7Srbslrntc prepamtion: I% of' pedln \\as prepared In hor~c ac~d-bra\ huner Thernrxturr was kept at S W "C' In the water hath and then hlendrtd ~ tth the help of thepohtron hnrnogenlmr It Has then passed through two la\er~xi cheese cloth and pHwas adlu5tul lo X 7\'ismit) w?: Vtscos~tr loss \\as delcrm~twd ~ ~ thz t (ktuald h \'~scornetcr 150 atInlenals of 30 rnln stantng from 0 to I KO mtn after preprlng thc reactton rnlvture7'n 4 rnl of the substrate. I ml of'the hullir and 2 rnl ofthe e w me source \\as&xiand ucrc ptpcttrd into the Ost\vald \'~womcter 150 I'hr elnu\ tlmr of therntxturc Has mncasurcd at ctcn 30 mlrl llltenal for 3 h and the reductton In \ tu.ostt>~zu e\prcsl*ul as perccntnpc loss In \ IXO\II! arrd cnlculated b) the lilnnula as gnenIn k:n&+PM( i4.3. I. EsrJmarlon of TR4 rcorlln~ snbsranm.:Ihgeatr:I 001 MTHA2 0 5 N IICI

Method: 3 ml of the reaction mixture Incubated for 3 h was pipetted out Into a 25 mllest tube 1'0 this. I0 ml of0.01 M 'TBA and 5 mi of 0.5 N HCI was added and placedIn ho~llng water hath for 60 mln This was cooled under running tap water and thevolumc of the wlut~on was adlusted to 18 ml with dlstrlled water. The absorbance ofthe supernatant was measured hetween 480 and 580 nm. The maxlmum absorbance ofthe wlutlon uas ohserved at 547 nm tnzyme-substrate mlxture drawn at 7ero time~ncubetron and boded ewme bas used as blank The actlvlty was expressed Inspeclfic ec.ltvi0 unlts (Me unit represents changes In the absorbance of O 00 u/h.4.4. ESI'Ih1:\'I'IO~ OF ('EI.l.1 I.OI,l'I'I(' EhZl'hlES: X solonr produces F,ndo-I .A-(Lplucanaw and I.\(+ 1.4-ll-glucanme \\hen groHn I ciaph's hroth4.4.1. Mrmurrmmr o/carban. mnh~l cellulere om'vi(v (Endo-Glucanme Cx): C,clea\cs carhn~! I n ~lh\ Icellul~~se randoml! (md+

VIscosIb mrawracnl: Ostwald vimmeter 150 slze was used to determine thevlscos~ty loss of cellulose substrate4 ml ol'carhox\l methvl cellulose. 1 rnl of the burner and 2 ml of enzymesource was plpened out Into the vlscomcter The contents were mixed b! drawng alrgentl? lhrough the large arm ofthe vlscometer Suctlon was applled to the small armand the etnux ttme of the mixture was determined at ever). 30 mtn lnterval for 3 h~ncubetlon 'The percentage loss In \.~xos~t) was calculated h) employing the formulaof the v~scos~ty assay of kndo-PMCi4.4.2. Measurement of fro-I.4-pGIucanase (Ct): Endo-fb 1.4-glucanax activ~tywas measured the estlmatlon of reduclng sugar released the hrealdosr-n of CMC\vtth dlnltro sal~r? Ilc acid reagcnt as de\eloped hllllrr ( 1972)bagrnts:I fhnttro YIIIC! IIC acid reagent Ig nf' 2 5 DNS. 30 g of sodlum potasslumlanaralc and 1 tr g of stdlum h)dro\t& (NaOll) \\ere dtssol\ed In water andma& upto I [K) ml2 Standard glucose 1 m%ml solullon3 0 5'0 CMC' d~swl\rrJ In acctats hulk!, pll 5 24 Scdlum acetate-accttc acld hull'er. pll 5 0Mctbod: trom the thrw hour lncuhatod reaction mixture of the pre\ tous ehperlment.2 ml of al~quots \bas added \\~th 2 ml of [INS and kept In a holllng \\orcr hath for I 0mln. t&\\-ere cooled and then dtlutd \vlth 10 ml ol'd~st~lled water T k orange redcolour forrncd \\as meusurrd at 575 nm In S!strontcsSper-trophotome(rr. \\hererespni alone was used as blank Standard cune \\as prepared h!ustng tno\\-nconcentrat1c-m of glucnsc solutlon 1hc. cnq mc a~?t\-tn bras esprmsrd as the amountof glucose released per ml ofthe -mecstract p r unit time

4.5. Esrlmarlon of CeNobime: 'The amount of reduclng sugars released from thecellohlase was used to assay the enzyme.Rapents:1 Sdlum acetate-acetic ac~d butrer, pH 5 82 5 mM cellohloseMetbod : 1 5 ml of the huller. 2 5 ml of5 mM crllob~ose and 1 ml of the enzymesource was taken m n tcst tuk and tncuhated at 30°C lor 2 h7-he reactlon waslennlnated h placing the test tuhe In a bolllng water bath for 10 mln. The amount ofglucosr. llheratcd h\ the ennme uslng 1)NS reagent was measured at 575 nm InSystron~s Spcxuophotomter (jlucax was used as standards 1.4' 171s-1'1 1)iKS:5. I. .See& used for Ihr pot u'pcrimenls: I'hc C'O: \ arlct! of (; mar h4r.n (I.)\\.asun-d Tor the pot1r.d m t.n,o studlea I'hc c\perlmcnts were carrlcd out In the School of1.1fe kwnws. PnMllc.krn Irn~\crsln I'hc pots \rere prepared according to theswndnrd prcx.cdur~.s Ir~pllcstm \\r.rc rnalntslncd tbr all treatments5.2. Prrpararlnn of ralr bawd formulutian of mlctobial antagonists: Talc hasedSorrnulatlom n i I' fluc~rc.rr.c.nstriiln5 \\ere prcparrd h the method of \'~dh\axloranand hlu~harnllan ( I*) P Rrrr,rrsccnr stralris were mult~plled In K1ng.s B broth Theh,th was autcxla\rrd st I 4 kgicni' fi)r 20 rntli h Itmpful ot' the hactcrla \\as~nc*.ulalr+d Inlo the txuth and ~nculwt~d In a rotaton shaker a1 I50 rpni litr 48 h stroom tcrnpcraiurc (2nd 4') 1hc hucter~al populauon In the broth uas aausted toYx I 04cfdmlIn the cax 01'7' kacrunlrm Icmpiul of fungal antagonist itas scraped out and~mulr~cd In che hmth and ~ncuhotd In the rotaton shaker at 150 rpm for 48 h Thefungrl m~crohul anmgon~si populatmn In thc both \\as adlusted to 6s10d cfdml

100 g of talc powder was taken In each polypropylene bag and pH wasatusled to neutral by addlng calcium carbonate One g of carboxy methyl cellulose(C'MC) was added to th~s, sealed and autoclaved at 1 4 kg/crn2 for Ih on twosucccsslve days Fort) ml ol 48 h grown rnoculum was added, mrxed under aseptlccond~t~on under Lhe laminar Ilow and the pol~propylene bag was sealed and stored atroom trrnpcrature (2812 "C) I he antagomst population was perlodlcall) estimated b)dllutlon plate mthod5.3. Prrpcyetlon of pathogen R. solani: Seken-&)-old rn)celral mats of R solanr,gro\\n on I'DA bere bashed thrw tIrnc3 under runnlng baler. bloned In the bloa~ngpnpcr and \cv~ghc.d He~pkd ponlon ol thc ~n)cel~um uerr tragmented fbr 3 mln ath~gh sped p)l\tron In X(K1 ml ol d~st~llud \rater 1 nton-X \+its added In the blendedmalcrlalI h~s peparcd mater~al \\as uwd Tor the ~nrrulatron of the pathogen, R solrrnrIn (; mu plants5.4. Pufhopnidl?. I& and au@: I.hc plhogmr~t! of K .rolani was tested on(; mar \arict\-C'o: Plants gro\sn from tk sexxis soaked In the dlstllled \rater \\ere~noculutrvf u~th K scdunt on 30 I)?\S (h!s alter w\\lng) h!spra! lng the pathogen(prcprrd iu ata~\r) hwtcc\\c*.n h IN)-7 30 Ah1 I'he Iw\cs ot'ths w!kn plants svere~njurrul wtth tk help 01' surg~cal n~vdle Control plants \rsrc spra!ed \r~th dtstllled-*- rnlun All ~hc plan1.i \rere rrnm~'dtatsl!. co\cr~d n~th holedpol?tkrw: bags splnklcd ~61th strr~lr. water on tk Inner s~de to malntuln hlghhuml,jtt) and kwundlsturh~ lor 24 h (\~)~I~cc~Ic'. .1(ti)2: ('t~itra. OOz; (tonvathi. z(H)I )Seten dn)s a hthc ~mulatlon, thc leases \+en. obsentd for the dlxaxdesulopmcnt 'Ihe pthogsn \\as rrlsolat~xl from the ~nli~tcul area of thc ~noculatdloaves and cnmpnml w ~th the orlgl~l ~solatc

5.5. Assrssmmr of the eflcacy of mfenl concmlrntfons of the an1agonLFIJ andphv oudracls:One kg of soybean seeds (COZ) was soaked for 12 h In 400 rnl of \ratercontnlnlng the talc hased product of antagon~sts adlusted to the respective dosage-100e/ Kg seeds F.?tcess water was dralned off and treated seeds were Incubated in thedark for 24 h aner uh~ch they were soun In control. sterllc d~stilled \rater was usedInstead of the prnduct Pot culture espcrlmrnt was la~d out In Cornplctel! Random~sedIk'slgn (C'RL)) w~th thrt~ repltcat~ons for cach treatment 30 seeds were soun In eachpnt'I'he voung lea\c7; of 30-&?.-old plants _een~nated from the abve seeds were~ncrulated wth R .cdunt h spra?.lng on thc Ica\es aRer the? Hpre Inlured with thehclp of surgtcal nwJles Intrulat~cln of H .ro/i~nt\$as done In the earl! hours 6 00-7 30 AM Conlrnl plants nerc spra\rxi ~ ~ the t wm h \olurne ofstenlc d~st~lled naterAll Ihe plants werc ~mmedlatel) co\ered ulth pl!thene hags sprinkled \rfth steriled~sltllrd uotrr on the Inner side so as to malntatn h~ghum~d~t\ and kepi und~stumed(br 24 h Altcr 24 h, plant extract mas spra\cd (31 IIAS) on the leaves The laveswrrc cnllcc~cd on IO* da! alter 1st spnl) of ph?tnextract for thc rst~rnatlon of' thevarious paramclen 'I'k kmnd spra! u.ns glvcn 15 da\.s (46 DAS) atter the 1st spra!lhen the Icavn ncrc collertcd on 10'~ da! alter 2" spra! and trcrc used forcst lmaltonIN I'II"0 Sl'l DIM:d EXPERIME.%lS WERE DESIG.KED .4S FOLLOWS:I Control (; nar plants mtsal l'm the srvds waLed w~th dlst~llcd Hater f'or 12 hovan~ghl

2 Infectton experlrnent G', mm plants ratsed from the seeds soaked In dtstllledwater were ~noculated w~th R solanr on 30 DAS (days after sowlng) and leftw~thout any treatment3 Stngle treatments4 (; mar plants ralsed from sewds soaked wlth one of the follow~ng mtcrobralantagontsrs Ihr I Ih ovcrnlght \\arc lntrulated \v~th K .rotan/ on 30 [)AS& 7' hor=tamrm ( l O;o)+ P fltrorr.rc.cns -CGNS straln ( 1%)+ P flrrorc.scrn.c- Pf?3 stralns ( I 04'o)4 ('ontrol plants tnnculatcd \ctth X sr>lnnr on 30 [)AS \rere spra!ed \\.tth 10°6cthanol ph\locutact-(' pr:anten onl\ on 31 IIAS (IY spra, ) and .)h I).AS (11"qpm\ )4 Ilual traatmrnts(; mar plants mlsd from the scds snaked \wth one of the follo\vingmtcrohlal antagonists were tnoculated \nth R solonr on 30 D.4S and sprayed\\lth I(E.o ethanollc ph! twstract oC(' Rrgunrro on 3 1 DAS and 46 UASa7' hcrunum ( I O O) + I ' ptgirnlra ( 10' O) ethanol e\Lmcth P pr~wc..~ceru stra~n-C'CiN5 ( loo) + (' pponrcu ( 10°e) r.thani>l eurdctcI' ,flr~wesc-enr stram- PC3 ( loo) + (' grgcr~?tt~o ( lonu) ethanol c.\tnct1 . d samples wre collc~tcul from control. ~ n f ~ and ~ t treat~xl ~ d plants on 411)AS and 56 IIAS for ttw rrtlmatton ot'\arlou% hlc~hcnitcal parameten6 41 I)AS unl 56 I)AS represent 10lh de! unrr I and I1 spm! olph~toestmct intrcatrul plants respctt\cl!61. ULKpce /wens@ Sludes: I'wnt) live plants In ew-h pnt under each treatmenthrrc selected nl random for rlrordtng Ihc data CM Ieal'sp~t tnctdcnce The tntenstt) of

leafspot dtsease tnfectcon was categond Into five groups based on the number ofdtscaw spots pr leafi(111) Medtum / 2-5 spots 1(I\ ) I 1co\.\(\ ) Vcn hcaq1 5-11) spots!jMore than lo spotsIhc d~scase tntenstt\ Has gncn numcr~cal for stat~sttcal analys (ntl*)I~ght=l.rncdtum-2, hca\> 3 and \en hea\-\ =J)6 2. MORPUO/.U(;I(:4L P.4Rdblt'TER.V:hL I. Shm Imflh: Shoot length a.as measured utth the help of scale and wetted62.2. ford leaf area Ihe area \\as calculat~d follo\\tnp the formula of KcmpI l W)) I hc total leaf aru &as ohtil~nrd h multtpl> tng t k total numhr of Ieatzs prrplantlotal Ical area .- I\ I3 \ ki \ NU'krc. I; length ol'tk lentH = hrcath ol'th' lent'I; -I Kcmps ('onstant (0 M for d~coth)N = Numkr of Icabes F r plant62.3. ~7ddpam~rn: 011 thc II snmpllng dn!. pcJs nerc rcmo\~d from tk control.tnfwtcd and treated plants and No ot pds and No o l ' per ~ pnl ~ \vent ~ recorded63. ELECTROL rncm.im6'E: FUII! e~pdcd meture leaves ww COIICC~~ andwashed undcr tap wtlm and lhcn placed In 6ml-ckwntsed \rater To estlmatc Ihc

amount of electrolyte released from the cell In the lnltial stage electr~cal conduaivly(IiC,) measurement was taken In the beglnnlng Then tubes were kept In dark at 25 "Cand measured at dlwerenr tlme Intervals (ECQ 1 5. 3.5, 7.5 and 22.5 h Followingthls. samples were autocla\,ed (EC,) and then cooled at 25 OC and the total electricalconduct~v~t\ was measured b!.uslng the following formula of Mohammed er a1(20G)(l:C, -l

Chlorophyll b (mglg) = (0.0229 x OD ,) - (0.00468 x ODw1)Caroteno~d= OD~MI- (001 14 x ODu,,) - (0.0636 x OUJI)The ptgmenl contents uere expre3st.d as mg/g fresh ive~ght of the leaf7.2. ('ARBOHYIlRATE.% 'l'hc reducing sugors. non-reduc~ng sugars. total sugars.starch. ptiennls and ntlrogen contents \\ere extracted from the oven dr~ed powderedleal' materialsExlmtian of .&lcohol Exlrart: Sugars irere extracted and atlmated follo\\lng themc-lkds of I.iw,~n~s and Shull ( 1937) and Nelson (1931l Fresh lea\.es \rere drled Inhot air o\sn 1111 11 was ahlc to k p~\\dcrrd~th the help of mortar and pestle Driedleaf samplr of I(W) mg \\as ho~lcd In 10 ml of XO 90 sth! I alcohol I'he homogenate\+m linl cmlr-d and then ccntri1'ugr.d at h(MH) rpm for If, mln The sup.rnatant \+assi~ca\cul and made upto 20 ml \ca\~th cth) I alcohol I'hls eitracl \\a$ used for quantltatl\ecxtlmatton ol carhoh\ drat-. ptw~~ols and nllrogcli r.ontcnt The rcs~duc \\as sa\ed for\larch cstlmatlon7.21. TOT.41. Sl'(i.4R.S: I'otal sugars \\errs csl~matcd I'ollo\\~ng the method ofI)uh~os rf a/ ( 1917)Hagent:I Anthrow reagent 2 g of anthronc \tos dissol\ed In 1 I. of cow 11?S0, Thtsreagent \+as prrparrd lresh . just klbrr u.3icthod: lo 0 Sml of alcohol~c e\tract. O 5 ml ol' dlstlllcd \+utcr \\as addedS~rnulunrnusl\. 1 rnl ol' cold anthrone reagent \vas odJcd Th~s \\.as hmtcd lilr I0 mlnIn a bnlllng srnvr hth 1'0 prevent loss due to c\npnratlon. thc test tubs \rere closedw~th glass marbles i'k tc-t tuhts wcrr cmlcd and the ahsorhaWc uas read at 620 nmIn a spcc~rophotomctcr against a ragen1 hlanL 'The standard gmph \\as drmn \\ ~ thknown conccntrntmn of glucose

Z2.2. REDUCING SUGARS: Amount of reduc~ng sugars was estrrnated follow~ngthe mahod of Wang er a1 ( 1997).Hcr~eat:I D~n~trosel~cylate reagent 1 g of 3.5-d~notrosal~cylate (DNS), 30 g of sod~umpotasslum tarlarate and I 6 g of sodturn hvdroxrde were d~ssolved In 80 ml ofdtst~lled water and made upto I(X) rnlMetbod: 2 (J ml of' alcohol~c extract was added to 2 (1 rnl of DNS reagent The tubesw~7e covered w~th glass marbles and kept In a bo~l~ng water bath for 10 mtn Thentho wcre ccnl~ul and drlutrd ir~th I0 rnl of crater I'he orange red color formed u.asmeasured at 575 nm In a spt*rtrophotometer agalnsl a reagent blank The standardgraph uas drawn w ~ th Lnorr n conccntntlon of glucose7.2.3.NCIN-REDU('IN(i S(fG4R.S: Non-reduc~ng sugar \vas est~mated lbllo\r Ing theme~hnd of I.mm~s and Shull (1937)The amount of non-rrduc~ng sugars *asdc~crm~mx! h the I'ollo\rtng (brmulaNon-reduc~ng sugar = 'i otal sugan - Free reductng sugars x 0 957.24. STARC'H: Starch was est~mstrd followrng the method of Mc Cread! er a1( 1950)WeqratsI Anthmw reagent Anthrow 20 rng tias dlssol\cd In 100 rnl of cold 95'0II?SCI*2 I'crchlor~c xtd (IY'A) i o I X rnl ol'd~st~llcul \rater. 52 rnl of cornmerc~al K-A(70%) ucrv rnl\vd 10 get 52'0 IY'AMetbod: l>~stilltJ wavr and tY'A (52s~) 5 rnl of each irere added to the res~due IcRcrl\rr alcohol~c extrclctlon I'hc tnl\turc \\.as ~ncuha~ed for 30 rnln and liltered 'Thetillmu was Nldc uplo 200 ml ~11h d~sl~llrd Haler In a volumelnc Ilash

To 0.5 ml of the above extract. 2.5 ml of distilled water and 5 ml of coldanthrone reagent were added and heated for 7 5 mln In a bolllng water bath The tubeswere ~rnmed~alely cmled to rnnm temperature and mlxed thoroughly In acyclomlxurc I'he blulsh green color was read at 630 nm against a reagent blank Inspctrophotometcr A standard cune was drawn wlth known quantity of glucose andthe quantlt? of starch was calculated by rnult~ply~ng the glucose equivalent present inthe sample h\ 0 97.2.S SlIC'ROSE: I'he sucrose content was est~matrd I'ollo\rtng the method of VanIlan&l(I%X)RageatsI Anthrone reagent2 30 "o hOtl%letbod: lo I ml ofthe 80°,~ ethanol extract 0 I ml of 30% aqueous KOH was addedand kept In a holltng water bath fnr 10 mln The samples were cooled and 3 0 ml ofanthronc reagent uas added and kep at 40 "C Ibr I0 mln 'The absorbance \\as read at620 nm (ilucose of knoun co~.c.ntr~uon was usad is standard7.3. h'l TR(hYEA1 ME T.4 BOl.IShI:7.3. I. E.STlMATION OF AMINO NITROGEN CONTENT:RagcabI C'ltralc. hut1i.r21 g criolric acld \\as dissol\cd in (Uml of I N Naoli and thesttinels uplo 500 ml n~th dtst~lled \\ater 'The pH was adlusted to 5 0h) addlng I N NaotVHC'I2 N~nhbdrin solulwn9 Solutlon A 800 nrg of stannous chlorldc ems added in S(W) ml ofCitrntc hutTer.ptl ( 5 0)

4 Solution H 20 p of n~nhydrrn was drssolved tn 50 rnl methvlcellosol\e9 Solut~nn (' 'To I ml of Solutron A. 1 rnl of Snlutron R was added3 Standard 'nc Lnn\\n quantll! nf glutam~c acid \\-as used as standardhlrtbod: I'hc pll 01' thc alcohcilcc c\tract \\as adlusted b> adding 0 1 N NaollIlfCI10 I ml oithc ah\ c cxlracl 1 rnl ol'nlnh?dr~n rcagent bras addcd 'I'hcn. 11 \+as heatedior 20 mcn and rcwlcd 5 rnl ol dtsulled uatcr uas added and the. absorbance wasmcasurcd at 4 75 rim7.3.2. .4MINO ..(('ID E.STlM.4TfON: lhe amino ac~d content \\as estcrnatediolh~\\lnp Ihc m-ttud ol'hlclnrc and Stein ( 1954)Rragnts:I 2 5 I I 2 4 UIIII I' r I a 2 5 I of 0 2 hl solutlon ofudtuni cllmtc k\crc rn~\cd and d~lurcrf lo a tnwl tolume of IM rnlJlrtlllrj \$u~cr and thc ptl \\a\ chrchcd In a pII meter: ( Itrtc WIJ u>IuI~>n 11) 2 $1) I IN g 1)1~.1tric acid \\as dlssnI\~xi In YIO ml clidlrl~llvrl wdlcr3 hdlum cclralc u~lutm>n 111 M I 2') J I p niwd~urn cttrav \\as d~sa)l\rd ~n 5(XIml ol dtctlll~tl Haler4 Nlnh\&tn u~lutu~ci 111 +*l 1111 rji1l2 h4 itlrdtc hulli'r cpll 501, (19 g ofstmnous chlnrdc \\a> djcd lour g (itncnh\drln In 5tNl rnl of rncth\lccllcw>l\c trnr n3rkxi 11,t k nhvc mi\turc The rawgent \..as stc>rrxi ~n arclirgrtalor at 4%'5 I)~lutant ullut~on I)ts~~llrrl rbrtcr and n-ptupanol twrc r'quull\ .Ickk.d

MctM: One ml of nlnh~drin solution uas added to 0 I ml of alcoholic extract andshaken well lo th~s, 0 9 ml of dtstilled uater uas added and the ahre mlxture uashratcd In a hoillng \rater bath lor 20 mtn and cooled under running tap water f i\e mlol dllutnnt wlulon was added to thc atxne mluure and kep for 15 rnrn Theahsorknce uas read at 570 nrnI he amino acid contenb of the sample \+err.dcfermlned ulth the hclp ol a \tandard cunc prcparud lor gl\ctne7.J. J. N1TR.I TE-NITRITE &.STIM,4 TI0.V Nitratc-nitnte conwnt uas r.st~rnatrdaccilrdtng to the mcthcd ot' U (wtlc\ ct ol ( I *IJ-hod:Atnut 50 mp of shade-dr~ed po\rdcrcd leaf mnterlal was btlcd for ID mlnIn 5 rnl of d~s~illd untrr (hw rnl nqu~>us e\tnct uas added to Y rnl oi SO I\ )\ )=aK wd wlui~on containrnp 0 2 ppm of C'uSO,Oor gram of sail rnlxture asdcscrttrd h Nclwn rr 01 c 1954) \\as added lo each ssmple lk wlt rnlrturc \\asmadc h mtwng ~homuphl\ the finel\ ground chem~cals \ v . IrX) g barium sulphate.75 g cilrs n-~d Ill g rnanpam sulpblc. -1 g sulphan~ltc nrld and 2 g llnc p\\dcrand 2 g I -~pln l arntncTub n~thoul ettracl scnd as Nan). l'ub crrntatntng t k asw mnturcurn shLm at Imr( thrtcc at 3 mtn tn~cnals and finall\ ccntrtiugd at ZIXW rpm forI0 mtn Ahforbnrc of the clca~ sufwmlnnt r\u read ol 520 nni agalna a rengenlNard1.m ntutic cwtmwwr. tk rpmw pu~dure r\m rcpca~c-d for atbother htch ot'wmpk, ~vnitttng /n. hlhk,, a d ('ubr,I hc ux~rnd run part tk q~13nlrl\ ot' ntlrtlcalone ptmt In thc s~rnfle I'hc itni taluc minus lhr. scxorkl ge\c lhc quantth ot'nitmlc p m t m thc ampla 1 % ~ nnnunt 111 nltcllc and nltritc \\err' colculat~d fnmrludud graphs trw pt-bss~um ntuatc and ~Jium nbtritc rr?ipmti~cl~

Reagents:I Ilye IlM) rng ol'C'tmrnass~c hr~ll~ant hlur (; 250 was dlssol\,ed tn 50 ml of959.0 cthanol a14 I(W) rill ol'conc ortho-phosphor~c acld Flnal \olurne \\as XHJrnl ~nadc up u~th d~st~llcd \\atrr It \+as stored In arnher hottlt' In thercfr~geralor2 Ihe concentratlrm I.lltcred d\e \\as d~luted to I 4 ratlo (d!c d1st11lr.d \rater)liir ussErtmctbn for prolein estimation: Ibrotr.ln has extracted and estlrnatrd followingthc mtkd 01Schnodcr t IYJSI 5(l1 rng o i lrrsh Iraf material from truunent wasuc~ghd 10 I ~IS 5 ml nf lo O. cold tr~choloroacet~c ac~d Has addtd on Ice. 'fils wask~mogcntwd w~th a pbl!Irnn and thcn alloued to stand Ibr 30 rnln and supernatanturn J~scarJrd I o the pllct, 3 n~l LII lo I cold I'C'A \+as added and rn11r.d thoroughl?in a r?clom~\cr It~~cntrilug~d st 25(KJ rpm for I 0 rnln and supernatant wasd1r.ardr.dIscpropnol ~oshlng \tar rrpilt~d thr~cr. lo lhc prtxlpllate. 5 rnl of 5 ObIY'A was wkkd, rni\rd a d hcat~d In a t*>lllng \rater hath ibr 1 i mln The tuks \rereccna~l'up~d at .3cxrl rpn tor 25 nun Ihc rc-s~dunl pellet \\as Jlw>l\ril In 3 5 rnl of 1) 1N Nmbll and ccntr~lugrd at itRl1 rprn tor 10 lnln I hc supernalant \\as used forprrblctn 1-tlmatlc1nMethod: lo 1 (I ml ol extra.!. 5 O rnl ol d~lutrxl d)r' uns added The content %\asrnlxcd wcll uwJ alloucd to &\clop the r-olcwr ior 15-25 rnln I'he red J\r turns hlueIhc Muc colour l o d uas mraswnf at 595 nm tn a sprt~omcer agalnst aMu3. Ihc JundPrd graph N* dmun w ~ th L~OUII cuncmvatlon of BSA

7.4. TOTAL PHENOL: 'l'otal phenol was est~mated following the method of Brayand Ihorp ( 1954)Ragmta.I Sodlum carbnate 2(P/. 20 g of udlurn carbonate HTIS dtssolved In 100 rnl ofdistilled watrr2 Poltn-C~waltcau rcugcnt C'cirnrncrc~al Fol~n-C'tocalteau sas dlluwd s~lhd~sttlled ~alc~ In I 2 ratlohlctbod: lo I mi oi alcoholic extract I rnl of lbltn-c~ocal~eau reagent and 2 ml of~(RIT 4tum carhrutte Hcrc add& and rn~wcd ucll in a cyclorn~xcr Thc mlsturr wasbld tn bntl~ng rratcr lor 1 mln and ccmled I'he solutton was d~luted to 25 ml ~61thd~st~llrd rrnlcr Ik intsnslt\ ol rcsultlng hlulsh \rh~tc color sas read at 6%) nrn in asprrrrophc~tomler apnlnst a reagent hlank A standard cunc \\as prepared rr~rhknown quantn\ ofcatrr-hol as phenol equ~\alcnt: 5. PR'IOUIE: Prollnc \ran\ e\tmct~rl and csttnwted according to thr. method of Hatest.rol (1973)WagrmkI Aqucou'.\ulpho UIIIC\IIC acd (YO) 3 grn oi sulphoul~s\l~c actd \\asrcogcnt rraq stahlr for 24 h rhcn stor~d at 4 "C'1 Shndsrd pmllnc s mp of pmltnc \ras dtswlred In lo ml of O I Nh dmchlot~ acidEatruUan: The m~dr~b of' Ihc leaf \cas m \ c d ad 500 rng of thc leaf ISWsasw~ghcd It w u hnmngm~rrd u ~th 10 ml of 3 ** sulphosalm ltc ud In a mv~u and

pestle The hornogenate was filtered through a Whatrnann No 2 filter paper Thepmcedure uas repeated wtth the resldue and the filterate were pooledMetbod: To 2 0 rnl of the filterale 2 0 rnl of' acld n~nhbdrln and 2 0 rnl of glaclalacetlc acld uac added I he tuhcs Here ~ncuhatcd for I h at 1(W)T on a water hathIhe tub unr trancferred on ~cc to tennlnatc the reactlon and 4 O rnl of toluene Hasadd4 and rnl\cd \~gorousl\ lor 15-20 \wands Ihe chromophore contalnlng tolueneuas asp~rntd from the aqueous phase It \\as alloucd to reach room temperature andthe absorbance uas measured at 575 nm A reagent blank -8s malntalned A standardcunc uas ohmtneJ ustng n know ronccntntlon of authentic prollnc: 1ht. prollne~onlenl uas e\prc%sed as mg 01 prollne vr gram frah src~ght7.6 I. AfE.4SI'REME.YT OF PERO..\'II).4SE .JCYll lT1': (E(' I. 11.1.7): Pc.ro\~daxac.tn tr? *-as msural h? thc ol'llarnpiorn ( 1962)m t s :I Phosphate hullcv (0115 hl) pl 1 (> 5? 4ropllol (0(XI1 M) 0 01 -761 g 01' p\ropllol was d~ssol\ed In I(K) ml ofphtnphate hinh3 Il\drogen pcm\lrk * D tl\drogen pero\~de \\as prepared b\ adding 2 rnl oi6% h\drogen pero\dc dcn 4 ml ot dlsltllcd uat~vHclbod: In I ml of 0 OUl hi p\ rogallol I X ml nl dlrt~llrul \rater \roc a&In accne and the ahsortawe HOF d~u~t~xlto 1er0 at 470 nrn Irnmedlalel\ 0 I rnl of ,Mo(0 SRW M) II:(h and o I ml of L-nnmc ucrc nddcrl I he contents \rvrc nw.cd uell andp l d III S~ttmnrs spctrophotonmcr Ihc change In the absorbance at e\en 30Ycond mien41 toc 3 mlnuta uas mcasuml SuttaMe contml u tth heat k~lled e m mem mmmrncd

7.62. MEASUREMENT OF POL YPHENOL OXIDASE: (EC: 1.14.18.1):Polyphenol ox~daxs actlvlp H ~ S measured by the method of Mana and Dlmond(1963)Rqrntr:I 0 2 M Phosphate hutl'cr pl{ 7 f)2

sample was added to a test tube wtth equal volume of e~ther -TBA and +TBAsolut~onSamples were heated at 95 "C lor 25 mln and after cooling, absorbance wasread at 440. 532 and h(Khm Malonl~aldehyde equr\.alents (nrnoles ml ') werecalculalcd a5IOhx(A-U)/ 157(KX)H here.A=(Aha12 + 1UA)-(Ah,.,+lBA)-(Absa~>- IBA -Abh,- IHA)I3=( (Ahrub + I UA - Ah,.) + I HA 1 \ (1 0571 17.7. SI)S-P:\(;E i.lectrnph~rrs~~ under knatur~ng conditions on \en~cal eels \+asprtormrd as de\crltwd In I acmnrl~ r I97111 uslng su~tahlc hufirsRmgraraI .-Ic.n/crm~r/~. 1rh.h ro/i~lron 1.~0"ul: 30 g ot'acn lamlck and 0 X p oi h~s acn lam~dcucrc Jlsu~l\~rl In IcMl ml ol'd~st~lled tratcr and 1-iltcrcd through H'hutman No 1liltcr pp-r 1 k tiltrate \+as \tnrsd In a hrl)\\n lnlttlr. at 4 "(2 Uercdvr~~ prl hufl~..r. IR I5 g (11' Ir15 ( I S M ) iras d~su>l\rd In I(K) ml oi d~st~llcd\rarer and t k p!l \rub adlustcd 111 X R utth conc IICI k u>lurlon \%as filtc'rcdand \t~~rcJ 314 Y'1 .S/nr&tn*. gt.1 buflrr: 3 01 g of 'I'r~s(05 MI uas dlssol\rd In 50 ml of d~st~llrd\rater unJ OK' pll \\it\;lJn~st~*l to 6 X ir~th conc ll('l sc~d The solutl~ln itastil~crrd a d stor~d at J "C'J.S/)S .~rdir/a,n(/P,u 10 g of SI>S \\as d~ssol\rd ~n lin rnl dist~lbd \rattr. fil~cr~dand utord a1 room lcmpmturc5 AP.5' (lOsrJ: I(I0 mg of ammon~um psr sulpharc Mas d~ssnl\~d In 1 ml of d~st~llrdwtn 'Ihc wlutton \\as preparc4 frcsh

6 PMSF (10 mM): 17 4 mg of phenylrnethyl sulphonyl fluor~de was dtssolved In 10ml of so-propanol 'I'hts solut~on was stored In alrquots at 20 "C.7 Elerfrophorests btrfir lpff 8 3) 3 02 g of Trts (0025 M). 14 42 g of glvctne(0 I92 M) and I 0 g of SDS (0 I%) were dissolved In I 0 hue of drstrlled waterRSI)S .wmple baffler: 'The huffer contams 0 M25 M Trrs (pH 6 8). 5 % SDS. I mMPMSF. I0 'i. sucrw. 1 96 (1-MkC' and 0 002 % bromophenol blue9 .Sfumrng ssdutton: 0 25 gm ol'coomass~e brtll~ant blue R-250 &.as dtssolved In 90ml 01' methanol and Hater (I I) and 10 rnl of glac~al acetfc actd and filteredthrough H'hatmann No I filter paper10 fk.~l~Ifllfl~ ..rol~rlrtm: W ml of methanol Hater (I I ) and I0 ml glac~al acetlc ac~dctcrc mrxcdE~trwtbn: I.L'B~ ~lmplr- \rcrc irrlghcd alter rcrnoblng the mtdrths and Hrrehumnpn~t~rj In 0 I h! phosphate hufkr (pi4 7 2) filtered through muslln cloth andccnu~t'ugcd a lo.(NKl rpm Tor lo mln at 4 'C''Thew sample uere used forclcctc~>rr-sts ~lthout lunhrr pur~ficatton I'he protan content \\as est~matcd w~(hslmllar p durc as m.nttonrd In and adlustrd around I XO pg In each sample(;el

mlnlne water was trmoved with the edge of papr towel. Over the polymedresolv~n gel, the nak~ngel was applfed 'Ihe well template (comb) was ~mmedtatelyplaced on the gel lo l'orm wells (care tvas taken to avo~d an\ trapptng of atr bubbles)After the pol!mcrt;ratton was complete. the 'lellon cornh uas remoted carefully andthe wells were tmmcd~ately washed wth Clonlsed waterto remove theunpol! r n r ~ acn ~ d lam~deThe composttlnn of the resnlrtng gel and the stack~ng gel are given belowSdwlon ResatVing Gel ( 1 0%) Srockim Gel (4%)- -- - - .Acn lamtdc. st~w). 5 O rnl 0 85 ml1 5 hl I rl\ IIC'I pll X X 3 75 mlo 5 h4 I rts IiC'I. pit t> X10 9.0 S1)S 1 sop1'Ik CLKtropLomk Hun: Ihr: protctn sample \+as then addd on to thc \rrlls Therwnorr hut%\\a% filled In hoth thc rlectrodr. chamhers and the clculrmk \\erecnnnccW to t k pwvr pack Ihe condtttons uerc %? tntttall! at W \' and 25 mAunl11 thc the front reached lhc rewlvtng gel and later al 100 V 1111 the b e imntreached Ehr ad Atb nmnmg. the gel uas rcmoked. \rashed in dlst~lld \ratu andwas sm~nod w~ Che ammassn hrllllant hlw R 250 solutmn USA nus cc~ckarophmscd along the sample roc thc thmn~nattonf the molwular ue~ghl

~ubhg.- The pl was lmmervd ow-ntght m coclmassle hrllllant blue K 250 rn amlxlurc of mnhrnol accllc acid marcr (4.5 10 15) and then destalnrd o\ern~ght In themlxm rr r tht chc-0 of fhr plant Mdd: I-rc\h ILZI\~\ ot ( p,,ycmfca \rere colleaed andwashcd under the lap Haln and allo\rcd In dn After dn Ing the m~drlh of the leaveswere rrmo\cd and cut Into \mall plcct! I e;l\c~ \rere kept n\rrnlght In chloroformwl\cnt to m r c wmc amunt of chloroph\ll content I.ca\es uere rernmed afterkanrlng the wlrent and drltxl twturm t-~Iler paper shorts Ikfore suh)wting theka\c~ for column chromatograph\ 11 bras crushed Ilghtlr'1%crrtract ohra~nrd was trstcd Tor the Tuncttonal group and to ldent~fi thestructure of the compound present In the plant extract17Cin Lper C'hrm1ograph.v (TLO:Thin h!rr prrp8ratbn: A slum of the stationan phase slllca gel In \rater applied too glass plate a5 a uniform thin lo\er b! means of a plate spreader The slum itassprd o\rr the glass plate carefull\ and allorred to dn h kreplng In the o\m( 100 T) Tlw th~chnc-ss ofthc slum la)cr rras marnralncd as I mm Thls bras done inorkr to acltratc the atwrtunt Saniplc \\as applied 10thc cIL~ TI C plate 31 ahlu~2-2 5 cm frrm the edge h\ mans oTa caplllnn r\lk Slllca gel '(i' plirc slrc 100-120mesh 31rc Has d for TI.('Owe tk plates srsre dn 11 \\as; wul for theulcnttf~ar~on of slnglc spot to ga thc purp compound h\ rk~Llng thc Rr ralueZepantmn (plate dc\.elcylmcnt) most commnl\ IaLes place In a glass mLthatconlllns the dcrclop~ng solvent (mohilc phase) to a kpth of ahout 1 5 cm 'lh~s \\asallowd to smd for somctmc uilh a Id on top of Ihc lank to ensure that the

atmosphere w~thin the tank became saturated w~th the solvent vapors (equ~l~brat~on)to avoid rrregular running of the solvent. whlch mtght result in poor separation Afterthe equ~llhrat~on the lid was removed and the separation of the compounds occurredas the solvent moved up the plates The system was kept undisturbedAfkr the elut~on of'the solvent along with the sample leav~ng 1.5-2 cm abovethe plates uerc renwved lrorn thc tank and allo\ved to dy The solvent front wasmarked k hrc dn tng Ihe plate was examlned in an iodine chamber to ascertain thenumhcr ol'compnunds present In the extract(b(unm ehr-tqgrqh.r:A column chromatographic system uslng a l~quid phaseconstsls ot thc column mohllr. phrtxl remolr and &li\rn sbstemf'wkiag the column: I hc column \\as c1ampc.d In the \ertical position and 11s lo\rerend \riu plugged with gliw \+ml I hc plug \\as brctted \\ith the solbent The slup ofthc ahorhunt and tk ujltrnt prcpar~d \\as purcd Into the column slo\rl> through thesidc 01- thc column I k glass column \+as yc.Lc-d t~ghtl! rc~th s~l~ca gel oi pnre SIXM)-?rM) mesh slrc dissol\cd in n-hcwne sol\ent The honom limb of the column nastumd opcn to remove ctcess of the sol\ent Ihe ahsorknt \\as allowed to rank Tonbtn o un~liwm column and that no atr hhhle to he present In the packing. thecolumn martirw 10 11m 'The length and diameter of the column wasmcaruredApplhfbr of the mmpk: lk sol\rn~ aln~\e ttic column uas drained Into columnM lo mlnlmix thc d~sturtrancs I'kn thc wmplc \\as appllcd carefull! on top of thecolumn A small \olunw of 1 k mm>hile *asc~ napplird s to \vash the linal traces 01'thr. wmpk Into the hd C'olumn nas tkn conwtd to the rrscnoir contnlnlng thernoh~lc w w (solbent) u1 that solicnt ahout (2-5 ml) \rns al~a!s present ahote thecolumn lo acotd rhc dq ing up ofthe columlr

t'olumn development and sample elution: The component of the sample applledwas separatedthe conttnuous passage of the mob~le phase through the column. Thesolvent as passed through the column unt~l the mnes due to the compound wereelut~xi out completel\ At the end of the elut~on. almost 10 fract~ons were subjected toS1.C' 10 find out the l'ractions contatntng the compound From the 'I'1.C it wasasccnn~ncd that 2-9 l'ractions of the leaf Here found to contatn the compoundShrrehrc. 11 Has suh~~~tcd Inr spectral anal)s~s 'I'hc collected Sractlon was thensuh~cxtcd lo c\ apratfon h\ using rotator? e\ ilporator The linal compound collectedIn this bra\ \\as taken for tunher studies like. varlous chemical and spectral studiesSdmMlit? t d for dive ftmctiom: Aller the c\tractlon and e\aporation the sample\\a\ dlswlrcd In ethanol tor ttw w~luh~l~t\ tr-stSpwtrrl aral?si.r: I'hc e\tr;r-t~d wmple \\a\ ageln suhlci-tcd to I IV. FTIK (Sh~rnapzuand I.'h~m&/u I:I-IK Nlt*I wries). '11-NMK,"c'-NMK and MS studies9. Sl'A'l'lSI'l('A1. .A\AI.\SIS: Slat~slir?ll anal!sis \\a5 ~rl'ormed using ow \\a\anal! \ISot r ariance (AN()\:I) tollo\\cd h> 1)uncan's Multiple Range TestI)~n'crc~ssc~n~i&~d to tV signlricant at p.0 05 apatnsl control group Data\rmc prr%ntCcl is mmai I $1) tc~ tr~pllcatzs All the hi~xx-krn~c'al estimations \rerecantrul oul In dupl~aln

IN VITRO STUDIES:Rrrdlrrl ~cefiol grmvrh: Screenrng studtes on the synergtstlc effects of the microbialantagon~st-l hrrrunum and phytwxtract of flve spies at Lhree wncentratlons onthe lnhibttlon of R solunr (72 h) were carrted out to find the most effectiveph\loextract which not onl) lnhlblu R sol an^ but also rather almulates theantqontst Kadtal rn?cellal gro\rlh stud\ \{as ohserved b? dual culture and pnlsonedplalc wchnqw Percenl inhlbltron of radlal rnqcelral pronth of R solanr IS presentedIn 1 ahle IPlant extracts of five spx~c? In different sol\cnts In cornbinatron nrthI hoc~mum \hnntd \an lng drgrec of tnhlbttton of rn~celial growth after 72 hAlthough nuixtmurn ~nhthition of H ~~~lrmr ( IWO)bras U\IIII~IILY~b\ th~' ~\UJS~S ofpctroleurn ether chloroform acetone and ethanol of P julr/lwu and chloroform.acdonc and ethannl leal cxrraar ol -1 rndrcn t b urn found to lnhlhlt the grot\* ofmlcrobml anqgontst( 7 Iwcrunum) Ijcnce. these plants Mere not fwthrrr studiedI caf cxtm of (' aurrculafa In 10% conc In all the four solvents incmb~nat~on u~lh fhur=f~~m hnd nui%rrmxtmum inh~b~tlon, ru. 83 7%. 72'te.32 3% and 93 6% cnmpad to :he 2 5% and 5% conc The pevokum ether.chlomfonn arnj cthanol exasrt of 0 suncram showed 76 7% tnhlhtion Whencornpsrcd to the OM leaf extract (C'(IYTI~JOIO and 0 -rum)mlvma. thesc n*onts Icu eff'bcttvc In the tnhtbttaon of Lhr R sdw The other hvo sdvuus-pmdcumether and chioroh we= not ucr) cflbctlbc as a w-rs mt m~sc~ble In water

'I'rblc I tffects ofantagonlst I' harrranunt and leaf extracts on the percentage of lnhlbltton ofii .\olrrrr/(ill 1 / I/ o >IULI! JProsopts julrJloro(S\c ) lkPetroleum etherChloroformAccloneIithanol( 'u.\sra uurr~.trlirrrr I. Pelrolcu~ll etherChloroformAcetoneIhhanolOcmtum sanc~um I. I1etrolrum ether('hl~iroliirn~Acetone1:thnolSI) : Means oftr~pl~catrI'abk 2 ~NX-IS of antajjon~sl Pwudomcmas stratnsCCiN5 and PC3 and leaf euracts on theprrcentage of lnh~httlon of R mkanr (tn vlfro stud) )1'123 + (' g~gcinrcu Petroleum ether 60 X (.H) 38) b8 4.H) 77) 70 S(tl 3 1 )C'hhoform 68 3 ( i I 41) 65 9(*1 18) 60 !(*I 59)Acctm 70qi183) 683(*184) 627(*183)lithanol XSO(iI44) RPI(i264) 9?3(*113)

Photograph I. ('ulture plates showing R. soloni, T. horziamum and dual plateculture of R soloni. T. horzanum.4 R. solani mycelial growth (3- da! old culture)BT. harzhnum rnycelial growth ( Ma? old culture)C' R. solani inhibition of m!celial growth of R. soloni underT. ham'anum treatment (+da\ old culture)Photograph 2. C'ulturc plate showing, inhibitioa ul mycdial growth ofR sdani under Z hanjanum treatment (5-daj OM culture)

As the ethanol leaf extract of C. gigontea was f o u n d U a ( L r mmicrobial antagonist-T. horzianlrm. it was subjected to further studies. Inhibitor).effects of C. gigontea were found to differ according to the solvents used for itsextraction. 'l'hat of('. grgontea In petroleum ether (2.5 to 10% conc.) extracts shouedinhibitor) eflect between 5 1 .I to 79% whilc acetone extract (10%) showed 30.2% to58%. 'Ihose of chloroform and ethanol extract in 10% wnc. were found to be 95.3%and 8% ~nhthit~on respectively. Since the chloroform extract IS immiscible in water.only cthanol extract was selected for further studies (Photo. I & 2).I5ascd on the abow result cxtract of (. grgunrru (In three conc ) Incomhlnatccm w~th t~i) stralnh of well hnilun bacter~al antagon151 P fluore,tens-CGN5and I'R3 was suhjected to studlcc agatn5t the tnhlhltlon of H ~olunr Percentage ol~nhthlt~on nf K rolanr bs CYiN5 In three conc of C grganrea shoued the folloa~ngpetroleum ether shoucd 62 2% 48 900 and 42%. that of chloroform shoued 68 7%65 2'0 and WO acetone shoued 78 1'0 75 3% and 73.0 and the ethanol enractc~hlhlted 79 900 78 and 70 tnhrhrtron H hlle that of PI23 and C grganreo Inpetrokum ether \houcd h0 8'0 68 4'0 and 70 coo ~nh~h~tliin chlorofc)rm extractcxh~h~lcd 68 3'0 65 900 and 60 1'0 acetone showed 70 9'0 68 3'0 and 62 7.0 andcthanol cxtracl shoued 85". 8g lo. and 92 3'0 prcsnt lnhlhlt~on ( Fable 2)Mcnnlna~ion of MIC 10% conc \+as found to he the MIC' (mlnlmurncimccnrratlon shoulnp highest rate of lnhlh~tlon of pathogen grou2h) of thephy~oex~rnct on the has^\ of the results ohtamed froni rad~al myxllal growthlnhthlt~tm Ilcncc. (' grguntto Icaf cthanc~l c\tract( 10'0) cimc uas used fr furlher mvrvo stud~es

I>cvc.lopmmr of antibiotic reslttant strains: Bas& on the anub~ot~c-rcs~sunt siramstudy. out of 6 stralns of heudomonas tested. two strains vlz . CGNS and Pt23 wereselected for m vrrro and m vrvo stud~esCELL. H'A1.L DEGRADING ENZYMES ALTIVII'Y: The actlvlty of pectlnolyt~cand crllulol~ t~c en0rnc.s produwd by H ~vlunr under wnuol and treatments-s~ngletreatment, elther with the antagonists or phytwaract alone and dual treatments-anlaponlsts along with C' grgmea has rstlmated and expressed as percentage of1 IworIt\ los5 (a+,) and In ~pec~fic actn ~h unlts (SAIJ)WI.I'METHI'L CAI.A

Fgae I E&ts of srgk and dual treatments on the activty of piyretttylplact~ronase (endo-PMO) of H solani in virro (%). - . . --.- - -0 30 60 90 120 150 180Time (in min)

T~blc 3 Elfcct of srngle and dual treatments on the acuvlty of PMG (exo PMG) ofR solanr In rn vreoIH solanr2 X .tnlunr + l horrranltm3 H solunr + C'( INS4 H solanr + I'C3hH solanr + 7' harrranunr * (' grpunrc'a'IJ. O 05 agarnsr mtrd (Man i SI))

(76 7 SAlJ) folloued by I'I23 (87 5 SAW), CGN5 (89.3 SAU) and 7' hurzrun~~n~(98 5 SAII) Among the dual treatments the least actlvlty was In the caw of enzymesource obtalned from PI23 + C glganiea (20 1 SAU), followed by CCNS -(' yrgan~ea (23 4 S4ll) and I' Irrrcrrrr~r~~rr + (' ptgc1111en (38 X S.AI 1 ) rcspctl\ el!PECl'lh ~I'lUhSt;l.lhllhASE (P I'E): I h~s mqmz cleaves pecrln randomly (mdo)or tem~nelly (exo), thereby reduclng the vlscosln of the substrate and producingI'BA reacllng substancesl'k actlvlt? olen~l''~1: prnduccd h\ H solant grown w~thout any treatmentand under treatments (single and dual) are presented In Figure 2 The e yme sourceobtalncd tiom X solanr showed rnaxtmum vtscoslh loss (74 12%) Among the slngletn%rments, that of 7 hacrammshourd the maxlmum r~~scoscp. loss (67 3 %).follo~rd h that of (' ~rgumrcr (62 3Z0o). RiN5 (59?,0) and Pf23 (56 12%) at theend of 180 mln 1.east actI\It! U ~ S M>~ICC~ In the dual treatments of' CGN51 ' xlgamca (230) and 1'123 * ( ' prgurlreu (21 229.0). lollotred b!T hocranum ti' flgm~ea (2.5 lo?*)1hc actlvco of eso-P1'1 Has expressed In speclfic acttvlp unlts (SAU)lable 4 All [he treatments lnhlblted thr actlvlh of rxo-PM(i at vmlng degreeIl~ghamount olrxo-Fl'll \&as relcaxd In the caw of enzyme source obtalned fromW sdmr groun ultk>ut an\ treatment (2202 SAlI) Among the slngle treatmentslcast actlr IQ ha5 e\hlbttrd h PC3 (24 2 SAlJ). folloued b! r horzramm (26 8SAII). CGNS (280 '0) and 1' gt~amca (36 8 SA11) 1.- actr\lt\ uas found In theenn mc wume oh~nrd from the dual culture of PC3 . C grprro 1 I I 1 SAll).followed h CGNS * (' grganrco ( 13 4 SAl I) and T kranwm * C grgamea ( 18 8SAIJ)

Flgw: 2. E&ts ol'smgk and dual treatments on Ihe act* oFpctn mrsehhse(endo-PTE) of R solani In vitro (%)

Tabk 4 Effect of slngle and dual treatments on the actlvlty of pecttn transellm~nase(exo Yl'f:) ofR solon: In :n rrtro.I R solunr ??02(*1 47)2 ff rolun: 4 I' ht~crunorn 26 8 ( *I 35)'3 R sr~lun: + C(;N 5 8 0 (i089)'J K .rolun: + t'P-3 24 2 (20 61 )*H H solanr + P12 3 + ( ' grganca I I I (*I 35).SAllchange tn the ahu>rhance at 5J7nm ot'0 OOl 'h'P.005 against contml (Mean t SLII

C

Figure 3. Elt'ects of s~nglc and dual lrcatments on the activit) of carbox),methyl cellulose (endo-CMC) of R. solani in vitro (%)

Table S Effect of single and dual treatments on the act~vity ofCMC endo P-1.4-glucanase(mdo C, ) of R solanr In m rivoEmdo ~1,4-gl~.nrre activity (ex0 Gis (SAU) In I&I H solunr 0 131 (10005)3 R solanr + CGN5 0 117(&001)4 H solanr + Pi?> O 127 (a 00-1)S H solunr + ( ' grgonleo 0 130(M00?)h H solanr + 7' hurzrunurn + (' grgun~ea 0 137(&001)7 H solanr + CCiN5 + (' grganreu o 112(dOO?~HX solunr + PI23 + ( ' grganl~>cSAli = mlcro g of maltose equnalent 11hera1edI-1*1141 05 against control (Mean * S[))

'I'ablt 6 Effect of's~ngle and dual treatments on the actlilt). of cellob~ase of R solanrm vrtro stud~esTrtatmtntsCcllobiasc activity (SAU)In ritro1 n ~ l~l~llll + ('( iNS4 R solanr + I'P3'PC0 05 apalnst control (Mean i SIN

IN VIVO STUDIES:Ulsense intensi(11: Leal spot disease Intensity caused by H. solunr on C; niux undercontrol, infection and treatments ISpresented In Figure 4. Highest d~sease Intensity(3 12) was recorded In the Infected plant, wh~ch was referred to as heaq to severet)pe 01' lnlcctlon Among the slngle treatlncnts, d l ~ Intensll) w 01 I horrronctm.CGNS. Pi23 and C grganreu were 2 66. 2 78. 2 61 and 2 37 respectively and referredas rnedlum lnf'ectlon fhe lo~est disease Intensity ~iis found In the dual treatments ofI'E.3 +

F~gure 4 Efl'ects ol'single and dual treatments on the disease intensity in Gmax infected b). R , solani'I'reatmentsWConvor • R sohm I T. hanianum.mCGN5 IPR3 C. giganteaT.harri.num +C ggdntse CGNS + C. gigan-a l PR3 +

Table 7 Effcci of slnglc and dual treatment on the total percentngc (%) ofelectrol)

MOHPI3OLOCI

No'fable 8 Effect of slnple and dual treatments on the shoot length and leaf area of (7 marrriicctcd b) I( .\olu~rr--Conditlonr- Sbwt lendhe) T-l &I;& (a')I" nmplirg ll* sampling lU sampling 11" aroplingr Pl?l 27 13 1X) 517) 3ROO(r0940)' 41 381*0 773) 59.20(+1.372)( ,~I&!OI~~~I ?JI!I:OOXSI ?~UOI:~~UJ)* 25.341:11~3~'41152(-1072t' 1 ho~ruftunr (' grprfrlirrr 10 18 I 1 5871 16 50 I:! 228) 49 10 It1 0101 71 25 1-0 566)1 I'E 3 i g~&!irfttv~f 5fl.Ifl,f09!!)* Sh 12(*0001)* 51 961-1 261 1 21344(*1 298f'I). 0 05 apalnr! control r Mean * SI))-~I able 9 I:llect of single and dual trcatmenta on I ~eld parameters (podsfplant and seedsipod) Inthc (; m~cr leases Infected b\ R solunr- - -- - - -- --6 No Tmhclltl Na of PodrlPlut No. ofControlInfected3.5 (iO 500)? 8 (i0.300)3 5 (+O 208)3 7 (M 126)3 7 (M 198)3 3(iO,l52)5 8 (i0.378)6.0 ( M 450)'b 4 (it) 2644).*P. 0 05 oplnst control (Mean * SD)

1'123 + C gigantea (29.50 with 5.8 seeds per pod), followed by CGN5 + C. gigaMeu(27.80 pods per plant with 5.6 seeds per pod) and T honianum + C gigontea (25.25pods per plant with 5.0 seeds per pod) (Table 9)PHOTOS\'NTHETIC' PIGMENTS: The total chlorophyll content In the I and I1sampl~ng dais varld under d~flerent treatments (lable 10) In Ihe control plant totalchlorophyll content mas 0 230 and 0 I94 mdg on I and I1 sampllng days M~n~murnamount was observed In the infected leaves (1-0 208 and 11-0 184 mgig) folloued bythe slnglc treatment of T horzranrrm (1-0 185 and 11-0 163 mgiml). CGNS (1-0 218and 11-0 176 mug), Pf23 (1-0 226 and 11-0 216 mgig) and C grgantea (1-0 2 13 and11-0 167 mug) Maxlmurn total chloroph>ll plgment was found In the dual treatmentof Pi23 + (' grgunreu (1-0 280 and 11-0 221 mgg), folloued b) CGNS + C ggantea(1-0 276 and 11-0 187 mug) and I hocranum + C grgantea (1-0 198 and 11-0 167m%g)Chloroph\ll a content durlng the 1st and llnd sampllng da)s are as follo~sI.ea\cs of the control plant sho\red 1-0 105 and 11-0 148 mg/g ihheras Infected lea\=shoued an !ncrcase (1-0 200 and 11-0 140 mg/g) Among the slngle treatments. PR3shoued the rnaxlmum content (1-0 194 and 11-0 189 mug). followed b) C grgmfea(1-0 162 and 11-0 132 mglg). CGNS shourd an Increase In the chloroph) I1 a content Inthe ilnd sampling da) than the laner (1-0 109 and 11-0 168 mglg) and T hanranum(1-0 152 and 11-1) 140 mgg) Among the dual treatments. PI23 + C grganrea andCtiN5 + (' grganrea shomed the same amount (1-0 162 and 11-0 I52 mug\. whereas.I' hrrrrtanunt + C grganfea sho~txI(1-0 156 and 11-0 I42 mdg)The amount of chlorophyll b In the leaves during the 1st and llnd sarnpl~ngdays showed the following: Control leaves shoud 1-0.132 and 11-0.084 mg/g

Table 10 Effect of s~ngle and dual treatment on the photosynthet~c pigments (m$g f \r ) in theIcn\cs of(; 11r~r.v inl;.~.tr.d h\ N .\O/NJIIC'(iN5I'C 1( plpunfrlr1 O2l1(z0003) 0 16?(s0004)* OOY5(:0000)II 0 Ih? (rU 003)' 0 I!? ($0 006) 0 OJ! (~0.02)In and lldsampltng days represent I and 56 DAS respecli\ely'PeO 05 against control (Mean * SL))

Infected leaves showed (1-0 077 and 11-0 034 rnglg) Among the stngle treatmentCGNS showed the maxlmum content (1-0 138 and 11-0 076 mg/g), followed by Pf23(1-0 121 and 11-0 116 mg/g), T hrzranum (1-0099 and 11-0077 mglg) and(' prp~~ntru (1-0 095 and 11-0 (J42 rnf/g) In thc dual treatment I'E3 t (- grgonreaexhtbtted the maxtmum amount (1-0 095 and 11-0 092 mg/g) followed hv, CGNS +(' grganlea (1-0 085 and 11-0082 mug) and T ha~~anum + C grganrea (1-0 082 and11-0 065 mg/g)Carotenn~d content durlng thc 1st and llnd sampling days are as follours:Leaves of the control plant showed 1-0.200 and 11-0 022 mg/g. wheras infected leavesshod an Increase (1-0 195 and 11-0 159 rnglg) than other aeatments. Among thestngle treatments. CGNS showed the rnaxtmum content (1-0.181 and 11-0 162 mg/g).I'ollowed hv i'f23 (1-0 171 and 11-0 186 mug). C ~~ganlea (1-0 181 and 11-0.062rng/g) and 7' hotzranum (1-0 137 and 11-0 I48 mug). In the dual treatments, CGNS +( ' grganiea showed the maxlrnurn amount (1-0 189 and 11-0 I62 mug) followed b> .7' hor=tunum + (' ~rganau showed (1-0 I84 and 11-0 173 mdg) and Pf23 +(' g~ganrra(l-O 181 and 11-0 162 mg/g)CARBOHYDRATE METABOLISM:TOTAI. SlMiARS: The total sugar content In the Icates of control. tnfected andtreated plants ts presented In the Table I Itllgher amount of total sugar \ra nottcedIn rhc leaves of the contml plants (1-70 2 and 11-85 58 m@g) Infected leaves showedthe maxtrnurn reduction tn the total sugar content (1-28 63 and 11-25 92 rnglrnl)Among the slngle mtmenls. PI23 cxh~b~ted the rnaxtmurn amount (1-56 7 and11- 60 8 mglg). followed by CGNS (143 1 and 11-53 7 1 mglg), C g,gantea (1-39 91and 11-4 1 63 rng/g) and T humranurn (1-33 6 and 11-46 92 mgtg) Among the dual

I'ablc I I hH'ect of single and dual treatments on carbohydrate (mug d u) content of G mu~nl'eclcd h R ~okmrTohl Snpn Reducilg S.$n Nos-redocia Samn,.lo Treatments ld 1 lM I" I J ~ r' IFumpliy urnding umptiug umpliug sampling umphgi ('(iN5 4.110 5371 3255 3885 1217 2068(?do 303)' (10 338)' (id? Is)* (iil206)* (i0 387)' (*I 080)'I"and 11'%~11n~ days represents 4 I and 56 DAS respect~vely'PcO 05 against u~nvol (Mean * SD)

treatments, PI23 + C. grgunteu cxh~btted the h~ghest amount of total sugar content(1- 81.93 and 11-90.1 1 mg/g), followed by CGN5 + C gigantea (1-76.92 and 11-78.32mglg) and 7'. horrianum + C. gigantea (1-53.63 and 11-65.81 mglg)REDUC'INC SIICAR: Marlmum amnunl of reduclng sugar uas not~ced In theIcav~r ol the control (1-50 2 and 11-54 79 mg/g) ( 1 able 11 ) The least amount ofreductng sugar was not~ced In the Infected leaves (1-24 31 and 11-22 6 mg/g) Amongthe s~ngle treatments. h~ghest content was exhlbrted b) Pf23 (1-40 35 and 11- 42 4mgg). follo~ed h CGNS (1-33 55 and 11-38 R5 mg/g). C grganfea (1-3095 and11- 33 8 l mg/g) and 7' harzranum (1-26 9 and 11- 33 46 mug) Among the dualtreatments Pf23 + C' grKantea exh~htted the hlghest amount of reduc~ng sugar content(1-50 V* and 11-83 32 mgg) follo~red b\ CGNS + (' g~ganteo (1-48 46 and 11-70 81m@g) and 7 horztonum(' prqanfca (1-36 8 1 and 11-55 05 mg/g)40\-WED1 ('I%(;S1lC4R: he htghrst amount of non-reductng sugar \\a?obsened tn Lhc Iascs of the control (1-31 51 and 11-33 5 mdg) Table 11 Infectedlcava sho\\ed the least amount of non-r~duc~ng sugar (1- 5 53 and 11-3 73 mg/g)Among the single treatments. PI23 chh~h~ted the htghest content (1-1836 and 11-20 52mgg), folloued b\ CGN5 (1-12 17 and 11-2068 mg/g). C grganrea (1-10 5 and11-9 51 mg/g) and f ho~ranum (1-8 I J and 11-15 13 mg/g) Among the duallraunsnts, mawmum arnount ol non reduc~ng sugar \\as shown b\ Pf23 +C' pigantea (1-29 42 and 11-37 81 mg/g). followed h\ CGN5 + C grganfeo (1-27 03and 11-8 9 1 mglg) and 7 hamanurn + C grguntea (I- 1 8 b6 and 11-8 29 mg/g)STARCH: The starch content In the leabe of control. ~nfected and treated plants ISpresented In the fable I?Max~mum amount of starch %as not~ced In the leaves ofthe mntml (1-60 67 and 11-86 12 mug) The lnlected leaves showed the hlghest

1 vblt 12 l:llec~ ol aingic and dual trcatlliunta trn thu ,larch and sucro,c (mgg rl \r) conicnlIn C; mm infected by H solani- - - - - - - --- - --StarchSucrose, No Treatments I* la 11"- - - -- Sampling "ld wmplin~ sampling: samplingI" and II~ sampling dr!s rcprcsents 41 and 56 DAS respct~\el)'l'

eductton In the starch content (1-31 03 and 11-28 91 mglg) Among the slngletreatments. Pf23 exhlb~ted the h~ghest amnunt of starch content (1-62 21 and 11- 66 73mug). followed bv 7' harzeanum (1-51 43 and 11-56 73 mglg). C grgantea (1-48 36and 11-57 51 mp/g) and C'('IN5 (1-44 68 and 11-59 18 m@g) Among the dualtreatments. Pi23 + (' gtRantea exhlhlted the hlghest amount of starch content(I- 78 34 and 11-82 hl mglg). folloued b\ C'GNS + (' gcgantea (1-64 38 and 11-70 %mgg) and 7 hncranitm( ' gtgantea (1-58 53 and 11-hO 48 me/%)SI'C'ROSk:: 'l'k sucrow content In the Ica\cs ol'control. infected and treated plants1s presented In the Table 12 The h~ghest amount of sucrose was observed in thelaves of the control (1-47 and 11-77 5 mp'p) Infected leaves showed the least amountof sucrose (I- "4 5 and 11-25 5 mglg) Among the slngle treatments. Pf23 showed themaxlmum amount (1405 and 11-56 h2 mglg). follo\+ed h\ T hcrceun~trn (1-39 5 and11-13 5 mgg). C'(iN5 (1-3695 and 1111 23 mdg) and C gtponrea (1-31.6 and11-17 92 mug) Among thc dual treatments. PI23 + (' gegantea sho~red the hlghestIc\cl of sucrose content (1-53 75 and 11-62 98 mug). follo\red b!CGN5 + Cxtgunrru ( 1-48 31 and 11-57 33 lnygl and I hactanum + C gtganteu ( 1-4 25 and II-54 67 mglg)hlTRWEh h1EI ABOLIS%l:AL11h0 hllOR(;Elrc: I'he ammo nltrogcn content In the Ica\e of conuol, lnfrctedand other treated plants IS prrtsented In the lahle 13 lhe hlghest amino nttrogencontent was rccordcrt In rk conlrol lea~es (1-82 23 and 11-80 13 mgg) The lowpstamount of ammo nluogen was ohsend In the infected leabes (I48 91 and 11-43 14mug) Among the slnglr vcauncnls. hlghe.1 amount was recorded In the plantsmaled u~th PI23 (1481 and 11-60 38 myg), followd b? CGN5 (14032 and

l'rhle I3 Efict ol's~ngle and dual treatments on the amno nltrogen content (mfig d \r ) InC; nrcu infected b!, R solanrS NoTratmcatrAmino ai&ogem mateatI" saapUas II* mmpliagICuntrcil2 InfectedI" and lld sampl~ng o@s reprexnl 41 and Sh [)AS mspect~\el!.

11-58.23 mug), T. horzianum (1- 57.67 and 11-54 18 mg/g) and C. gigantea (1-48 13and 11-45 44 mg/g). In the dual treatments, Pf23 + C. gigantea showed the highestamount of ammo nltrogen content (1-73 58 and 11-70.15 mglg), followed by CGN5 +(' giganleu (1-70 17 and 11-66 73 mdg) and T hurzranum + C' giganfeu ( 148.33and 11- 6.5 2 1 mglg)AhIINO ACID C'ONTEN'T: The amino ac~d content In the lea\es of control.fnfeaed and treated plants 1s presented In the Table 14 Leaves of the control plantshowed the maxmum content (1-32 13 and 11-37 63 mug) A decrease was found Inthe lnfectcd lea\= (I- 18 93 and 11-14 Yh mug) Among the slngle treatments, Pf23shouvd the h~ghest contmt (1-24 33 and 11-30 84 mglg) followed b). CGNS (1-25 6and 11-30 R1 mdg). I humunum (1-22 37 and 11- 28 32 mglg) and C grganfea(1-20 58 and 11-28 Ih mug) Among thc dual treatments. Pf23 + C gigantea shouedh~ghest content 11-30 IY and 11-35 h? mglg) followed b)CGNS + C g~ganrea(1-28 32 and 11-34 41 mug) and 1 hactanum + C glgamea (1-27 63 and 11- 32 73mug)NI-I'HAI'E-NI~I'HITE ESTIMATION:NIrrafe conlmr: Ihe nltrale content In the lwes of control lnfecled and treatedplants 1s pwxnled In the l'ahlc 15 I'k nitrate content was lound to k h~ghrsr In thecontrol (1-1 3 41 and 11-1 1 13 mug d \r) Infected Ieates shoued the loutst amount 01nttrate contmt (1-5 32 and 11- 4 I mdrnl) Among the slngle ucatrnenrs. Pt23 sho\redthc maxlrnum (1-7 87 and 11-7 8 mug d u ) followed h\ CGN5 (1-6 89 and 11-6 32mug d H 1. C' giganleu ( Id 89 and 11-5 89 mg/g d u ) and T h~~onum (1-6 57 and11-5 9 mug d H) Among the dual trraunents. PC3 + (' gtgonrea shoued the highest

Table 14 I3lecl of single and dual treatments on the total amino ac~d content (mdg d w)In (i nras lnfcctcd b) K, solanrS NoTnrtmentrAmino acid contentIammpliag p m p l i ~Y PC3 . ( ' gcgunrrir 3U 19 (k0 33) 35 62 (*I 26)I" und lid umpllng da,, rcprcscnt J I and 56 IIAS rcsp.cll\cI)*P< 0 05 aplnst control (Mean i SLII

I ahlc 15 I:ll'kcI ofs~ngle ilnd dual treatments on the nltrate-nltrl~ estlrnatlon (mdg d M ) contentIn C; rnm ~nfected hv R solanr- - - - - . -S. No TrahcrtrNilrate eamtllltNitrirc ~~tcatPUIII~~X -" 11~mmp~4-- PIwI@I~-- - --~PmmplingIControlI" and 11" sempllng da!> represents 4 1 and 56 L)AS respectively

value (1-10.28 and 11-9.58 mg/g d.w). followed by CGNS + C. gigantea (1-941 and11-8.3 mg/g d.w) and T harzianum + C. gtwnrea (1-8 17 and 11-7.12mg/g d w)Nitrite contmt: The nltrlte content In the leaves of control, Infected and treated plantsIS presented in the 1 able 15 1 he nltrlte content ha5 lound to be hlghest In the control(1-10 18 and 11-9 37 mug d u ) Infected leaves shoued the louest amount of nltrltecontent (1-3 4 and 11- 2 86 rngiml) Among the slngle treatments. PR3 exhlblted thehlghst amount of nltrlte content (I-h 81 and 11-5 67 mg/g d \r). followed b!T hocranrrm (1-5 61 and 11-5 mg/g d u). CGNS (1-5 43 and 11-4 8 mg/g d u) and(' grgoruea (1-5 3 and 11-5 mdg d \r) Among the dual treatments. PQ3 + (' grganreashoued the maxlmurn talues (1-9 JI and 11-7 9 mug d u). lolloued b! CGNS +(' grgonteu (1-8 1 nnd 11-7 7 mgg d \r J and 7' hcranum + (' grganreo (1-6 9 and11-651 mgigdu)PWOTEIh: 1 k protetn content In the Iwes of control. ~nfected and treated plants ISprrscnlcd In tk loble Ih I.ea\es of the control bhoued the maximum content ofprotctn (I- 8 M and 11-9 mgg) 1.cares from the Infected plants shoued a s~gn~licantrecluctlon In the protein content (1-2 23 mgig and 11-3 76 mgig) Among the slngletrca~rncnb. 10uesI content \+as, obsened In (' gtganreo (1-363 and 114 M mglg) andI horsrunum (1-3 93 and 11-5 41 nidg). follourd h CGNS (1-5 93 and 11-6 49 mug)and PC3 (1-6 37 and 11-7 hJ mpg) In the dual treatmc>nL\. Pf13 + (*grgatireushourd the maxlnium proteln content (I- 8 22 and 11-92?), follo\red h CGN5 +(' RlgUNPU (I- 6 h9 and 11- 7 39 mglg) and T harranrrm + C' .rgonreu (1-5 7 and11- 6 39 mglg)

Table 16 I:l'Vcct of s~ngle and dual treatments on the proteln (mgg f \r I contentIn (; mar Infected b) H solanlProteinI" umpli~g 1 1 sampling ~I Control 864(H)05) 9 00 (10 31)2 Infected 2 13 (id 04)' 3 76 (HI 07)'3 1 /IUIZIUIIIIIII 3 93 (*u 0 7 ~ ~ 5 JI (dujp4 C(iN5 S 93 (a 08)* 6 49 (dl 25);5 IT3 6 37(& 17)* 764(M??)*h (' pr~onrcu 3 63 (io 09)* J 64 (dl 05)'7 i hocru~tum . ( ' ~~ganrra 5 70 (0 If))* 639(iC,05)*U ('(;NS . ( ' grgunrr4[l 6 69 (HI 1 1 )* 7 39 (a 07)'9 PI? 4 * ( Krpunreu 8 22 (dl 12) 9 27 (+20131I" and ll*f~rn~ltn~ duts represents 4 I and SO DAS respt.ctl\el\'1'. U 05 aplnst control (Mean i 31))

PHENOL: The phenol content In the leaves of control, infeaed and treated plants 1spresented In the Table 17 Control leaves showed mlnlmum phenol content (1-26 41and 11-28 94 mglg) There was a sharp Increase of phenol m the Infected leaves(1- 59 4 and 11-69 3 mdg) than the control and other treatments Among the slnglcueatments Pf23 sho\red the hlghest value (1-339 and 11-36 7 mdg). followed byCGNS (1-30 33 and 11-31 8 mdg). C grgantea (1-29 51 and 11-30 3 mg/g) andT hr~rzrantrm (1-37 16 and 11- 31 42 mug) Among the dual treatments PO3 +(' giganlea shotred the mahlmum content (1-3701 and 11-40 87 mug), follo~ed b!CGNS + ( ' grgantea (1-35 82 and 11-38 93 mug) and T hamum + C grgantea(1-32 8 and II- 34 96 m@g)PROI.IWE: 'The prol~ne content In the leaves of the control. ~nfected and aeatedplantc 1s prcsentcd In the Tahle 1 R Control leaves showed mlnlmum content (1-2 99and 11-3 41 mg/g) I-here Bas a sharp increase of the prollne In the Infected leaves(14 11 and 11-7 11 mug) than that of control and treated ones Among the singletreatments. C' grgantea shosrcd the least amount (1-1.5 and 11-3 98 rng/g) followed hl\'.1'123 (1-3 28 and 11-5 76 mglg). C'iiNS (I-? 7 and 114 9 mug) and 7' harzran~rm (1-2 2and 11- 4 71 mug) In the dual treatments. PI23 + C grganteo showed minimumcontent (1-1 23 and 11-2 XI mug) followed h> CGNS + C gtganrea (1-1 8 and 11-3 3mug). 7' horzrunrtm + (' gtgunfcu ( 1-1 73 and 11- 2 7 mg'g)PEROXIDASE: The perov~dase act!\ tt\ In the lea\es of control. ~nfected and treatedplants 1s presented In the hble I9 1.eaves of the control plants showed the lowestprov~dasc: actlvlt\ (0041 and 0 053 ulmln) &hereas leaves of Infected plants showedhtghest perox~dase actt\~t) (0 119 end 0 I22 ulmln) Among the slnglr treatments.nvyrne ectl\ln u.as found to be lowest In P123 (1-0078 and 11-0 089 dm~n).

Table I7 EfYect of s~ngle and dual treatments on the phenol (mdg d w) contentIn G nlax ~nfected by R solant1 Control 26 4 I (d 27)? lnlected 59 40 (d 49)'3 7 hat=rununr 27 36 (i0 52)4 CGNS 30 33 (*I 58)'5 I'C3 33 90 (3Ll 180)'h (' grganreu 29 51 (HJ 95)'7 7 hutz~utrun~ . (' gigantea 32 80 (M 96)'8 CGNS . (' ~tganrea 35 82 (d 82)'9 PC3 , (' gtguttreu 37 01 (M 62)'I" and lldsampl~np dsts rrprexrnts 4 I and 56 [)AS respecubel)*P

'Table 18 Eflect of s~ngle and dual treatment on proline content (rngig f.w)In (; nrax Infected hy R solunr.IC'untrol? Infected3 7 hueranurn4 C(iN5b (' grgunreu 1 53 (HI 07)' 3 98(io05)*7 1 /~arorrtunurn . (' grgunrer, 1 73 (HI 05)' 2 70(+0 10)'8 C'GNS ( ' grgunteu 1 8 (f0 06)* 3 3 (M 08)'9 I'Q3 . (' grgunrea 1 23 (M 07)' 2 81 (K, 06)*I" and lld somplrnp dn\\ rcpre\cnt\ 4 I nnd 56 I11C respect~\el\*Ib

Table 19 Effect of slngle and dual treatments on perox~dase (PO) actlvlty (ulrnln)In (; mm lnfccted h) R solanrS. NoTrertmenbPere.Muc activity (dmin)- - I" trmpli.0 fl samplingI' and II*' sampl~ng da\s rcpresnt 41 and 56 DAS repectl\el\*I3.ll 05 tlpinst convol (Meun * SI>)-. - . . - - -- -- --

followed hy CGNS (1-0 082 and 11-0 093 dmln), I harrratium (1-0 086 and 11-0 095dmln) and C glganfea (1-0 100 and 11-0 100 u/mln) In the dual treatments, thelowest actlvlt) has recorded In the PC3 + C grgawea (1-0 058 and 11-0 068 ulmln),lhllo\r cd h\ C(;N5 + (' prganrcu (1-0 063 and 11-0 075 utmln) and T harzrantmt +(' gtpantea (1-0 074 and 11-0 080 dmtn)POI.)' PIiENOI. OXIDASE: The polyphenol oxldase actlvlt!. In the leaves ofcontrol. Infected and treated plants is presented in the Table 20 Control leavesexhtbltcd lo\rfat act~vity (14.026 and 11-0.031 dmin) while that of infected leavesshowed the maxlmum actlbity (1-0 135 and 11-0.143 utmtn). Among the slngletreatmcnts. PE3 shotred the least actlblt) (1-0 073 and 11-0 007 dmln), followed by('(iN5 (1-0 077 and 11-0 102 ulmln). T harrranttm (1-0 098 and 11-0 106 dmln) and(' pryantea (1-0 103 and 11-0 118 LI/~I~)Among the dual treatments. lotvest actl\.tt>tras found In i'E3 + C grganreo (1-0 062 and 11-0 070 dmln). followed b) CGN5 +( ' Rlganrea (1-0 (166 and 114 074 dmln) and 7' harzranrrm + (' glganrea (1-0 078and 11-0 OM dmln)1-IPID PEROXIDATION: 1.1p1d peroxidatlon is an Important indicator of oxidativestress and IS eccompanad h\ the formatton of aldeh\dtc llpld hydroxidedrcompos~t~on product such as malonadlaldehyde (MDA) whtch IS expressed asnmoledml'lI.tpld perovldatlon actlvlt) 1s presented In the Table IThe amount of ltpld pero\ldatlon actlwl? In the leaves of control plants was(1-4 55x10'" and 11-2 h5hxl0'" nmoles/ml'l) li~ghcst rate of Itptd p.ro\~datton wmS ~ Oh H ~ the ~nfected leaves (1-4 77 XIO'"and 114 26 x10'" nmoledml) Among theslngle treatments, lowst rate of l~ptd peroxldatlon was found In PI23 (1-1 19x10'"and 11-0 I 1x10''' nmoles/ml'l). followed h) CGNS (1-1 38x10"' and 11-2 ?OSIO'"

(''I'rblr 20. Effect of single and dual treatment on the plyphenol oxidase-PPO activity (ulmin) inG mar infected by R solmiControlInfected7 Iia~ranurn('(iN5I'P- 3(' glganrea7 hacranum +gtganreaC'(iN5(' glRanIPc7PI23 . ( ' gtganrea- .-- ---IU md ll*uirnplln~ dn\s rcprcwnl JI and 56 [)AS repectl\el\*I1- 0 05 aplnst control (Muan + SD)

Table 21. Effkt ofr~ngle and dual treatments on l~ptd peroxidation (nrnolesiml)in the G, mpr leaves ~nfected by R. solaniI Control 455(N00l)2 Infected 4 77(N012)*3 7 hocran~tm 121 (N001)*4 C'Ghj 138(N00l)*5 PO3 I 19 (M002)*b i' grgun~ru 0 51 (N 001 )*7 1 hutzranum (' g~gan~ea O 98(*O001)*U c'(ih5 9 (' grgunl'a 1 09 (+dl 003 )*9 I'O3 * ( gigantea ORS(+dltOI)*--I' and 11" snmplrng - 41 1)AS and 56 DAS respect~\el)*Pc0 05 apalnst control (Mean i SD)

nmoles/ml). 7'. harzianurn (1-1.21 X I 04') and ll-0.13~10'"" nrnoles/ml") and('. gigonleu (14.5 l x 10"' and 11-1.49 x 10"'nmleslml-I). Among the dual treatments,Pf23 + C'. giganlea showed the minimum rate of lipid peroxidation (1-0.85~10'" and11-0.6531 ~10'"' nmoleslml"), tbllowed by CUNS + C'. gigantea (I-1.09~10"" and II-2.72~ 10'"' nnioles/ml.') and T. harzianum + C, giganrea (I-0.98~ 10."'and114.17~ 10'" nnmles/ml~l).PROTEIN ANALYSIS THROUGH SDSPAGE: Studies on the effects of thesingle and dual treatments on the extracellular protein was analysed through SDS-PAGE in different treatments (Plate 1 ). Banding pattern showed both qualitative andquantltatlve diflerences. On comparing the patterns of soluble proteins with standardmarker. new hands of different molecular weight were detected. The number of totalprotein fractions varied from a min~mum of 10 bands in the control to a maximum of13 hands In the treatments. 1'123 + ('. ~~ganlea and CtiNS + ('. giganrea. Out of 13hands 10 bands were common to all the conditions. Infection with R, solanr theC' lane, showed two hands In the range of 67 kDa to 68 kDa approximatel?. F and Glane. representing C'(jN5 and P123 treatment showed a new band of 26 kDaappox~matel~ other than the existing one. whereas E lane Has not ven visible.Anwng the dual treatments. 7'. hur:ron~rrn + (' grganrea in the H lane showed a bandof 15 LDa. Ibllo\r.ed h! C'(iN5+ C'.gigantra and Pf23 - C. giganrra in the 1 and Jlane show lng 68 and 70 Ll)a hands respectively PI23 - C. grguntra showed oneextra hand also of 35 Llk approxinutely (not ven clear).IUENI'IFIC'A'I'ION 01; AC-I'I\'E PRINCIP1.E: The spectral studies from theFI'IK. '11 NMR and 'I(' NMK are represented In the Figures 5. 6 and 7

Plate 1. SDS-PAGE analysis of single and dual treatments induced proffractions in G. m a infected by R. solanikDa A B C D E F G H I Jlane ALane B1.ant ('1,ane D1.ane Elane FI.ant (;i.ane H :Mr. Standards('ontrolinfected with R solattiSingle treatment nith C gigonteaSingle treatment with T. hanianumSingle treatment with CGNSSingle treatment with Pf23Dual treatment with T. hadanurn+C giganrealant lLane JDual treatment with CGN 5 + C gigmeaDual treatment with P N + C gigantea

Figure 5. FTIR SPECTRlIhl OF A("TI\'E PHlhC'1PI.E FRO11 I.EAF ES'I'WIZ

Figure 6. 'H-NIR SPECTRI:hl OF ACI'II'E PHIN('IP1.E FROM LEAFEXTRACT OF C gigantea

Figure 7. "c-~hl~ SPECTRl!hl OF A

0 urn-* nu - Y I2& a 7 ,*a -i:5:6 Ci!Z? v:999 s::3r 3:ar. 3:.,. ., -,I, -

DISCUSSION

DISCUSSIONThe present studv brlngs out the rndlv~dual and synerglstlc eft'ects of mlcrob~alantagonists and phytoextract on R solanr lnfect~ng C: max The main objective of thlswork 1s to find out the Ideal svnerglstlc comblnatlon of antagonist and phvtoextractthat IS not onl) beneficla1 to the hoct plant but also not detrimental to the antagonrstor phytoextract mutuall\The morphological parameters studied include height of the plants, leaf sijrr.number of pods pr plant and seeds per pod n.hile blochemica1 parameters includepcctlnolyt~c and cellulolyt~c enpmes (in vrrro). electrol).tic leakage. photosyntheticcontents. carhoh!drate. nltrogen content, proteln. phenol, proline. anttoxidants andltp~d pemxldatlon (m vrvn) lsolatlon and ~dent~fication of the active principle of thephytneutract \$as also done1.112' I'ITROSTI'DIES1.1 bdbl m!rrlial emn.th: In 1~rro screening stud~es on the leaf extracts of(' uurrc-trlulu. 0 wtrct~mnt. 4rndmcu. P jlrl!Jloru and C grganrea which were alreadyrcponcd Ibr thor lnh~hlton artn ~t!, on K solunr and thermostable nature (Kurucheverr at. IW7) \rere canled out to confirni the~r lnhthlton eflicts on R solanr incornhlmtlon \r ~th I' Itrrrrrotrttnr Anal! sls on the effects of all the leaf extracts of fi\.eplants along \\lth 7' hart-rrrntott rc\cals that c\tracts In ell solvents sho\+ed lnh~btton.pc>tcnt~al On thc has~s of the h~ghat ~nhihlton actlvlty on R solanr and its non-tnhlktlon (lfthr' anlogonlst (' grgmrea leafextract was selected for further stud~es Inhe prernt stud?, combtnatlon of 7' hucronrm (1%) and C grgameo in petroleumether (2.5 to 10% conc.) extract showed ~nhib~tor?. effect between 5 1 .I to 79%, that

-- --of chloroform (10% conc ) showed 95.3%. acetone showed 58% and ethanol extractin 10% conc. showed 89% of inhibit~on. That of 0. sanctum inhibited moderately.while those of C, auriculara and C gigantea inhibited more effectively.In virro studres were also carried out to find out the inhib~tory effects of C.glganrea leaf extract in combination with two strarns of CGN5 and Pf23 ofP.seudomnas Juorescens which hebeen knoun as the most effective m~crobialantagonlstr.Rate of rnh~h~tron under CGNS + (' grgunrcrr and Pi23 + C g~ganteo leafethanol extract var~ed from 70 9%. 78 5% to 70 8% and 859'0. 89 1% to 92 3%respcrlvel! In three concentrations (2.5%. 5% and 100/0) Combination of C g~ganreaextrdct rn other soltents also performed well but prtrnarrly due to the ~rnmisciblenature of other solvents In water and based on the h~ghest percentage inhibitor) effert.( ' gt,qunrea ethanol extract \\as selected for further m \.rrro and In \,rvo stud~esSeveral reasons have ken anr~huted to the lnh~brton actlvrty of mrcrob~al andothcr agents erther slngl or In combrnatron A cr~trcal survey of the l~terature revealsthat ethanol extract of C'gtganfea (10%) along w~th T horzranum and that of(' gtganreo In comhrnatron u ~th P .lluorescens were found to be most efficaceous Inhr~ngcng out the rnaxlmum ~nh~b~t~on of mycel~al gro~th of R sol an^ Several reasonsare attr~buted to the tnh~b~top acttvtr! of m~croh~al agents and phytoextracts e~thers~ngl! or In combrnat~onI lnh~brtron ma!be due to the presence of ant~hrotrcs in the biocontroiagents Resca cr a1 (2001) continned that the ~nhihrtron of zoospore germlnatlonof I' hetar hy Pserrdomtmas treatment \\as mainl! attr~huted to the action ofantth~ol~cs Maurhofer rr 01 (1992). Barea cr (11 (1'498) and Bhan and Sahalpara

(2001) confirmed that the ant~biotics produced in the Pseudomonos culturefilterate was the rnaln reason in the inhibition of the spores of Pythrum ultrmum.Glomus mosseae and C. fulcatum.Significant inhibition in the ascosporeproduction of C',inaeqrrulis was brought by the antib~otic actlon of 7'richodermuspp. (Clotola. 1987; Carlssa el ul., 2000). Effective inhibit~on of R, solani by T.vrrrde and 7'. horrianum treatments has been reporled by Roy (1997). Duky(1098) establ~shed that the antibiotics of 7: vrrrde were respons~ble for the~nhiblt~on of spore germlnation of 'Ihuncrrephorus cucwneris causlng web blight ofhorsegram. Ant~brotrcs of G'. vrrenr and Trichodermu spp. act as the marn factorsIn the suppression of sclerotral productron of R soluni (Howell, 1987). Wilhlte eta1 ( 1994) concluded that the sporang~al germlnation of Pyrhrum sp was inhibitedb) the actton of vlr~d~n antlb~otlcs. It Has considered as the main mechanism forthe lnhlh~t~on of sclerotla 01' S. rovsrr (Iien~s er a/.. 1983, Papavrm and Coll~ns,IW). In I

plant. Pseudomonas antibiotics were fbund to be the main factors in the inhibit~onof mycelial growth of Septoris spp. (Levy et a/., 1992).2 Inhibition may be due to the presence of antifungal inhibitorysubstances other than antibiotics in the biocontrol agents. The suppression ofpathogen growth by antagonistic treatment was mainly attrtbuted to the actrvity ofseconday metabolites including antibiotics present in the culture filterateMukhopadhyay (1994) and Mukhopadhyay and Kaur (1990) reported that thetnhihiton, substances (antfungal in nature)present in the culture filtrate ofTrichoderma spp and C;.virens were found tn be responsible for the inhibition ofmycel~al growth of Oaysponrm and R solanr Sneh et a/. (1984) reported that thesuppresston of the chlarnydospore gcrmtnation of Fztsarrum onsporum wasmatnl) due to the pnduct~on of ant~fungal compounds In the Pseudomomculture S\radling and Jeffr~es (1998) stated that the fung~static effect ofP.~cudomc~nas against the conidla1 grrmtnatlon of Bottyrrs crnereu, the cause ofgre! mould of strawkrries, was due to the antifungal compounds present in theculture tirondonit et a1 (1947) reported that the m vrtro inhtbitlon of mycelialgro\r.th of X solant. A cochlrudes. P belae. .4. cucurbrracearum and' c>q,.spnlm bras ma~nl) attr~butcxl to the actton of ~nhib~ton substances presentIn the culture filterate of' 7' hurzrunum Scarsellent and Faull (1994) reported the~nh~bttion of m).celial grotrth of R solanr by the culture filterate treatment of T.hacranwm3 It may k due to tte hyperparas~ttsm and rnycoparasittsm Duhey and Patel (2001 )suggested that the hyprparasitism of 7hrhodPnna spp acted as the matnmechantsrn In the inhib~tton of myceltal gro~zh of 77tanatephorus cucumens. Thework of hpak Kumar and Duke!. (2001 ) on the management of collar rot of pea

evealed that m vrrro rnyceltal growth of Fusarrum solanr was effecttvelycontrolled by the mycoparasittc actlon of 7 vtrrde, T hrzranum and G vrrensSirntlar finding was evtdent from the work of Anahosur (2001) on the lnhlbttionof growth of S rolfirr by of Trrchodermu spp and ti vrrens Gupta er a1 ( 1 YW)showed that the h)perparastttsm of 7nchoderma spp and G vrrem on Stheobromae Involved the cotllng of the hyphae by fonntng hooks, haustoria orappressorta lthe structure ~hich led to wrinkling, bursttng and collapse of thehyphae of pathogen Stmtlar report has avatlable on the hyperparastttc actron of7nchoderma spp tnhtb~tlng the hqphal growth of H solant (Chet er a/. 1991,Benhamou and Chet, 1993) and S rolfirr (C'het er al. 1978 Upadh~a) andMukhopadhla! 1983)1 It may he due to the synergtstlc effects of rntcrobtal antagonists and chemicalfungtc~des Jayaraj and Ramabadran (1909) showed that In the dual culturestudtes. Rhcohrlrm and Trrchrderma cornhination showed a sltght inhib~toneffect In both 'Thts report 1s substantiated b> the work of Setht and Subba Rao( 1968) and Jayarw er a1 (1994) w~th tuo strains of Rhcobrrrm In combtnationwith T vrrrde and 7' hu~ranum rn vrtro The h!phalttp of F, solanr f. spl\~ct~persrcr. I.' solanr f sp phareolr and Penrcrllrirm drgrtantm showed Iysis due tothe rll'scl ol' the wmb~ncd treatment of chitinasr and P-1.3-glucanax purlfiedfrom the pods 01'1'1.~11n1 .TN?II.II~I~ tvhtch acted s!nrrg~st~call! (Frl~h 1988) Marloner UI ( 1994) showed that the synthes~s of hydrolyt~c mcmes and peptaibols from7. hmranum is supposed to he tr~ggered hy Rorn~tts crnerea (medium containingthe cell walls). and both act synergisticall! brtth chlt~nase and P-1.3-glucanase inthe ~nhibttton of fungal spore germtnation and hyphal elongauon Di er 01. (1993)reported sim~lar type of synergism hetween an endochitinase and diketoptperazine

antlb~otlc (gllotoxin) from the blocontrol fungus C;lroladrurn (Trrchoderma)vrrens, closely related to 7 huntanurn The lnvestlgators reported that synergismresulted in the weaken~ng of the cell wall of B cmerea, whtch lmproved the rateof diffusion of the ant~blotlc through the cell surface5. lnhibltlon may be due to the synergistic effects of chemlcal fungicides and phytoproducts Effect of different concentrations of fungicides and neem extract onmycel~al growth and myceliogenic gemmation of sclerotla of S. sclerotionrm hasbeen brought out by Quais el a1 (2004). Mancozeb (75%) and neem oil (25%)showed slmtlar gronzh as that of control. while treatment with bavistin, topsin-Mand sa~laxyl-M% inhibited the gro\ilh considerably Treatments with ~arious conc.of' iunglcides (I,10. 50 and 100 ppm) afler dlpplng the sclerotla in the neemestract (25%) reduced the germination of rlerotia slgnificantl!. Pnddar er a1(2fH)O) dctermlned the most effe'ftve carbendazlm tolerant stralns of T hacran~tmfi~r the use In management of w11t of chlckpea disease caused b!F on.sporiimI:i\c mutants (four liom LIV and one from y-irradiations) of T harrianumtolerated 2 ppm of carknduim In the m vrfro stud~es There Bas four to elghtfold Increase In the tolerance of 7' hur:iunrrrnu,hlch was dekeloped from UV andy- ~rradiat~ons to urknducrn (2 ppm) and showed enhanced antagonisticpotCntlal agalnst the pathogen In the m vrrro condltlon.In addltlon to thc ah\e reasons enumerated h!the researchers so far thepresent stud! adds one more reason I e colnblnatlon of mlcrob~al antagonist andph)tcx\tract for the first time lnhlbltlon of H solani m irrru m~ght be due to theprcscnce of antlblotics in the microb~al antagonlst and antifungal substances ofph\toextract It non suggested that the highest rate of lnhlbitton of pathogen In the

-dual treatment is due to the fact that the inhibition of the pathogen by antagonist wasfurther enhanced by the phytoextract (C.gigantea)CELL WAI,I, DEGRADING ENZYMES ACTIVITY: The fungal pathogens areknown to produce different tspes of pectinolytic and cellulolytic enzymes. which arethe main agents for disease development Most phytopathogenic microorganisamsproduce pctinase which hreak the linkage between the galacturon!lmoieties inplymen of galacturon~c acid Though the present studies on the cell wall degradingenzymes were done in vrrro a comparison hith the following in vno studies leads tofollowing critical discussionKeduct~on ol' the ennme actlvltles leads to the loss of the potentla1 ofXsolunnr to dlssol\e the ccll \vall of host and thus the growth of R solunr \\,as~nh~b~ted The actlvlt! of tso-ISTI: was reduced to the maxlmum level In the s~ngleand dual ircatments compared to that of control (R, solanr culture alone). \+hilt that ofother mnmes shoued moderate act!\ II!.compared to that of control and that of Exo-CMC was last reduced Among the slngle treatments, phytoextract sho~ed leastreduction of all enqmatlc actlvltlcs and whlle Pseridomonos (PC3 stra~n) esh~bitzdh~gher reductlon In the case of dual treatments. comb~nat~on of Psertdomonas-Pi23strams and phyrwxtracl hrought mlnlmum percentage of actlvit~es of all the enzymes'l'hc ah\e find~ngs rcveal that all the ennmss were not lnhlh~ted equally b!thehrmontrol agcnts 1.1herr IW, the emme actlvrtles are reduced to van,lng degree b)the slllgle and dual treatmen1 and o\ er all reductlon of enz).me actn it~es Has found totw h~gher In dual treatment due to the suppression of enzyme actlvltles b!mlcrob~alantagonists and ph? toestrdct In success~ve manner These ohwn.at~orrbuhstant~ate thefollowng lindlngs

Chakrabartl and Basu Chaudhary (1979) observed that the pectinolytlc andcellulolytlc enzymes produced by Fusarrtim spp, dissolve the mlddle lamella anddiffuse into the xylem vessels resulting In xylem parenchyma maceration, mlddlelamella distortion. vessel blockage and disease symptoms. The pectlnase enzyme isproduced prlmar~ly to dissolve the cell wall of the host and to reach the Interlor tissue.Durlng colonlzatlon 11 secretes cellulase to attack the primary and secondary cell wallresulting in thlnnlng of cell wall followed by disintegrat~on. Durlng the lnfectlonprocess, cut~nolytlc. pectlnolytlc and cellulolytic enzymes are produced by pathogensto facilitate 11s penetratton Into host tissue through the cuticle and cell wall. Zimandc1 a1 (1995) fbund that the activity of pectinolytic enzymes such aspolygalacturonase. pectin-methyl-esterase, and pectate-lyase produced by B cinerea~nfkcting bean leaves was less In the presence of the antagonist 7: harzranum T-39.According to them. the antagonism of that spec~fic isolate is partly based on its dlrector ~ndlrect ett'ects on the enzymes production by the pathogen during the lnfectlonprocess Cam-Canchola er a1 (2W) observed that extra cellular enzymes of L'srrlagospp transform the polysaccharldes of the host. Zea mqvs, which are used by thepathogen for the~r growth and development Cell death of the host caused by thepathogens was attributed to the pect~nolytlc enzymes wtth d~tlerent pH opttma.~soelectrlc points and thelr mechanisms of substrate cleavage (Rwslnk. 1971 : Ruesinkand l'h~mann. 1965) Aktar and 1)imond (1960) reported that the cellulolytlc enzymesstxreted b!busurr~rm spp attack the prlman and secondary ell wall of the host(tomato) and d~slntegrate them The degraded products may get Into thetransplratlonal stream, whlch block the vessels Ieadlng to the forrnatlon of HIIIsymptoms Vclrvhagan er a1 ( 1999) found that the extracellular chitlnase and P 1.3-glucanase of Psetrdc~nronas culture lnhihlted the chitin and glucan present In the cell

wall of R, solani. Riathilagam (1999) found out that the non-volatile antlb~otics ofT. viride treatment effectively suppressed the lytic enzymes of C, capsici (in vitro).Pugazhenthl (2001) confirmed that the suppression of lytic enzymes of('ollefotrichum spp. (in virro) was malnly due to the antib~ot~c action of7iichoderma spp. Decreased actlvity ofthe pectinolytic enzymes of varlous Fwsurrumstrains causing leaf spot and leaf bl~ght In mulberry leaf due to the fung~toxic effect ofcxtract of' Pongumra prnnara and Aradirachta indica was reported earller (Ciupta eta/., 1996) Similar repon on the effects of bark extract of Barringzonra ce~vlanica on('~~n.ularra spp. and ('. gloeosporrodes were made earlier (Palanakurnbra et 01.. 1996).Chltlnase and [)-I .3-glucanase purlfied from the pods of Plsum sarivrrm wereused syncrglstlcall! to degrade fungal cell walls of two Fusarium spp vlz , Fusarrumsolanr I'sp phaseolr, a non-pathogen and I.. solanr f, sp. pis;. pathogen of pea.Chltlnase and PI .3-glucanase given alone or In combination showed d~fferent resultsIn both 'tcsarrrrm spp In I.'. solanr f sp phoseoli treated hith chltlnase and P-1.3-glucanase erwmes alone showed the release of wluble duclng olrgosaccharidesfrom the cell walls Treatment with chltlnase led to the release of N-acetyl-P-glwosarnln~dase ol~gosacchar~des (GlcNAc): hohever comblnatcon of both releasedmore GlcNAc ol~gornen than the ch~tlnase glven alone In F, solanr t sp. prsi. celluall uas ponlally degraded hy the ahwe treatments Amount of'reduclng sugars andthe (jlcNAc ollgomen released was 30% lower than those of I.' solani f sp phoseoli.Agaln the purlticxi chltlnase and1)-1.3-glucanase were testtd ~ndlv~dually or Incombtnatlon Ibr detcmllntng thelr etl'ectlveness agalnst elght dlti'erent fungl (Fel~x1988) It was confirmed that e~ght fungal sptrles treated \r.~th chltlnase alone ~nhlhlcedI' vrrrde \rh~le, pl.3-glucanase ~nh~hlted F solunr t: sp. prsr while cornhlnat~on ofboth uvre ctli.ct~\.e In lnh~hltlng all thc c'lght fungl tested

Marion el al. (1994) brought out the importance of cell wall hydrolyticenzymes, such as chitinase, b-1,3-glucanase and protease and antibiotic-peptaibols(trlchor~lanines) from T. honranum In the Inhibition of various phytopathogens.According to them, ch~tinase and 8-1.3-glucanase enzymes cooperate synergisticall),and were more eff'ect~ve in comb~nation rather than the single enzyme. Lorito et a/.(1993) showed the potential of cell wall degrading enzymes produced bylrrchtwlermu and C;lrocladtrmr In the control of fungal dlseases The ant~fungalactlvlty of purllied endochit~nase, ch~tohiosidase. N-acetyl-p-glucosaminidase andglucan-0-1.3-glucos~dase, e~thcr alone or in combination on the spore germinat~on (orcell rcpl~cat~on) and germ tuhc elongat~on In nine dlfirent fungal species wereconfirmed Strong ~nh~b~t~on of all the pathogenic fungi is ohwved, except inI'.vrhrrmr. \rh~ch docs not have ch~t~n as structural component in the cell wall Thedegree of ~nh~hrt~on was proponlonal to the le\el of ch~tin In the cell wall of the targetSung1 C'omh~nat~ons of the purllied enzymes resulted in a slgn~ficant Increase In theant~fungal act!\ ~t) ([)I ef 01. 1993. I.or~to er a1 1994 a, b). Espec~ally h~gh synerglst~cellkts Here Ihund when glucan 1.3-P-glucos~daws and ch~t~nol!t~c ewmes kjereu.wJ In thc comb~nat~on 'l'h~s com,sponds to the effecls ohxned when p-1.3-glucanasc and cndc~hlt~nasc ~solated tiom plants \rere comb~ned (Fel~x 1988) Ck LaCru er rrl (1995) Sound out that 7' hucrunr~nr straln CbCl' 2413 produced two 1.6-1)-glucanasc. Is(wn4 mes of \\ h~ch one \\as 43 kI>a \\h~ch\\as supposed to k purlfiadto homogene~t> When th~s ennmc \\as used In comh~natlon \\~th other cell \\,alldcgrad~ng cnLlmcs of I Inrr=run~rtrr, such as 1.3-0-glucanax and ch~tlnase. Ith)drol)xd lilamcntous fungal cell trail and tnh~h~ted the growth of the fung~ studledv12 . HON?.IIJ c~rrrcreu. (;rhhcrrllrr.f~qrAi~rf~~. P/!l~lOplhOr~ qsrttgac and Soccharonr~~rescerec'rsrue

Dlsense Intensiy Studles: Significant suppression of disease intensity in the presentstudv was attributed either due to the synergist~c activity of antihiotics/antifungalcompounds produced by the antagon~stic microorgan~sm and phytocstract or b) thehvperparas~t~sm on the pathogen or hy ISR (tnduced systemic resistance) in the hostplan1 caused h! bnth agents which comhat the pathogen infectionIi~ghest disease intens~ty Mas seen in the Infected plants (3 12) In all theslngle and dual treatments. the dlseasc intensity was reduced compared to the Infectedones Plants treated \v~th 1'123 + C' g~ganreu exhih~ted lowest drscase Intenslt) (I .I b),follo\ved by CCiN5 + C' ,qigunteo (I 92) (I~ght ~nfection) and T, hor=ronum + C.glgonreo (2 30) (mcdlum infection) In all the single treatments (CGNS. 2.78: P123.2 61. (' g~gunrea. 2.37 and 7: har;:ianrtm. 2.66) med~urn d~sease Intensity wasobserved I'he present study galns support from the follow~ng stud~es Manoranlithamer a1 (2001 J ohsened that the ant~h~ottcs of Pseudomonos lnhib~ted the symptomfonnatton of'damplng ott'caused by firhtrtm spp. In tomato. Singh (2000) suggestedthat the reduced pa po\vdev m~ldew s),mptom format~on tn the Pser~domonassprayed plants was ma~nly due to the ett'ect of ant~fungal compounds wh~ch ~nhib~tedthe toxln product~on ol' the pathogen thereby reduc~ng the symptom formation.S~mllar elTect ot'ant~b~ot~cs of heudomonas spp. In reduc~ng the symptom formatton\+as repfled by Sleesman and Leben (1976) In southern maize leaf bl~ght. Spurr( I981 ) and Spurr and Knudsen (1 985) In ('ercospora leaf spot of peanut. Keel er a1(IW) In black root iot of tobacco: Dult'y and Defago (1997) in F~~surrrtm crown rotof tomato Ile Meycr and tiolte ( 1997) found that the reduced Bornfrs lnfect~on byPseudomonus treatment In tobacco was ma~nly due to the action of s~derophores(pyoverdln. pynchelln) and saltcylic ac~d V~dhyasekaran er al. (1997) showed that the

seed treatment followed by foliar spray with P. ,fluorescem in rice might help In itsextensive colonization on the phylloplane.In the seed treatment. P.,fltrorcscens moved from the applied area to the leavesand inhib~ted the blast d~sease symptom formation. The foliar spray helped to boostup the quantlty of' the colon~zation thereby reducing the symptom de\elopmentCameron et a1 ( 1994) repnncd that the bacterial lipopolysaccharidel ol~gosacchar~deactivates the host defense reaction In the Arabidopsts spp and thereby reslsts thes).mptom format~on caused hy cucumber mosaic \,irus.S~rn~larl!. ant~blot~c actlon of 7hchudernru spp and G. vrre11.5 \\as el idcntliom the work of Uube) (2002) in the control of French bean collar rot caused b)Xbuta~rcolu. tio\+ell (1991) In the control of fi-rhrtim damping off of'conon. Iio\\elland St~panov~c (1983, 1995) In the control of symptoms caused b) P tiltmturn.K solant and I.e\\~s and I'apa\~zas (1991) and Howell er ul (1993) In the control ofdamplng on' of cotton caused b> R solanr. Slmilar report Has made h\ Corke andtlunter (1979) In prunlng \\ounds of apple caused h). .\'ectrra spp Elad and Kapat(1999) found that the rnh~b~tlon of Bor~tts crnerea by T hatriantrm \vas malnl), dueto the actlon ot' pratease enzyme which lnhib~ted the protein synthes~s of the pathogenthcreh! reduclng the sun~val of the pathogen In the host plant Slm~la repork+crce\ ~dent from the trorks of I:lores et ul (i9Y7) and Koch (1 $99) Bael, et ul (1999)rcpon~vl that the r~ducrd d~.sease Intensrty caused b) R solani In T wens treatedplants was ~na~nl) due to the actlon of ch~t~nase. The chlt~nase produced b> I' wrens&stro)ed Xsalunr cell ivall and its I).t~c enzymes thereby inhib~tlng the symptomdevelopment U~dar~ and tiundappagoal (1997) explained the role of competltlon Inthe control ofp~geonpea u.111 hy 7' ~,tride treatment. The colon~zatlon of Trichudermu

spp. created a competition for nutrient and space with the pathogen. The rapidmultiplication of Trichoderma spp, utilized all the essential materials. which wererequired for the growth and survrval of the pathogen. Due to the starvation for foodand space the surv~val and symptom formation of the pathogen was drast~callyreduced. He further concluded that the synergistic action of ant~b~otlcs. competltlonand mycoparasltlsm of Trichoderma spp. was collectively responsible for theeffective control of the dlseasc.1 he present stud~esupport the le\r that the reduct~on In the dlsease lntenslt)m~ght be due to e~ther ant~blot~c and ant~fungal compounds produced b) the m~crob~alantagonlsts or due to the comptlt~on m\coparaslt~sm or both In the case oi7 harr~unltm and Pteirdontnnas treatments Reduced disease ~ntens~t) In those treatedurth C glwnrea alone ma, be due to the ant~fungal compounds and slgn~ficantreduct~on In dual treatment m~ght be due to the s\nerglstlc erects of the m~croblalantagonlsts and the ph\ toextractIianare er a1 (IW) sho\rjed the successful lntegratlon of biological andchemlcal control of Botrytls gray mould (BGM) caused by B crnerea in chickpeaThe double alternate treatment of \ ~nclo;ml~n + T virrde followed by T viride alone.and then vlnclozolin + T 1-rrrde was followed In the experiment. ivhich proved to bevey cff~~tlve than e~ther of the indlwdual treatments The treatment reduced thed~sease severih. and Increased the yeld This stud!, also hiphllghts two points,F~rstly. fungcc~de use can be reduced to half and secondly due to the integmtion of thehlo and chemical fungic~des, the h~ofung~cide seems to hr. more effective due toseveral dtfferent modes of action against the pathogenic fungi

Studies on the effects of talc based formulation of T. viride on seedgerminat~on and seven fungicides in the control of the angular leaf spot disease inFrench bean (Phaseolus vulgaris) caused by Phaeoisariopsis griseola (Rajappan andYesurala. 1999) showed that T Wideas seed treatment Improved the seedgerminat~on and the angular leaf spot was effectively controlled by the comh~nat~on offbl~ar spray of' 7' vrrrde with mancozeb.Kajappan ct a1 (2000) gave an alternat~ve suggestion for the less usage ofchem~cal funglc~des hv replacing certaln chem~cal fung~cides by the neem 011 (NO)based two emuls~fied concentrate (EC) formulat~ons vlz., NO 60 EC, NO 60 EC (C )and one neem o~l + pungam (Pongamra glabra ) 011 based EC fbrmuation, NO + PO60 IiC' (C) Neem and pungam 011 based EC formulations were found to be Inferlor tofung~c~des hut superlor to control In reduclng the disease Incidence and lncreaslng theyield In the 1:rench bean (f'rulgarts) Infected by Xanthomonas campesrrrs causinghacturlel bl~gh~ Lemanceau et a1 (1992) were able to control Fusarlum wit diseaseb) comhlnlng a non-pathogen~c antagonlsttc straln of F, owsporum w~th P, purida.which d ~d not reduce dlxtlsr severlt) on 11s o\cn. Intrgrat~on of Trtchoderma spp, andtilroc.lad~um ~ ~ l'ung~c~des t h like th~ram, captan, benomyl and vttavax gaves~gn~licantly h~gher disease control In sekrral crops (Mukhopadhyay, 1994. Vyas.1994. I)ubcy. 2000) Dube) (2003) found that G, virens and 7. viride alone showedsuperlor perthrrnance over the fung~c~des Comhlnatlon of G virerts T vlrrde +vlovah (iungic~de) + Rhcobrwrn again followed by G virens T virrde + vitavax gavemaxtmum plant \~gour and minimum seedling mortalit). and disease intensit). in bothurd and mung heans Among the two antagonists used. G virens and its comh~nationsshowed hener performance and superiority over 7' viride and its combination in theeffective management of web blight of urd and mung bean diseases Tiwar1 and

- - - - -- - -Mukhopadhyay (2003) brought out the Integrated approach of fung~c~de-vltavax w~thvarlous preparations of G vrrem In controlling ch~ckpea root rot and collar rot causedby S rolfirr. R solanr and F solan1 Appllcatlon of carboxymethvl cellulobe (CMC)w~th C; rvrrens In comblnatlon 141th bltatax (6 rllrens + CMC + \ italax) pro\idedmaxlmum protection than any of these agents glven alone or in d~fferentcomblnatlons Mukhopadhyay er a1 (1992) reported slmllar result In the control ofchlchpa and lent11 u~lt complcx b\ the comhlnatlon of chemrcal funglc~de. 11tavax~lth antagonistSlmllar s~tuation is encountered In the present studies wherethe reducedd~sease severity was more effective under dual treatments. Among the singletreatments. those of rnicroh~al antagonlsts showed almost same dlsease rntensitywhich ~ d slightly s less than ~nfected ones while that of C gigantea showed lesserdisease Intensln. 'fh~s mlght be due to the spra!of phytoextract after 24 h ofinoculation of R solan1 wh~ch may be responsible for preventing the pathogen fromrroch~ng the leaf tlssues Llnder treatments of antagonlsts and phytoextract. thesynergistic elfecls wcre found to differ in \.arious treatments PC3 + (' pganteatreatment sho\r,ed hlghest reduction in disease IntenSlh The reason m~ght he that theph\-toextract might ~nhibit the ph)~slolog~cal processes of the pathogen which arealready lnhihlted to certain extent by the antagonistMORPHOLOGICAL PARAMETERS: Shoot length of control plant showed 28.8cm(l) and 15 2 em (11) and lnlkctrd 21 . I I cm (I) and 28.20 em (11). folloued b) singletreatment \rith PI23 (1-27.27 and 11-38.3). CGNS (1-2713 and 11-38 cm).7' kcranurn (1-26.21 and 11-32.12 cm) and C. grgan/ea (1-25 12 crn and 11-29.8 cm).'l'he mastmum shoot length was found In the P123 + ('. gigantea (1-50.4 cm and

- --11-56 12 cm) and CGN5 + C' g~gantea (1-33 2and 11-36 16 cm), whlle that ofT. horzronum + C.'. gigontea (1-30.18 cm and 11-46.5 cm) was almost the same as thatof control.Among the s~ngle treatments shoot length of those treated w~th mlcrob~alantagonist was always more compared to that of C' grguntea All the dual treatmentsshowed almost the same shoot length on I sampl~ng day However. the d~fference wasmuch pronounced In the II sampling day Maxlmurn shoot length was observed InPI23 + (' grganrea treatment 7 hese stirnulatory effects m~ght be due to the comb~nedeftect ol the abncc on the hlgher rate of synthesis and actlv~ty of shoot apexI.eal'area ofthe infected plant was vey much reduced when compared to thecontrol and treatments In the I and II sarnpllng days. Among the slngle treatment.1'123 showred the mavlmurn SIX. followved by CGNS, T haclanurn and C' g~guntea.In the dual treatments PI23 + (' grgantea, showed the maximum size compared toCGNS * (' glguntea and T hucronttrn + C'. gigunrea. These stimulator! effectsm~ght bc due to the synsrglstlc elTect of the abnve In brlnging the h~gher rate ofs\ nthetic activit) in the difirentiatlng leavesPods per plant and seeds per pod showed the same increasing trend as that ofthe other hiochem~cal parameters in the I1 sampling day. Infected plants showed andecrease in the number of pods per plant and seeds per pod compared to the plants of'control and treatments This is due to the negative Interactton of host and parasite hj.which the mtabnl~c act~vlt~es are lnhlb~ted Among the single treatments. Pr23showed an enhanced result followed bv CGNS. T horrionum and C. grgomeo In thedual treatments. PI23 + C. &anleashowed an increase in the yield parametersfollowed b\. CGNS + C. giganlea and 7: harzionum + C. giganrea. The increase in

the yield can be attributed to the stimulation ol' biochemical actlvltles resulting Inbetter source sink relationship and growthMadhu and Wajld Khan (1994 a) brought out the efTects of two symbionts-Glontus culendonrr~tnr and Xhizobmrmr spp on Cignrr mungo and also assessed theImpact of' 502I'he above two spccles elther slngly or In comblnatlorl sho\\edIncreased plant growzh pirameters Ilke, length. fresh and dry welght of shoot and rootand also Increased yleld parameters (number of pods per plant and number of seedsper pcbd) and Increased In the root nodulatlon. According to them, due to thecomhinatlon of treatments there has an Improvement in the numtron ol' themycorrhliral and nodulated plants and they were less affected by the SO2 exposure.I'rlya er ul ( I9YY) carried out the stud) to determine the Influence of' VAM fungus-Glornus nrosseue. Rhrzobruttt spp. and I hcronurn ~ndlvlduall) as well as Incombinatlon on Acocru nrlo~tca Co-~noculat~on with VAM t'ungus and Rhrzobrurnspp resulted In maslmum plant groulh (shoot length, shoot dr) weight, root length,root dn weight) and lncrcase In nodulatlon. nodule number, nodule weight and O/OofVAM lnlkct~on than the other slngle and dual comblnatlon Co-~noculat~on of VAMfungus and Hlrcohrrrm pp had shotvn a slgnlficant growth and nutrltlonal uptake In Anlk)/lc.tr Slmllar results \+ere reported h!, Bag!.arai er a1 (1979). Varma (1979). Disone/ 01 (1993). Mandal and Kaushlk (1995). Bala el a/. (1995) and Mehrotra (1995).Studies on the eff'wts of fl! ash. elther separately or In combinatlon ofC; coler~d~~nrc~otr and Rhrohilrrtr spp on hlackgram showed an increase 111 the gro\rthpramcters Ilbe. shoot length, fresh and dr! weight of shoot and root (Madhu andWqld Khan. 1999 h) The!suggested that combination of treatment might bekneficial and more effective than the single ones. Jayaqi er a1 (1994) reported theincrwe in the yield and total hiomass with two strains of Rhizobitmm in combinatlon

w~th 7 vrrrde and I hanranum I'he compat~b~l~ty and even an oc~s~onal synergismof Rhrzobtum and Trrchoderma combtnat~on have been reported (Haman et at,1981) Bharatwal (1970) reported stlmulatory effect of T hanranum on the growth ofMe11loru.s ulba and M rndrcaPartner and Dada~af (1999) reported Increase InIncrease In the actlvtty of ch~ckpea follow~ng the comblned ~noculat~on ofPseudomonas and Bacrllus sps w~th XhrzobrumP .Punre.rcen.~ and P purrdo have been reported as successful seed inoculantsI'or blo control of scvrral plant pathogens (Weller 1988; Dileep Kumar and Dube19%'. l~uilTe/ ul. 1993: Drleep Kumar, 1998) Dual treatment of chick pea seeds~8th the s~derophore produc~ng tluorescent f3eudomonas straln RBT13 and anant~h~ol~c producing H suhsttlrs straln AFI. Increased shoot length, root length, freshand dn weight and ! ~eld In so11 tnfected \rtth Ftlsarrum ngsponlm (D~leep, 1999)Successful rr~ults of crrrncrulat~an. h!seed hater~i.at~on uith Pseudomonus strainsand Xhcc~bium has k n alreadj reported b) Chanua) eta1 (1989) In Lens esculenfaand Prstim safrvunr. Grlrns and Mount ( 198.2) In Phaseolus vulgarrs. D~leep Kumar er01 (Z(W)I ) In I' surrvnr,t and I'olonmko cr a/ (1987) In so) beanFor the first tlme the prrsent stud!hr~ngs out the s)nerg~st~c effects ofantagon~sts and ph\roextrict on thc Ieal'slLe The Increase in the leaf SIX might beduc to the ~ntian~emrn~ of all nie~hol~c ncrl\~t~c.s tnclud~ng photos>-nthesis b) thecclmh~nrd trratment of the mlcroh~al antagon~sl and ph!.toestract.ELE(TWOI.\'~l'j(' I.EAliA(;E:Control plants showed the mlnlmum electrol)~tcleakage. Inlkltd Icabt- sho\vcd hlghcr electrolyt~c leakage (69.77%). Among singlemrments. 7: k ~ranum and ('. gigmlrJu shotred sl~ghtly Increased leakage wh~le the

two Pseudomonos stralns reduced electrolyt~c leakage sl~ghtly Kate of electrolyt~cleakage was reduced In dual treatments wlth T harzranum + C ggantea and CGN5 +C grgunrea, whlle Pt23 + C' grgantea exhlblted mlnlmum leakage Hlgher percentageof electrolvtlc leahage %as found In the I1 sample wh~ch ~nd~cates the on set ofsenescence of leaves Interestrngly the electrolyt~c leakage was always h~gher In thelnltlal stages In all the treatments (s~ngle and dual) and reduced after 22 5 hrs on I andI1 sampling da\s I h ~ \ suggests that the mlcrob~al antagonlsts and phytoextractmalntalns the stabtllt! of the membrane sem~permeab~l~ty by ~nh~b~tlng R solanr Indue course oft~me from 1 5 h to 22 5 hElectrolyte leakage has been ~mpllcated in governing resistance/susceptibilityIn man!host plants (Hrtsset and I'aulln, 1991. Whalen er at. 1993) SeveralInvestigators have reponcd pcrmeablllty changes durlng pathogenes~s In \,ariousplants (Vtdhyasehharan. 1997) Tholtho~ Slngh er at (2001) found that fungal toxin orI& enzymes contributed to the hlgher electrol~~t~c leakages from the infected riceseeds According to them. the actlon of fungal toxln on the host cell resulted in thedlslntegrat~on of semi permeable membrane. \r.h~ch led to higher leakage graduallyresulttng In the loss of sernl-prmeab~l~ty nature of the cell membrane Increase inelectrol>.t~c Ieaknge In the Infected condition finds support in the work of Khirbat andlaloll (2000) In tlrc Iea~es of resistant \,arlet! than those of the susczptlb~le varlet? ofchlckp ~nlbcted wtth ."lscoch~'/u rahrerAccording to Padma Singh ( 20) the damaged cells of onion leaves infectedh\. .4lrrrnar1a spp lost the~r ab~lity to accumulate the rnetaboltc solutes, which wererequired for the normal growth and development of the cells Similar reports weremade by Cook and Stall (1968) in (' annrlm leaves infected by .\bnthomonas and.

Wheeler and Black ( 1962, 1 Y63), Samaddar and Scheffer (1968) and Schefler andSamaddar (1970) reported that h~gher electrolytic leakage in the Helminthosporium -infected cells was mainly due to the toxlc effect of victorin.Reduced electrolqtlc leakage In the treated plants m~ght be elther due to theactlon ol antrhrotlcs produced b) the anlilgonrstrc rnlcrobes, ~hlch lnh~b~t theestahl~shrnent and acuvlt) of the pathogen or the) Induce cell to develop systemlcresrstance. whlch restst the pathogen In\aslon Thus the host cell 1s protected from thedamage caused h) the pathogen 7'h1\ lindrng \\as suppnned b) the uork of Abad eta1 ( I ')Vh) that mlnlmum electrol\ t~c leakage rn the treated tobacco leaves was marnlydue to the actlon of PR protein. osmotln Similar repnrt was made b) LIU et a1 (1994)In potato plantBICX'IiEMICAI~ ESTIMATIONS:PhotmynIhcrlcpigmenr conlmr: l'he Ilght reactlon of photosynthesls IS a direct resultof photon ahsorptron b!pigment molecules called chlorophyll Any change in theplgment contmt wuld be reflected ~mmed~atrly on the photos) nthetrc etficac), of theplant and suhsequcntl! on 11s grouzh and !,~eld. Leaves are the primar) organs forlight Interception and photosynthesls In plants Hence the analyis of pigment contentIn the leaves of control, infected and treated G mar plants uas carried outfhe quanllt! of chloroph! II plgments end carotenoid content itas lesser In the~nl'rctrd Ica\es when compared to those of control Among the slngle treatments, Pf23treated leabes shotred an Increase over other treatments on both sampling daysAmong the dual treatments, PR3+C glgantea rxh~blted rnaxrmurn chlorophyllplgments and carotenotd content illd samples showed decreased trend In the

photosynthetic pigments In the leaves of control, infected and treated plants. 'I'hismight be due to onset of senescence of the leaves. The lower photosynthetic rate inthe infected leaves of maize plant inf'ected by Helminthospor~um turcrcum causing'Turclcum leaf blight (7'1.B) d~sease might be due to the necrosls and chloros~s In theInfected leaves (Pant er a/., 2001). According to Magyarosy etal. (1976) one possiblereason for the reductton in the photosynthesis rate in the leaf blight Infected plantsmay be the suppression of ATP formation in non-cyclic photophosphorylation as inthe sugar beet powdey mlldew disease. Another posstbility seems to be the endproduct ~nh~bition by starch and other carbohydrates accumulation in the tnfectedtissues Decrease due to ~nfect~on could be attributed to the inh~bltlon of synthesis ofplgments or the rapld photo-oxidatlon of chlorophyll. Tu and Ford (1968) observedthat heat? reduct~on in the photosynthetic acti\,lty In maize due to virus infectionmight be due to the actlvlt\ of the pathogen. wh~ch lnltiated chloros~s and necrosis inthe ph\Iloplanc Accord~ng to Pant er a1 (2001) the reduct~on ~n the photosyntheticacll\lt\ In malx leaf (suppressed ATP format~on) was ma~nl! due to the toxic effectof the pathogen It mlghl also be due to the synthesis of ph!totox~c substances hy thepathogens involving the ~nteractlon of h) drogen In the presence of chlorophyll.Illcner ( 1963) explained that the reduction in the photos! nthetic acti\,it) \\asma~nl\ due to the induct~on of chlorophyllase act~vily by the pathogen or its toxinColletoun. a tostn produced h) C cap sic^ reduced chloroph!,ll pigment content andphotos) nthet~c act~\.it! In the infected turmeric laves (Suhharaja, 198 1 )I.east reduction in the chlorophyll content was noticed In the dual treatmentcompared to the single ones Co-~noculat~on of two symhionts - C calendonicum andRhi;.obirmmspp In the presence of SO2 increased the chlorophyll content whichultimately promoted plant gro~zh of I' mtmngo (Madhu and Wajid Khan.1999 a).

Llndow (1984) recorded least reduction of pigment content in the Pseudomonas-treated leaves of pear infected with fireblight disease. The main reason was that theantibiotics produced by Pseudomonas inhibited the activity of the toxin produced bythe pathogen. 'I'he treatment rnighl suppress the activity of chloroph~ llase or it mightproduce the needed organic acids for the host growth and development Studles on theeffect of ash and crrlnoculat~on ofG calendonicum and Rhizobium spp elther singlyor In comhlnatlon In hlackgram showed en Increase in the chloroph\ll content andyield (Madhu and Wayd. 1999 b) Antibiot~c actlon might be another reason for theleast reductton of chlorophyll In the I'.~errdon~onas-treated rice leaves infected by Rsolnn~ causlng sheath hllght (Mew and Rosales. 1992). Similar observations weremade h! 1.1ndou er a1 ( 1%).Puse). ( 1996) and Zhou and Young (1 996) in fireblightand frost Inlun of' pear and scab dlseasr ol' apple respectlvel)The above findlngs substantlate the ohsen,ations made In the present study.Induction of chlorophyll under (-' RiRanrea slngly or In combination with theantagomst might he due to the antlfungal substance present in the phytoextract whichacted In the presence ot'thc latter more ert'ectlvel!Total ccnbohydrares: Varlallon tn the chlorophbll plgrnents led to the changes In thecarboh\drate conlrntInfected lea\es shoired reduced amount of carboh\dratecontent (SPA) compared to that of control All the scngle and dual treatments shouedmore amount of carhoh\drate 11 . total sugar. reduclng sugar non-reduc~ng sugar,sucrose and starch content) than the ~nfected ones Among the slngle treatments. Pt23shoued the mawmum amount oF the total, rsduclng and non-reduclng sugars,followed by CONS. (' giganlea and T har=ranum, whereas. maxlrnum amount wasshown b) Pt23 followed b) CGNS, 1 har:ranuniand C gigan~ea Among the dual

treatments, Pt23 + C. gigantea exhibited maxlmum amount ofcarbohydrate lollowedby CGNS + C. ~iguntea and T, hrrrzianrrm + C. giganrea.Idower quantlty of'carbohydrates In thc lnfected leaves is gunerall!bel~evedto be due to the use of the carbohydrate rnctabolltes in the host tlssue by the pathogenlor 11s mel;rbolrsln and growl1 I'hrs 1s supported by the work ul Chakrabany et 01.(2002) In cotton plants Infected w~th grey mildew, disease, Agrawal et a1 (1982) inturmeric leaves lnfkcted with leaf spot disease and Khode and Gahukar (1995) inch~ll~ I'ru~t ~nf'ec~ed w~th (', gloeosporrodesLlebnath et ol. (1998) repned that thedecreascd starch content In the Brussico leaves lnfected w~th Albugo spp was malnlydue to the tact that these substances were ut~llzed by lhe pathogen for ~ts growth anddevelopment Padma stngh (2W) lbund that the loss of carbohydrates content In theonton leaves lnlctrd by Alternarru spp were due to heavy loss of these contents fromthe lmpalrld membrane whlch are used b) the pathogen for ~ts grohth Slrnilarohsenailon was made earl~er In ch~ll~ tiu~t tntectzd b) C.'ollerorrrchum spp. (AshaI W. Ciornath~ 2001 )Ihc least rcduct~on In the bloagents sprabed leatt.5 ma) be due to theinduction of the treatcul plants (SK) to ut~llle the carbnhbdrates for the b~os\nthes~s ofphenol~c compounds In turn these phenol~c compounds ucre used for the defensercaction agalnst the pathogen ~nfkctlon Th~s tinding substant~ates the \rorls of Ne~sh( IYM) and Llpadh\a\ and Muhhopadh?a\ (1986) on Trtchodernma spp emplo)edegalnst sheath hl~ghl fungus In rlce plantt3nctcr11at1on ol' thc seed u~th the antagonist and the ph>toe\tract treatmentuas effecu\c In enhancing the carboh!dmtc content under dual treatment dur~ng the Iand II sarnpllng dais ll~gher synthes~s ofcarboh?drate content In the dual treatment

compared to the slngle one can be due to the synerglstlc actlon of both microbialantagonists and phytoextract resulting In the enhanced chlorophyll synthesis and C02assimilat~on by the leavesfinhanced quantity ofcarhohydrates in the single and dual treatments might bedue to the lnductlon of some resistance action ~n the host plan^. Ka~avel (2000)reported higher amount of carbohydrates in (' capsrci-infected Caps~cum fruitstreated w~th Pse~~domonas and Trichodermu. Phys~ological changes In the leaves andrcwls of ('ocu.7 t~~cc~!feru, infectcd h!l'lt~~foplusmu. causing lethal !ello\r.~ng(LY)d~sease showed decreased rate of photosynthes~s and carhohydratelstarch In the root,and Increased carhhydratedstarch content in the leaf It suggests the inhibition ofsugar transport In the phloem leading to stress in sink tissues and development ofvisual systems of 1.Y (Maust. 2003). Reduced sugar transport could be caused byph\slcal hlockage of sleve tubes hy P~roplasma. deposition of callose or othermater~al In response to Ph\aoplasma infection or reduced phloem loading of sucroseSchilTer and M~rwha (19h8) reported that the depletion of starch content In the rust-~nfected bean leaves was mainly due to the activation of starch hydrolyzing enzyme(am\lase) hy the fungal metahl~tes Application of foliar spray of m~neral nutrientsand ethrel ~ncrrxised the total sugar content. reduclng and non-reduc~ng sugar and thetotal soluble sol~ds and decrased the ac~d~ty In the peach plant. The combinat~on ofCI:FA+ C'a (NO,)+/Ins(), was the most cfli~u\,e one followed h! CEPA and ZnSO,i ('I:f'Athan the rest of cornh~natlon e~ther s~ngl!, or In comhinatlon (Koul andMuthm. 1999)Nittogen meiabolism: The leaves are the major sltes of utilization of nitrates. Thenitrates that are ahorhtd hy plants arc reduced to nitrltes and then irnmed~ately to

- --ammonia Finally they are converted to ammo ac~ds and protelns The nltratereductase IS the key enzyme In nltrogen metahol~sm, which converts nltrate to nltrlteMaxtmurn reductton In the nltrogen metabol~sm-amino nltrogen, ammo ac~dcontent and nltrate and nltrlte content was found In the leaves of plants Infectedby R. solani Plants under stngle and dual treatments showed htgher nltrogen contentunder d~fferent combinations. Among the single treatments. Pf23 showed the highestamount of nitrogen content followed by CGN5, T. hanianum and C. gigantea.Among the dual treatments, PR3 + C:. giganlea showed the maximum contentfollowed by C'CiNS + C'. gigunrea and 7 harztanum + C. gigrmrea.I'rasad (19x1) reported that the heav) reduction In nltrogen content Inbanana plant durlng 1Lngal pathogenesis was due to the fact that the pathogensurv~ved at the expnse of host nltrogen pool leadlng to the enhancement ofdisease de\elopment, uhlch brought do\r.n the protetn and total nltrogen contentIn thc host trssue Onlon lea\ cs rnkcted b! ;Ilrrrnurto spp, showed less amount ofnltrogen content due tn the d~sruptlon of cell structure along \kith enhancedproteol\t~c cnz.\me actn it! (l'adma. 2000) 'The present finding was supported b)the work of Khare and 1.akpale (1997) and Padmanahhan er ul (1973) In so) beanand cllrus Icic\ es ~nl'ccted h! .\irtrrlrortrona.r sppKno~lton and L)a\r.son (1983) runoned that the comblnatlon of Psetrdomonclsand krank~a spp treatment ~ncreased the nltrogen liutlon In the treated plants ascomparcd ti1 thc health) control plants 01.Abnrs rubra Slm~lar enhanced nitrogenmetabolism duc to tire appllcatlon ol' IJsectdonronas uas reported In ch~ckpa (Parmarand Ihdanral. 1999). In P \*rtlgar~s(Cjrlms and Mount. 1984) and In soybean (Dasht~er 01. 1998)

Protein Content: Leaves of control plants showed maxmum proteln content Lowestamount of proteln content was observed In the Infected leaves Among the slngletreatments. Pf23 showed h~gher amount of the proteln content followed bv CGNS, Thornanurn and C'grganrea Dual treatments showed hlgher amount of protelncompared to that of s~ngle treatments Pf23 + C grgantea showed the highest amountof proteln followed hv CGNS + C' grganreu and 7 hanranum +grgantea Thesame trend was seen In both I and I1 samples H~gher amount of proteln In I1 samplesuggests that more tlme 1s ava~lable for the biocontrol agents (single and dual) to actupon the pathogens or Induce the rm~stance In host plant agalnst pathogenM~n~rnum reduction In the proteln and ammo ac~d content was rnacnly due tothem breahdotm h\ proteol\tlc cnnmes of thc pathogen wh~ch enabled the pathogento use the host proteln as rource of nltrogen and ammo ac~d for ~ts the~r growth and&\elopmentTh~s 1s suhstant~ated h\ the find~ngs of Chakrabart~ and BasuChaudhan (1979) In samower tr~lt mused b\ Frrsarri~rn spp. 140\rrell and Krusberg(1%) In alfalfa and pea plant ~nfccted tr ~th Drnlendus spp and Chakrabarty et a1(2002) In cotton plant Infected ~ ~ gre\ t m~lde~ h d~seasel o\rest amount of' protetn Has not~ced In the Infected plants hen comparedto those of treatments I he reason ~n~gl~t be that Infected plants to ut~llzt: the ammoac~ds of the protcln and the sugan of the nucle~c ac~ds for the s\nthests of phenol~ccompounds which acted as the defense agents agalnst the fungal lnfect~on Thepresent finding substant~ates the tcorh of Veeramohan er a1 (1994) that the decreasedprotein content of chlll~ Infected filth Alrernarra solanr mlght be due to tklr activepanlclpatlon In the s) ntks~s ot'phenol~compc~unds

- -Highest amount of protein obtained In dual treatments in the present study isin conformity wlth that of Dashti et al. ( 1997) who reported the combined effect ofSerratia liquefac~ens 2-68 or 5'. proteamaclans 1-1 02 and Bradyrhizobrum japonicumIn lnaeastng the prote~n content in soybean Madhu and Wajid Khan (1999) reportedthe Increase in the protein content In the blackgram seeds which was treated withC; coledonrcum and Xhrrobium spp, along with SO? treatment. This combination wasmore en'ect~ve than e~ther of the VAM or Hhrzohrtrm treatment given alone Likewise,Increase In the proteln content was noticed in the co-inoculation of G caledonicumand Hh~robrum pp along wth the fly ash assay (Madhu and Wajid Khan. 1999 b).The Increase In the proteln content In the host plant under single and dual treatmentscompared to the Infected leaves IS anrlhuted to the fact that the antibiot~cs producedh! the antagonist Trrchodermu spp Interfere wth the proteln metabol~sm of thepathogen lniectlng C capsrcr (Frlh et a1 , 1977. Rajath~lagam and Kannab~ran. 2001).Prote~n content nas more In all treatments compared to the control In wheat seedl~ngtreated H lth the crude estract of (' globosrrm. \r-h~ch was applied as pre-~noculationspra!.post-~noculat~on spray or \{.ere applied twice as pre- and pst-pathogeninoculat~on Increase In the protein content In the graminamus hosts. such as wheat(Sock er ul . 1990). maw (Nasser er ol . IW) and barle) (Ilq er 01.. 1989) wasreport~ul lo be due to the act~vlt) ol'thr [)-1.3-glucanase Kloepper (1980) reportedthat thc plant gro\r.th promoting rhlrohacterla (PGPR) treatment recorded h~gherproleln content and total ammo actds In rice and \\,heat plants and high sugar contentIn sweet potato and sugar beet fh~s was malnl! due to the suppression of the diseaseand lncrcasr In the actl\atlon of en/) mes that were in\[3ollrr (1985) tras of the vlen that protci~ls are associagolnst Sung1 and bacter~a by their acllon on the cell.. ,

Gornathl and Gnanaman~ckam (2004) reported the role of polymethyl galacturonase~nhlbttlng protelns In the degradat~on of cell wall enzymes An Increase In the protelncontent IS supposed to be ~nd~catrve of ~mmun~zed system In the plant In those plantsralsed from the seeds treated wrth mlcroblal antagonists, the m~crobtal antagonlstsprevented the establ~shment of pathogen and showed h~gher values of proteln whereasIn those sprayed wlth C glgantea extract, after inoculat~on of pathogen phytoextractwas not as etTectlve as the antagonists In preventing the establ~shment of pathogensHowever. C gtgunlea extract rn comb~nat~on wlth erther of the antagonlsts showedh~gher values of proteln thus showlng thetr potentlal to ~nh~blt the growth ofR solunrlunher which Has already ~nh~b~ted by the antagonlstsPhenol and proline: The phenol and prol~ne compounds act as adaptive mechanismIn the host plant agalnst the fungal ~nfectlon. 'There was a sharp increase of phenol inthe Iea\.rs of the lnli~ted plants than those of control and treatments Among thesingle trt%tments. 1'123 shoued the least amount of the phenol and proline contentstbllo~ed h! C(jN5. (' g~gunteu and 7' har~anirrn. Among the dual treatments. Pf23+ (' gtgunteu sho\\rd mtnlmlum amount followed CGNS + C' gtganteu and T.bctunrrm + (' g~gunleuAsha and Lannablran (2001) recorded h~gher phenol content In the C, capsicttnlhted Icu\cs of chi111 'I'hls \$as supposed to be due to the hindrance of thegl!colysls h! the actlvlt! ol'the pathogen. \vh~ch In turn act~vated the pentose path-)leading lo the format~on ol'4-carhon compounds for the synthesis of phenols. Jaypaland Mahadevan (1968) found that sharpl!. Increased phenol content In the infectedplants mlght he due to the fact that the accumulat~on of phenols In the infwted tlssuem~ght come from the sunoundlng health! leaves In order to resist the advancement of

the pathogen towards the other healthy cells. Increase of phenol and prollne might bedue to the induction of systemtc resistance In the host plant due to treatments Theover-product~on of phenolic compounds reslsts the advancement of the pathogentowards the other healthy cellsSim~lar tindlngs have ken reported hyRamamoorth? and Samivappan (2001) that the h~gher content of phenol In thePsc~idomonus-treated ch~ll~ plants Infected rrlth (' capstcr was malnly attributed tothe fact that the phenols are fung~tos~c In nature and thetr accurnulatlon Increasedthe phys~cal and mechanical strength of the host cell wall result~ng In the lnh~blt~onof pathogen Invaston Anderson and Guerra (1985) found that Pseudomonastreatment Induced I~gn~licatlon of the cell wall of bean leaves wh~ch In turn reslstedthe In\aslon of the pathogen Ueckman (2000) reported that the accumulatlon ofphenol reststed or reduced the w~lt s!mptomsh\ Inactl\atlng the pathogen~cennme act!\lt\Phcnollc compounds are mosll? cons~dered as one of theImponant hlochem~cal parameters for tnd~catlng the degree of d~sease reststanceand also that the acsumulat~on of total phenols IS usuall? more In resistant genotyeas compared to suweptlhle ones (Arora and Wagle. 1985) Fol~ar spra? of the cruderxlract of (' g/ohfsum 1nereawd phenol content cn the uheat plants (B~suas el of,2003) and rag1 plants (Vtdh!asr.Laran. 1997) tnfected h\ Ifelmrnrhospnrr~cmr.itrhc~~ilrrn and I/ rerromeru rcspectttel?AN'I'IOX IDAhT ENZI MES: Peraddme (PO) and Po(vphend addme (PPO):llsually plants respond to a pthogcn h mobll~rlng a comple\ network of' acmedefence mwhan~srns ].he acttve ox)gen sprcrs (AOS) such as superoxlde anmn.h)drogcn perovldc and h\dmx)l rudtcal are generall\ produced In the plants as aresult of ~hc rnctabolcc processes that mke place In chlomplasl. m~tochondna andp l m membrane-l~nked electron transport system Dunng the ~nfcct~on pmcess the

pathogen/or ~ts actlvlty lnlerfered with the electron transpon system thereby resultingIn the leakage of electron These electrons altered the structure of the molecularoxygen result~ng In the productlon and accumulat~on of AOS wlth~n the cellGenerally the plants produce many ant~oxldant and low molecular welghtenzymes such as superox~de d~smutaw. (SOD), catalase (CAI ). pcrox~dase (1'0).ascorhate peros~dasc and glutalhrone reductase Increase In the ant~ox~dant enqmeactlrlty (PO and PPO) was found In the R. so1an1-~nfected leaves The enhancedant~osldant enL\me acttvltles In the ~nlicted host tlssue m~ghtK due to ~nducr~on ofsystemic reslstancc In response to the pathogen infection These enzymes partlclpateIn raptd detox~ficatlon of reactl\e os!gen spe~~es (superos~de anlon ( 0, ). h!drogenperoi~de (Ii?O:l. s~nglrt ox) gen (.Oil I Into tr-ater The rap~d con\erslon lnhlb~tedthe toxic cll'ect caused by AOS to the host plant. Asha and Kannabtran (2001)suggested that the h~ghcr 1'0 and I'M) actl\.lt) In the ch~ll~ leaves ~nfected u~th Ccap~tci nus ma~nl due lo the enhanced resplraton rate Induced h) the pathogenactlvlt\ S~m~lar tindlng tvas made h\ Ciomath~ (2001) tilgher actn 11) of PO andI'l'o In chocolate leafspot d~uase trss due to actlon of I!ttcenFme actlvlv, of thepathogen. wh~ch ~nvolved In the actlvatlon of the latent PPO acflvlh. of the hostplant S~rn~lar Iindlng tvas made In perenn~al negrass Infected \vith endoph\-t~chacter~a (Naflaa et a/. 1999). In anthracnosr disease of cucumher (Zhang er a/.19%) and In pearl rn~llet Infected \c ~th Sclrrosp~ru spp (Sreedhara cr a1 . 1995)Phenollc compounds and related oxldatite enn mes are rnostl! cons~dered as oneof the Important h~whem~cal parameters for dlsease reststance (Arora and Wagle.198s) Sh\+rt et nl (20.4) reported that there \\-as s~gnilicont drfference In theproductlon of laccase. polypheno1 nsidase and pemsldase enzyme not onl!Indifferent life cycle stages hut also there was least activit? of all these enzymes when

treated wlth the mvcel~al extract of species Bacterial blight of conon plant caused byXanrhomonas axonopodrs pv malvacearum (Xam) was studled for the PO actlvlty andphenyl antllne lyase (PAL) by the use of chernlcal like sal~cyllc acld (SA) andblologlcal ~nducers, a non-pathogenic mlcrmrganrsm. P Jluorescens (l'f) elther aloneor In cornblnatlon In the suscept~ble and resistant varletles of the cotton plants A totalof nlne lsalates of pmxtdase were observed when d~fferent Inducers and thelrcornb~natlons %ere ~noculated 1 he reststant and susceptible cotton showed differentIsoennme profile (Padrnaja et 01. 2004)Trrchoderma spp and whlle rot fungl-Lentrnula edades were grown In thestertl17Pd wheat straw u ~ th or without 7rrchudem spp The antagonlsrn bemeen theI. edodes and irrrhrdrrma spp changed the pattern of enn me acttvltles In the u h tstraw substrate i.here was a st~rnulatlon In the laccase and Mn-dependent perox~daseactlvlr\ due to the dlm~nlshed growih rate of L edudes lncrense In the laccase actlvrt)w-s due to the oudat~on of pol\ phenol at the contact between the m\ cella of the twoantagonlrts (Sa\ole and Mata IW)Inc'rmsc. In thc concentratlon of malond~aldeh\& (hlDA) trhlch 1s ~ndicatr\eof o\t&t~\r. Ilp~d metahllsrn Has okned In thc ~nfected Ieates compared to that ofcontrol end uutments There 1s a slgnlficant Increase In the actlrlt? of llpldperoudatlon In thc Itro\csrwcn Ing dual treatment MDA Ie\el in host cell membraneIS oncn found to Increase durtng pathogenesis In panlcular. thc damage of llp~dmembrnnc can empllf\ celluler tcl\lclt\ h\ the fonnntlon of llp~d htdmpero\ldes andthctr tortlc aldeh\& degrodatlon product Scandalloc rr a1 (1997) found an lncrrax Inthe MDA concentratlon due to the ahlotrc gtress. 14 hlch led to the accu rnulatmn of11:0> In the cell wall and plasma membrane chang~ng the cell uall smture Theahlotlc and hmt~c stress \+as supposed to cause damage h the productton of ROS

particularly OH through Fenton reaction. Fe + H202 + Fe + OH+OH, due to excessof Fe. The ROS-superox~de anlon (02), singlet oxygen ('02), hydroxyl radicals (OH)and hydrogen peroxide (14202) are produced due to fungal infection. Their funct~on isinterdependent and also dependent on lip~d peroxidat~on Gonner and Schlosser(1993) reprted the Increased level of MDA in the oat leaves inoculated w~thDreshslera avenue. Increased level of MDA In the infected cond~t~on was observedby Kauss (1990) and Kosen and Halpm (1990) and Sharma el a1 (2002) Whenfungal pathogens such as P&thopthora spp (Hodges et 01.. 1999) and P,vrrcularraoncae (Cionner and Schlosser. 1993) invaded host tissue of potato and rlce differentoxygen rad~cals. such as. 02-superoxides anion were produced Ilsuall) Singletox!gen ( 0 2 ) is grncratud during pathogenes~s Maix plants Infected by H solanr heretnvrsttgated for the study of accumulation ol' MDA In the suscept~ble and resistantvarlrtles Suscept~hle \.arletles of ma1z.e showed more accumulation of MDA than theresistant \arietres (Sharma el ul , 2002)1.u and H~gg~ns (1998) reported the ~nvol\ement of actlve oxvgen species-AOS In defcnw response of tomato to a race-specific el~citor of ~'lodosporrvrnfitlvttm AOS mav he ~n\ol\ed in the ox~dat~on of memhrane lipids that results Inproduction ol wcral antifungal compounds inltlatlon of cell \$all I~gn~ficationreactlon d~rect lnlun to pathogens and s~gnal transduct~on H! unghuan er a1 ( 1995)demonstrated that the rap~d t1?01 genemtlon In elicited cells of Puerarru lobara couldhe regarded as a plaus~hle in~t~al def'ense strategk prlor to the appearance of de novonnthesiuld defense mater~als I hese AOS ma!act directl! as toxins agalnst thepathogen Pasw.hmil er 01 ( 1998) reported the pass~hle role of ROS (Ch and H202) Inuruslng inh~h~tion of fungal spore germlnatlon br, leaf d~ffusates of res~stant ncecullivao La hlast Perox~dation damage of the cell membrane lea& to leakage of

cellular contents also (Leopold and Vertucc~, 1986). Toxin generally induceselectrolytic leakage from the cells.PROTEIN ANALYSIS TIiROl~CIt SDS-PACE: Plants defend themselves againstpathogen~c fungl hy producing fung~toxic substances such asphytoalexlns,patklgenes~s related (f3K)-pmlte~n, oxldized phenols and several other componentsMlcrohes, m~crob~al metahol~tes, plant extracts and chemical agents have been knowto Induce reslstance In crop plants against pathogens (Kuc 1995. Ogla andOrtrestksorskaya 199.5 and Ste~ner and Schonbcck. 1995) These ohservat~ons ~mpl)that certaln h~ochern~cal alterations In the host m~ght he associated w~th defencemechan~sm and enhanced gro~zhOut of 13 hands 10 hands. were common to control. infected. s~ngle and dualtreatments Ixa\.cs of plants ~nfected wth R solanr showed w o hands ~n the range of67 klh to hR kIla appri>x~matel\. whereas those treated slngl? u ~th CGNS and Pf23shorwd a ncu hnd of26 kDa approx~matel\ other than the exlstlng one Among thedual treatments. 7' ha~runtrm + (' grgnnrea shotved a band of 45 k1)a. follo\red h!CtiNS+ ( ' ~rpanleu (68 kDa) and PI23 + C' grganlea (70 kDa) handsCrude extract ol'('haeromrum glohosrrnr was used to Induce reslstance agalnstArp)Iurr.~ sorokrnrunu Prclfil~np of soluhlt: proteins was done In B sorokmrana todetcrm~ne ~l' an\ protetn 1s assw~at~ul u.~th resistance. Proteins of I I0 kDa. 105 kDa.3.5 klla and 32 hl)a \rere resolved h! SIX-PAGE anal>sls All these protelns \reremlsslng In Ihc casc of weds treated \\ ~th the crude extract of(' glohos~rm Absence ofneu prote~nlln treated planu ind~cated that the crude extract could not rnalntain 11seWectivcncss for longer durat~on and 11s b~o-effican. decltned gradually in the

seedling raised from the treated seeds (Biswas et ol., 2003). According to Antonieel a1 (1980) pathogenesis related proteins (PR-protein) are involved with defense inhost plants to pathogens, Induction of systemlc induced resistance in tobacco afterlnoculatlon w~th P. foboci was indicated by an increase In the concentration of PR-protein (Tuzun er O/., 1989) Bera and Purkayastha (1999) reported that there was agreater number of PR-protelns compared to the non-~noculated kitazin-treated rlceleaf sheath Infected bv R, solonr. Compar~son of the proteln profiles of healthy andInfected leaf sheaths revealed that ~noculated leaf sheath produced more proteln bands(I6 bands) than the non-~noculated leaf sheath (I I bands). They tried to study amult~component coordinated defence response of rice plants against R solonr.II~EN1'11;1

substance obtained from the leafextract of'the C. grganfea IS taraxasterol- a triterpenecompound.Reports on single treatments with P. .fluorescem.Glrocladium, andHhizohrum. Trrchoderma spp were shown to inhibit the growth of pathogens areava~lable whrch support the advantages of the biocontrol agent (Kannaiyan, 2001.Manoran,jitham et 01.. 2001: Ramamoorthy er a/, 2001) According to Cook (1991,1993) In almost all the biocontrol systems. a single hrocontrol agent is applied withthe expectation that it w~ll control the targeted d~seasds) under various conditions.however. such broad spectrum antagonrsts seldom, if ever. exist and differentantagonrsts must be used to assure adequate level of disease control under variouscondrtrons llse of' antagonist mrxtures to hroaden the spectrum of activlh has beensuggested for many vears, but onlv a Itw studla have been conducted Mrxture ofantagonrsts has three main ad\,antages (Wolcrech. 1998).I It may broaden the spectrum of actrv~ty2 11 may enhance eflicacy and reliabilrty of b~ocontrol, allow~ng areductron rn applrcat~on rate and cost of the treatments.3 11 allows the comb~natron of \arrous brocontrol tram trrthout resortingto genetlc engrneerlngOut of the three dual treatments PI23 + (' grgontea was the best combrnatron Thestncrgistrc actron strms to be \en cffecrr\e in rnhrh~t~ng R solanr ~nfectlngC; marSimilarl? slnerglstlc elkcts tvhrcli \vcre hetter than thc slngle treatmentswere rep~nrd I.umanc.eau and Alah>u\ette (1991) and Leeman et ul (19%) hawdemonstmrcvl that co-rnoculation 01' lluorcscent Psesertdornonus spp \r~th roo1colon~~lng fungi always results In a greater and inore consrstent suppressron of'

disease than the application of a single microorganism. Lamanceau and Alabouvette(1991) isolated stralns of fluorescent Pseudomonos spp, from the suppressive soil thatwere able to improve the eflicacy of biological control achieved by the appl~catlon ofa straln of nonpathogenic F oq~.sporrrn~ could achieve hiocontrol of Fusarium \liltsCo-operation between Introduced Pseudomonas and indigenous antagonistic fungimay also wntr~hute to wilt suppression Whether a commercial product based on twodifferent microorganisms will bc pnsslhle and is accepted is still an opn debate, butone may expect the commercialization of two products that can he used in associationto Improve hiologlcal control of Fusarium wilts Indeed, most biological controlagents are target specific and, therefore, are only effective against a specific pathogen'l'h~s propen? represents an advantage from an envlronrnental polnt of view: ~t 1s agreat disadvantage for the grosrer \rho has lo control several plant pathogens In thesame crop Cc+~noculat~on of..lcremoniicm rirrrltrm. F oqxponrm or I'erricillitmleconrr ~lth Psertdomonos spp stralns H'CS358, WCS374 or WCS417 s~gn~ficantl)suppressed dlscase. compared ulth the control treatment in pot b~oassay Enhancedhlolog~cal control of plant pathogens uslng t\%o or more antagonrsts has beenexpenmentall! dcmonstratcd for a number of pathoqstems ~nclud~ng take-all ofwheat (I'lerwn and Wcller. 1994) and post harvest deca) of apples caused b)H crncrcu and P expunsum (Jan~s~eu~cz. 1988) Studies were made to drtermlne theetlcu.1 ol' .~lsprgr//rrs jlm~ir.r. Lprcocc.um pirrpttrarcens, P r~ermrcrrlottcm andK nrgrrcun.~ and ,\I mrogrirru on the germlnatlon of wed of tomato plant Plants weretnoculated \r~th d~tl'crcnt ~ncwulum Ic\el of lung1 ellher separatei) or In comblnat~on\v~th H solcrnr and A l mcogtirtoOut (if four fung~. former thrw shoned sl~ghtlncrcase In the percentage of srxdllng emergence uhen ~ncorporated In so11 togetherw~th X solunr, compared to X solonr togcthcr ~ ~ At t rncogni~u h Thls stud! hrlngs

out the fact that these fung~ are able to dilute the adverse effect of both the pathogens,R. soluni and M. incognrtu (Rekha and Saxena. 1999)Fel~x (1988) demonstrated that the chltlnase and p-1.3-glucanase Mere able toinhlh~t fungal gromth when treated synerg~st~call), but ~nd~v~duall) Mere not soeffecllveControl of pnrcden mlldeu of pea caused b) tnsrphe prsr b) seedbacter~mtlon mith P uenrpmosu and I'fltrorestenr alone and In combination wasfound to hc more cfTectlve than an aer~al sprat u~th the cell suspension of antagon~stsor ncemml-a product of neem (A rndrcu) at difterent concentrat~ons and the seedhacterlzatian rr ~th P uenrgmoso and P /7uorescens alone (S~ngh. 2000)As evident from the above dlscusslon. all the slngle treatments and dualtreatments shonrd lnh~h~t~on of R solanr over a wide range (Flom charts I & 2)Among the slngle treatments. Psescrrdomonus PI23 straln 1s confirmed as the bestlnhth~tcln agent follorved h> Pseudom~nas CGNS sualn. 7' har=iantrm, andph\tcx.stract ((' .g~,qan~ea) The lnh~b~ton elfect \\.as found lo Increase t\\o fold ormore In the dual treatments 'The anal\s~s of the results reveals that of the two strainsof 1' fluorescens shou lng h~ghrr ~nh~b~ton potent~al. P. /luorescens (P123) showedmaxlmurn lnhlh~ton actlt.it) under hth slngle and dual treatments due to the~rgenetlc constltutlori ~hlch lnh~b~ted the mz\matlc and other metabol~c actnltles ofthe pathogen as follox.s9 It ext~~h~tcd maklmum inh~h~tlnn ni' radial m)crlial growth ofthe pathogen(In 1.11ro)

- - - --*f. It rnhrbrted actrvrtles of' the cell wall lyttc enzymes such as PMG, PTE,CMC and cellobiase of R, solanr more effectively in the dual treatment thanthe single treatment (in virro) and thus srgnificantly reduced the growth ofR. solm1.9 It enhanced the amount of photosvnthet~c prgments. carhhydrate. nitrogenand proteln In the leaves of the treated host plants*:aIt induced the svstemtc aquired resistance (SAR) in the host cells by therelease of ant~b~otrcslanufungal compounds and developed different defensemechan~sms agalnst pathogen rnltctron such as thickenrng of cell wall,reduced clttctrolyt~c leakage. s)nthesis of ROS and PR-protein In the hostussues and b! sustainung better source stnh rrlatconshrpSo far the studies on the inhibitron of the pathogen~c fungi and bactena broughtout the syncrgisttc eN'ms of the comh~nation of two or more fungi or bacterra(mucrohral agents), comhinatron of antagonust and ph!.toproduct on the control of thepathogen 'fhc present study hrings out that the synerglsuc en'ects of combtnation ofmrcrohral antagonrst and phytoextract on the control of an) pathogen for the firsttrmeIt IS Intercstung to ohsene that ph\tcwxtract alone inhib~ted K solunr at a lesserrate hut shoucd hrgher rate of rnhrh~tuon of the groirth of R. solunr uhen rt Gasapplred wuth mtcroh~al antagonist e~ther stinultaneousl~ as In m vrrro condut~on orwhen 11 was sprayed on the leaves of the plants after a gap 01. t~me (m \*tvo) Thrsunrque and hrgher ~nhihrton elTect due to the comhrnation of m~crohial antagonist andphytoextract suggeststhat the hrgher rate of tnhrb~t~on ofR solanc might take placeIn two successive stages In the tint stage. ~nhihitlon of R solanc to certarn level uas

ought by the microbial antagonist which was further enhanced by the phytoextractsuccessively.The higher values of various parameters obtained in I1 samples compared tothose of I samples reveal the cumulative effect of the phytoextract in bringing downthe inhihition of R solan; furtherThe conclusion dra\tn from the present stud! is that maxlmum control of Rsolunr ~nfecting ti mar can be brought b) the dual treatment of Pserrdomom~ Pf23stram and leaf extract of(' g~ganreolunher stud~es on the effects of' microbial antagonists in combinat~on withtarassterol. active prtnc~ple obta~ncd from (' gtganreu would bring out the ~nhibitor)potential ofthe active principle of the ph!,tnestract

SUMMARY

SUMMARYThe present study bnngs out the synerglstlc effects of antagonlsts(7 harzianum and Pseudomonas fluorescens - CGN5 and Pi23 stralns) andphytoextract (C grganfeu) on the inh~b~t~on 01' Rhuoctonra solanr Infecting Clycmemm for the first tlmeMIC (mintmum lnhlb~tor) concentratton) of leaf ethanol~c extract ofC grganteu %as found to k IO?h conc ~hlle that of the mtcrob~al antagontsts was1 % concIn wrro experiments include the culture of R solanr In Klng's mediumamended w~th antagonist and phyuwxtract slngly or In comblnat~on. In vnvo studieswere camled out In the potted plants Control plants of Ci mar were sprayed w~th thesame volume of ster~le dlstllled water Plants were Infected by inoculating w~thpathogen on 30 DAS. Plants under slngle treatments comprised of control andinfected plants grown from the seeds soaked In rach of the rnlcrobial antagonist for 12hn and control and ~nfected plants sprayed w~th C'. grgmreo extract on 3 1 DAS.'l'how under the dual treatments Included ~nfmted plants gronn from the seeds xmkedIn rach of the mlcrohtal antagonlsts sprayed wth the phytoextract on 31 DAS and 16[)AS. 'I'k laves new collcctd on loah Jab after la (46 DAS) and 11" (56 DAS)spra) ol' phytocxuact Sir thc esttrnation ot'thc \,arious parametersAn anah st\ of results obtalned from stngle and dual treatments brlngs out thefolloutng facts* lJn&r m rVrtro condtt~ons all the slngle treatments \$ere round to show 70-80% oflnhlh~tlon of mvcelial gmwth of R solanr All the dual treatments (T hocranum

+ C. gigantea. CCiN5 + C. giganlea and Pf23 + C, gigantea ) showed highest rateof (85-95%) of inh~b~tionthe radial mycelial growth of R. solani.a The actlvltres of pectlnolytic and cellulolyt~c enzymes were found to he lnhibrtedto varying degree by both single and dual treatments The rate of enzymaticactrvlty was less In the dual treatments due to the synerglstrc effects of antagonistand phyloextract 'I'h~s reduced actlvity resulted In the ~nhib~tion of growth of R.solanrThe leaves of those plants in both single and dual treatments were comparativelylarger in size than those of control and infected plants Leaves of dual treatmentwlth Pf23 + C lpRonrea plants showed maxlmum leaf area. Number of pods perplant and seeds per pod were also found to he more In the dual treatments.esptrlally In PI23 + (' glgantea because of enhanced metabol~c actlvltres andhefter source-sink relatlon4 Maxlmum elcctrolyt~c leakage uas noticed In the leaves of the ~nfected plantscompared to the plants of control Single and dual treatments, showed lesser rateof elecfrolytlc leakage Comhination of Pseudomo~s Pf23 strain and C giganteaHas found to show mlnlmum electrol~trc leakage under s~ngle and dualtreatmentsi 'I'he chlorophyll plgments and carotenold content were redud In thc ~nfectedIcaves compared to those of control Among the slngle uratments. PC3-treatedleaves show~d an Increw than other treatments. Among the dual treatments.IBf23+(' giganfeu cd~ibrted maskmum chloroph!,ll pigments and carotenoldcontent followed hy other two dual treatments, llnd sample showed decreasedphotosynthetic actrvlty tn the leaves of control. lnfrctad and treated plantscompared to thox of I sample. whrch m~ght he due to aglng of Iea\'es.

+ All the single and dual heamrents showed more carbohydrate content than controlplants and infected ones. Of all the treatments, dual treatment with Pt23 +C pganfeo exhibited maximum carbohydrate content+ Leaves of control plants showed maximum protein content, followed by the dualtreatment with PI23 + C gigontea showed the h~gher content than the infectedplants under single and other dual treatments+ Lower level of nttrogen content was observed In the leaves of Infected plantscompared to that of control, slngle and dual treatments The dual treatment (Pl23+ C' gcgantea) showed h~ghest amount of nitrogen content+ Phenol and prollm content were found to be highest In the rnfected leaves becauseof the stress caused by R solonr Lower content was noticed In the plants ofcontrol, single and dual treatments+ Ant~ou~dant enzymes vir.. peroxldase (PO) and polyphenol oxidase (PPO) showedan increase In the Infected leaves Ihose of control, single and dual treatmentsexhlhitd lower amount L.east enzyme actlvlt). Bas recorded cn Pt23 +C' grguntea treatmen1+ Production of pathogencs~s rclatcd protern (PR-proteln) In both the treatments andIn the tnfrctd condit~on can k attrlhuted to the delinse rractlon tn plants tootercomc: the h~otic stress Released ROS detoxifies toxlns of pathogens andinduces SAK In the host plants ROS is generall) ~n\ol\ed In the oxldat~on ofmembrane lipids that raulls In production of se\aral antfungal compounds,initatmn of cell wsll I~gnlficatm nsctmn. direct Injury to pnhgens and s~gnaltransduction

4 Active principle In C. gigantea leaves was identified as taraxasterol (triterpenecompound) which was slmilar to that obtained from the latex of C, procera plantsearherUnder slngle treatment phytoextract alu8a)s showed lower values ofmorpholog~cal and blochem~cal parameters compared to those of mlcroblaluntagonlsts Among thc latter Pseudomonas ,l]uoresrens Pf23 straln exh~b~tedmawmum values+ All the dual treatments showed h~gherate of lnhlbltlon of R solonr compared tothat of single treatmenb Among them. cornblnatlon of Pser1dnmonasf7uoresrensPI23 straln and C' grganreu was found to control R solanr more effectivelyIt IS lntrrestlng and lntrlgulng to note that whlle the phpoextract was lesselTect~vc In slngle treatment 11 was found to enhance the ~nh~b~ton. actlvltles In thepresence of mtcrob~al antagonists In dual treatments This might be anrlbuted tothe synergtstlc actlon of ph!mestract tvlth m~crohlal antagonists in hrlnging outh~ghcrate of lnhlbitlon of the pathogenr litgher rate In lnhlbltlon of Rsolanr and h~gher values of ph!s~olog~calprameters In the I1 samples suggest that 11 spra! of ph!toestract could sustaln ~tsoptlmum concentration In the host tlssuc thereb) enabllng the host plant not onl!to reslst the pathogen hut also Increase the metahollc actn IIICY rcsultlng In hlgher\ lcldr f'hytochemlcal analysis of the leal'estract of (' glgcmrcu rc\eals that the actiieprlnc~plc IS tarassterol - a lrltcrpcne compound[lam ohtnlwd from the present stud) confirm that there IS not onl) as~gnlficanl Increase In the defence octlon of the host plant agalnst R scdanr but also

enhanced physiological activities and biochemical processes In the plants under dualtreatment ultrmately resulting In an higher yield.tispec~ally among the dual treatments. cornblnatlon of Psetrdomonos/71rarescens PI23 straln and (' grganrea was found to be the most eflicaclouscomblnatlon for the maxlmum control of R solanr lnfectlng Glvctne maw

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LIST OF PUBLICATIONSI. Asha AN, Kanna biran B (2001 ) Effect of Llutttru metal leaf extract on theenzymatic and nucleic acid changes in the chilli seedlings infected with('olleto~rrthrim cupsicr. Indian Phytopo~h. M(3): 373-375.2. Asha AN, Knnnabiran B (2001 ) Changes in the seedlings of Capsicum anmmI.. infecting with ('o//crotrrrhrrm cu/~sicr (Syd.). Butler and Bisb). in response toleaf extract oflh~~irametel. M. I;eohios B(1): 65-66.

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