Journal of Tropical Biodiversity and Biotechnology
Volume 07, Issue 02 (2022): jtbb67359
DOI: 10.22146/jtbb.67359
Research Article
Growth and Physiological Attributes of Rice by the
Inoculation of Osmotolerant Rhizobacteria (Enterobacter
flavescens) under Drought Condition
Hasna Dyah Kusumardani1, Triwibowo Yuwono2, Diah Rachmawati1*
1) Departement of Tropical Biology, Faculty of Biology, Universitas Gadjah Mada, Jl. Teknika Selatan, Sekip Utara, Yogyakarta,
Indonesia, 55281
2) Departement of Agricultural Microbiology, Faculty of Agriculture, Universitas Gadjah Mada, Bulaksumur, Yogyakarta, Indonesia,
55281
* Corresponding author, email: drachmawati@ugm.ac.id
Keywords:
drought stress
Enterobacter flavescens
‘IR64’
osmotolerant rhizobacteria
‘Situ Bagendit’
Submitted:
04 July 2021
Accepted:
02 February 2022
Published:
01 June 2022
Editor:
Ardaning Nuriliani
ABSTRACT
Rice (Oryza sativa L.) has mechanism for morphological, physiological, and biochemical self-defense in response to drought conditions. The ability of osmotolerant rhizobacteria to develop association with plants suggests that it could be used
as an inoculum to support plant growth under drought stress. The purpose of this
study is to determine the response of ‘IR64’ and ‘Situ Bagendit’ to the inoculation
with osmotolerant rhizobacteria under drought conditions. The experiment had 3
treatment factors: 2 rice cultivars ('IR64' and 'Situ Bagendit'), 3 drought treatments
(25%, 50% and 100% field capacity), and 2 types of rhizobacteria treatments
(without inoculation and with inoculation using osmotolerant rhizobacteria
(Enterobacter flavescens). Plant growth was measured in terms of plant height, number
of leaves, number of tillers and panicles, and percentage of filled grain. Physiological and biochemical parameters, namely chlorophyll, carotenoids, proline, superoxide dismutase (SOD) peroxidase (POX) and ascorbate peroxidase (APX) were
measured. The inoculation of osmotolerant rhizobacteria enhanced ‘IR64’ and
‘Situ Bagendit’ growth (plant height, number of leaves, tillers and panicles) and
increased the percentage of grains in ‘IR64’ cultivar. Proline content, SOD, and
APX activities were all increased by osmotolerant rhizobacteria inoculation, however, carotenoid content was decreased. Plant growth, physiological and biochemical responses of both cultivar to drought were enhanced by inoculation with osmotolerant rhizobacteria.
Copyright: © 2022, J. Tropical Biodiversity Biotechnology (CC BY-SA 4.0)
INTRODUCTION
Water availability is crucial in sustaining growth and development of plant.
Climate change has reduced the availability of water for agriculture. Drought
can affect various cellular, biochemical, and physiological attributes in plant
(Bouman & Tuong 2001; Mundree et al. 2004). This stress can cause metabolic changes in plant by controlling osmotic pressure and creating free radicals as ROS (reactive oxygen species) (Bhattacharjee 2005; Halliwell 2006).
Plants develop non-enzymatic or enzymatic oxidative defense system in response to free radicals (Miller 2008). The non-enzymatic defense system can
take the form of antioxidant production, while enzymatically it can take form
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J. Tropical Biodiversity Biotechnology, vol. 07 (2022), jtbb67359
of enhanced SOD, POX and APX biosynthesis (Wang et al. 2005).
Rhizobacteria are microbes living around plant roots and playing a crucial role in plant growth. Several species of rhizobacteria have been identified as having a role in promoting plant development and crop yield (Loon
2007; Elango et al. 2013). Changes in water availability and environmental
circumstances will have an impact physiologically of the soil microbial population, which will alter cell osmolarity and osmotic pressure on rhizosphere
microorganisms (Miller & Janet 1996). Some rhizospheric microorganisms
have developed osmotolerant systems for survival. They are also able to synthesize organic compounds in the cytoplasm that act as osmoregulators or
osmoprotectants during osmotic stress or drought stress by producing proline compounds and/or glycine betaine (Csonka 1989). According to (Munro
et al. 1989; Kunin & Rudi 1991), microorganisms and plants produce glycine
betaine, an osmolyte that helps them endure osmotic stress. Osmoprotectants help rhizobacteria to adapt and to survive under salt and drought stress
by keeping the osmotic potential of cell greater than that of their surroundings.
The ability of rhizobacteria to develop association with plants in the
root system of plants opens its potential as an inoculum for plant cultivation.
In a drought stressed environment, such an association, is likely to improve
plant development. The addition of rhizobacteria isolates is known to stimulate plant growth by increasing P availability and nitrogen fixation (Gholami
et al. 2008). It is also suggested that betaine produced by rhizobacteria reduced on the root surface is able to the root solute potential, resulting in a
flow to the rhizosphere in a way that the rhizobacteria can survive under
drought stress conditions. Glycine betaine is an osmolyte that is not found in
all higher plants, but is produced by a variety of microbes and plants to help
them withstand osmotic stress. Some bacteria, such as Cyanobacteria, Eschericia
coli, Salmonella typhimurium, Klebsiella pneumoniae, and Azospirillum sp. generate
glycinebetaine, a significant osmoprotectant (Yuwono 2005). Enterobacter flavescens is one of the rhizobacteria that can create an osmoprotectants, such as
proline and/or glycine betaine compounds, in response to osmotic stress or
drought stress (Csonka 1989). Osmoprotectants help rhizobacteria adapt and
survive in environments that are stressed by salt or drought by maintaining
the cell’s osmotic potential higher than its surroundings. This is accomplished in Escherichia coli via osmoregulatory processes that result in a cytoplasm that has an appropriate osmotic pressure and is conducive to enzyme
action. E. coli cells accumulate potassium ions and activate system for the
transport and synthesis of several organic osmolytes compatible with metabolism in response to osmotic stress and a decrease in cell turgor pressure,
preventing cell dehydration and stabilizing enzyme activity in high ionic
strength solutions (Munro et al. 1989). Plants accumulate glycinebetaine and
proline during osmotic stress suggest that these two compounds have the
potential to counteract the inhibitor effects of osmotic stress in bacteria. Proline and glycinebetaine interact with proteins in a unique way that protect
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them from denaturation in the presence of high electrolyte concentration.
These two chemicals are simply inert compatible solutes that help cells maintain turgor in high-osmolarity environment (Csonka 1989).
In Indonesia, rice is the most widely consumed food. However, rice
productivity is decreasing due to problems such as salinity and drought
stress. Currently, there are many superior rice cultivars with increased higher
productivity compared to local cultivars. In the face of drought, the use of
suitable and adaptable superior cultivars could be a viable option for rice production. In specific growing condition, superior variety seeds have a high purity, high growth percentage, and high yield potential. ‘IR64’ is a lowland rice
cultivar that can be planted in irrigated or swampy rice fields, while ‘Situ Bagendit’ is a rice cultivar that can be grown on dry land or paddy fields
(Suprihatno et al. 2009). ‘Situ Bagendit’ is known as upland rice which
demonstrated resistance to drought stress compared to rice ‘IR64’.
In response to decreasing availability of freshwater, alternative sources
are required by improving plant adaptability in drought condition. For
drought survival, several rhizospheric bacteria have developed osmotolerant
system. Rhizobacteria’s ability to form associations with plants in the root
system of plants makes it a viable inoculum for plant cultivation. ‘IR64’ and
‘Situ Bagendit’ were used in this study to determine the plant response under
drought conditions with the application of osmotolerant rhizobacteria. This
study aims at establishing the response of 'IR64' and 'Situ Bagendit' to the
inoculation using osmotolerant rhizobacteria under drought condition.
MATERIALS AND METHODS
Materials
In this work, osmotolerant rhizobacteria E. flavescens was taken from Faculty
of Agriculture, Universitas Gadjah Mada, Yogyakarta, while two rice cultivar
(Oryza sativa L.) ‘IR64’ and ‘Situ Bagendit’ were collected from the Indonesian Center for Rice Research (ICRR), Indonesia.
Methods
Osmotolerant rhizobacteria inoculation and application of drought treatment
The study was carried out in the Greenhouse of the Sawitsari Research Station, Faculty of Biology, Universitas Gadjah Mada, Indonesia between July
2019-February 2020. The experiment involved three factors and was conducted in a completely randomized design (CRD). The first factor was the
rice cultivar with two rice cultivar (‘IR64’ and ‘Situ Bagendit’), the second
factor was inoculation using osmotolerant rhizobacteria (E. flavescens) and
without inoculation, and the last factor was three levels of drought at 25%,
50%, and 100% of field capacity.
Seed sterilization was accomplished by soaking the seeds in 70% (v/v)
ethanol for 5 minutes, followed by submersion in 0,2 percent HgCl2 for 4
minutes. The seeds were rinsed six times with sterile distilled water following
surface sterilisation. Rice seeds were inoculated with osmotolerant rhizobac-3-
J. Tropical Biodiversity Biotechnology, vol. 07 (2022), jtbb67359
teria inoculants in a volume of 5 mL (108 cfu/mL) prior to planting. During
21 days, the soil’s water-holding capacity was sustained at field capacity. Following that, the soil moisture was changed to achieve 25%, 50%, and 100%
field capacity. Each treatment’s field capacity is maintained by watering every
three days till 12 weeks.
Plant Growth Parameters
Plant height, number of leaves, tillers, and panicles were measured weekly
during treatment. The distance between the longest leaf tip and the plant
above the soil surface was used to determine plant height.
Physiological Responses Measurement
Physiological characteristics were measured using the (Harborne 1984) method with a few adjustments on chlorophyll and carotenoid content. Cold acetone solution (3 mL of 80%) was used to homogenize a 0.3 grams leaf sample pulverized in a mortar. The spectrophotometer (Thermo Scientific
GENESYS 10 UV Scanning) was used to analyze chlorophyll content at 470
nm, 645 nm, and 664 nm multi-wavelengths and the results were expresses in
mg.g-1 FW (fresh weight).
Proline content analysis was measured using (Bates et al. 1973) method. Leaf samples of 0.25 grams were pulverized and homogenized in 5 ml
containing 3% sulfosalicylic acid solution. The sample was mixed with ninhydrin reagent (ninhydrin, acetic acid, and phosphoric acid) and glacial acetic
acid in a 1:1:1 ratio, and then heated in a water bath (Memmer GmbH +
Co.KG.WNB-7) at 95 oC for 60 minutes. The solution was cooled to 25o C
and reacted with toluene to form two layers. The absorbance of the solution
at 520 nm wavelength was compared to the standard proline curve to measure proline levels.
The activity of superoxide dismutase (SOD) was measured using the
(Marklund & Marklund 1974) procedure. Fresh leaf samples (0.5 grams) were
frozen in liquid nitrogen and crushed, then homogenized with 0.01 M phosphate buffer (pH 7.0), 1 mM EDTA, and 1% PVP, then centrifuged for 20
minutes at 4° C at 15000 rpm. A reaction mixture was prepared, consisting of
2 mL Tris-HCl Buffer at pH 8.2, 0.5 mL 2 mM pyrogallol, and 2 ml ddH20
were combined with 0.5 mL supernatant. The generated test mixture was
compared to a blank solution) containing pyrogallol) at 325 nm at 3 minutes
intervals by spectrophotometer (Thermo Scientific GENESYS 10 UV Scanning). The oxidation data for pyrogallol were gathered every minute for 3
minutes and utilized to determine auto-oxidation of 100%. The results are
given in units per milligram of protein (1 unit is the amount of enzyme used
to inhibits 5 percent of pyrogallol oxidation per minute).
Ascorbate peroxidase activity measurements were carried out following
the (Nakano & Asada 1981) protocol with modifications. Enzyme extract
(100 µL), EDTA 0.1 mM (400 µL), 0.05 mM sodium phosphate buffer at pH
7.0, 0.05 mM ascorbic acid solution (400 µL) and ddH20 (1.5 mL) were
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J. Tropical Biodiversity Biotechnology, vol. 07 (2022), jtbb67359
mixed together. The mixed solution was then added with 400 µL of 3%
H2O2 solution followed by incubation for 60 seconds. Furthermore, a spectrophotometer was used to detect the decrease in absorbance at wavelength
of 290 nm with a time interval of 3 minutes (Thermo Scientific GENESYS
10 UV Scanning). APX enzyme concentrations were calculated using the exclusion coefficient (€ = 2.8 mM-1cm-1). The amount of APX that oxidizes
one nmol per mL of ascorbate per minute is defined as one unit (U). The
activity of the APX enzyme is measured in U.mg-1 protein. A mixed solution
without enzyme extract is used as a control.
The determination of peroxidase (POX) activity was performed using
the modified (Kar & Dinabandhu 1976) protocol. Sodium phosphate buffer
solution (500 µl of 0.05 mM) at pH 7.0, pyrogallol solution (10 µL), and 250
µL of 5 mM H2O2 solution were put into the tube, followed by 500 µL of
enzyme extract and incubated at 250C for 15 minutes in a water bath. The
tube was filled with 5% H2SO4 solution (250 µL) and gently shaken. The purpurogallin absorbance was measured at 420 nm with spectrophotometer
(Thermo Scientific GENESYS 10 UV Scanning). The same process was used
to make a blank solution, but no enzyme extract was added. The activity of
the POX enzyme was measured at 420 nm (A420).
Data Analysis
Homogeneity and normality of the data were analyzed using one-way ANOVA and followed by Duncan’s Multiple Range Test (at 95% confidence level)
using IBM-SPSS version 16.0.
RESULTS AND DISCUSSION
Results
Growth Performance
Treatment under different water availability (100%, 50%, and 25% field capacity) resulted in significantly different plant heights in the two rice cultivars
treated without and with the inoculation of osmotolerant rhizobacteria (E.
flavescens) (Figure 1). The rise plant height during the vegetative phase, and the
plant does not tend to grow taller after it enters the generative phase. According to Figure 1, plant height increased until the 5th week in rice ‘IR64’
and ‘Situ Bagendit’ grown at 100% field capacity, and it remained steady until
the 12th week. The increase in plant height remained until the 6th week in the
50% field capacity treatment, whereas in the 25% field capacity treatment,
plant height constant at 8th week. Plant development is more ideal at 100%
field capacity because the water needs are more adequate than at 25% field
capacity. This situation also permits plants to perform better in their growth
phase and enter their reproductive phase more quickly. The establishment of
tillers and panicles was faster with 100% and 50% field capacity than with
25% field capacity treatment.
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Figure 1. Effect of rhizobacteria inoculation on plant height of rice 'IR64' and 'Situ
Bagendit' for 12 weeks under drought condition A: 100% field capacity, B: 50%
field capacity, and C: 25% field capacity.
According to Figure 1, the highest increase in plant height was found
in ‘Situ Bagendit’ with inoculation using osmotolerant rhizobacteria in the
100% field capacity treatment, while the treatment without inoculation using
osmotolerant rhizobacteria resulted in the lowest plant height. In comparison
to ‘IR64’ with and without inoculation using osmotolerant rhizobacteria at
50% field capacity treatment, ‘Situ Bagendit’ with osmotolerant rhizobacteria
inoculation produced the maximum plant height. ‘Situ Bagendit’ produced
higher plant height than rice ‘IR64’ at 25% field capacity treatment.
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Based on the data obtained during 12 weeks of planting, it is observed
that 'Situ Bagendit' plants produced higher plant height compared to
'IR64' (Table 1). Treatments of water availability (100%, 50% and 25% field
capacity) resulted in considerably varied plant heights in two rice cultivars,
both with and without rhizobacteria inoculation. Under 25% field capacity,
'Situ Bagendit' plants without rhizobacterial inoculation produced higher
plant height compared to 'IR64'. This proves that ‘Situ Bagendit’ is more resistant to drought than ‘IR64’. The inoculation of rhizobacteria to each rice
cultivar gave maximum results in supporting plant growth, both at 100%,
50%, and 25% field capacity.
The number of leaves was significantly different in rice 'IR64' and 'Situ
Bagendit' between treatments with and without osmotolerant rhizobacterial
inoculation. The number of leaves in two cultivars differed significantly between treatments of 100% field capacity with 50% and 25% field capacity,
while there was no significant difference between treatments of 50% and
25% field capacity. The highest number of leaves was found under 25% of
field capacity with osmotolerant rhizobacteria inoculation to 'IR64' and 'Situ
Bagendit', while the least number of leaves was found in 'Situ Bagendit' rice
at 50% and 25% field capacity without rhizobacterial inoculation (Table 1).
Different level of water availability also resulted in different number of
tiller and panicle in the two rice cultivars treated without and with the inoculation of osmotolerant rhizobacteria (Table 2). ‘IR64’ at 25% field capacity
without rhizobacteria inoculation had lowest average number of tillers. Both
cultivars of rice generated the same number of tiller in each field capacity,
regardless of whether they were inoculated with rhizobacteria or not. “Situ
Bagendit” produced the most tillers at 100% field capacity without rhizobacteria inoculation treatment (with average 5.33 tillers), which was not substantially different from the treatment with inoculation. When comparing treatments with 50% and 25% field capacity, the number of tillers ‘IR64’ and ‘Situ
Bagendit’ at 100% field capacity with or without rhizobacteria inoculation
resulted in the highest number of tillers, while the treatment with 25% field
capacity resulted the lowest number of tillers.
Table 1. Plant height and leaf number of rice (Oryza sativa L.) ‘IR64’ and ‘Situ Bagendit’ at week 12 at 100%, 50%, and
25% field capacity.
‘IR64’
Parameter
Plant Height (cm)
Leaf Number
Field Capacity
100%
50%
25%
100%
50%
Without
Rhizobacteria
79.50 b
75.00 c
61.67 e
11.00 p
8.67 q
With
Rhizobacteria
87.33 a
76.33 cb
65.00 ed
8.00 q
11.00 p
25%
9.00 q
11.67 p
‘Situ Bagendit’
Without
With
Rhizobacteria
Rhizobacteria
85.33 a
87.33 a
77.00 cb
78.00 cb
68.33 d
65.00 ed
11.00 p
8.00 q
q
7.67
8.67 q
7.67 q
11.67 p
Values having same letter (s) in a row and column of each parameter was not significantly different at (p ≤ 0.05) level of
significant by DMRT.
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Table 2. Number of tiller, panicle, and percentage of filled grain of rice (Oryza sativa L.) ‘IR64’ and ‘Situ Bagendit’ at
week 12 at 100%, 50%, and 25% field capacity.
‘IR64’
‘Situ Bagendit’
Without
With
Without
With
Rhizobacteria
Rhizobacteria
Rhizobacteria
Rhizobacteria
100%
3.67 edc
4.67 cba
5.33 a
4.33 dcba
Number of
50%
3.33 edcb
4.00 edcb
3.00 fe
4.33 dcba
Tiller
25%
2.33 f
3.67 edc
3.00 fe
3.33 fed
lk
ji
i
100%
3.67
4.67
5.33
4.67 ji
Number of
50%
3.33 kj
4.67 ji
3.00 ml
4.33 kj
Panicle
m
ml
ml
25%
2.33
3.00
3.00
3.00 ml
q
p
s
100%
65.72
78.62
32.48
61.02 q
Percentage of
50%
47.12 r
46.90 r
17.47 ut
34.80 s
Filled Grain (%)
t
r
u
25%
19.54
46.25
10.80
21.32 t
Values having same letter (s) in a row and column of each parameter was not significantly different at (p≤ 0.05) level of
significant by DMRT.
Parameter
Field Capacity
‘Situ Bagendit’ produced the most panicles with 100% field capacity
treatment without osmotolerant rhizobacteria inoculation with average of
5.33 panicles. The results were similar to those obtained with rice ‘IR64’ at
100% and 50% field capacity by inoculation with rhizobacteria, as well as
with ‘IR64’ at 100% and 50% field capacity by inoculation with rhizobacteria.
The lowest average number of panicles was found at 25% field capacity (2.33
panicles) was recorded in ‘IR64’, which was not significantly different from
the inoculation treatment with rhizobacteria, and with ‘Situ Bagendit’ at 25%
and 50% field capacity without rhizobacteria inoculation and 25% field capacity with rhizobacteria inoculation. The average number of tillers and panicles produced by both rice cultivars was about the same. The number of panicles produced by ‘Situ Bagendit’ at 100% field capacity treatment with rhizobacteria inoculation was higher than the number of tillers, whereas the number of tillers was higher in ‘IR64’ and ‘Situ Bagendit’ at 25% field capacity
with rhizobacteria osmotolerant in comparison to number of panicles.
‘IR64’ under 100% of field capacity inoculated with rhizobacteria resulted in the highest percentage of filled grain (78,62%), while the lowest percentage was found in ‘Situ Bagendit’ under 25% of field capacity without the
inoculation of rhizobacteria. Water availability at 25% of field capacity resulted in the lowest percentage of grain compared to 100% treatment and 50%
of field capacity. The osmotolerant rhizobacteria inoculation also gave a significant difference in the resulting grain compared to the treatment without
rhizobacteria, both in rice 'IR64' and 'Situ Bagendit' on treatment of 100%,
50% and 25% of field capacity.
According to Salsinha et al. (2021), with the increase in drought stress
level, the proportion of growth parameters (plant height, and number of tillers) decrease significantly (p<0,05). Salsinha et al. (2021) also shows that
drought-tolerant cultivar (Boawae 100 Malam and Padi Merah Kuatnana) was
significantly different from Ciherang cultivar. In this study, the analysis of
growth shows that ‘Situ Bagendit’ is drought-tolerant cultivar with high sus-8-
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ceptibility to drought stress. Drought stress affects food crop development
and yield. Due to a lack of water, the development process from vegetative
to reproductive phase has also been slowed that also damage cell membranes,
perhaps leading to cell death. Plant morphological growth, like height, number of tillers, panicles and grains was reduced in the presence of drought
stress (Farooq et al. 2009). Drought during vegetative phase can inhibit the
growth of leaves and roots. Drought stress during flowering and grain filling
reduced grain yield considerably as compared to the control. The decrease in
yield at the flowering stage is largely due to the decrease in the number of
grains per panicle. Stress during the growth stage can decrease assimilation
translocation to seed which decreases seed weight and increases the percentage of empty seeds. The success of seed formation depends on the availability of assimilates translocated to the seeds. The source of assimilates for seed
formation comes from the flag leaves. Flag leaves contribute 45% assimilation for the formation of rice seeds (Abou-Khalifa et al. 2008).
Physiological responses
Plant uses photosynthetic pigments primarily for light gathering and the creation of reducing power. Although chlorophyll a and b are susceptible to soil
drying, carotenoids play a complementary role in helping plants endure
drought (Farooq et al. 2009). The highest chlorophyll content was found in
rice ‘IR64’ under 50% field capacity with rhizobacterial osmotolerant inoculation. ‘Situ Bagendit’ under 100% field capacity resulted in the lowest chlorophyll content compared to the 50% and 25% field capacity treatment, as
well as with the ‘IR64’ plant treatment (Table 3). Drought stress altered the
ratios of chlorophyll a and b, as well as carotenoids. Drought stressed cotton
(Massacci et al. 2008) and sunflower plants (Kiani et al. 2008) were found to
have reduced chlorophyll content. As a relative water content and leaf water
potential diminish, the foliar photosynthesis rate of higher plants is known to
decrease. According Salsinha et al. (2021), drought tolerance is higher in rice
cultivars with lower chlorophyll reduction percentage (Hare Tora and
Boawaw 100 Malam with 9.37% and 7.56 % respectively), however Pak
Mutin, Gogo Jak and Padi Putih Maumere had the biggest fall in chlorophyll
levels indicating a high drought sensitivity. As water content and leaf water
potential fell in higher plants, the photosynthetic rate is decreased. The
measurement of chlorophyll a and b shows that when the amount of water in
treatment was lowered, the amount of photosynthetic pigments was similarly
reduced, affecting the morphological and physiological processes of the
plants (Usman et al. 2013).
Drought can cause carotenoid damage due to free radical activity. According to Martinez-Ferri et al. (2004), Jaleel et al. (2009); Du et al. (2010)
and Anjum et al. (2011), enhanced biosynthesis and free radical destructive
activity, as well as increased conversion of carotenoid pigment into other
chemicals such as ABA, might cause a decrease in pigment content, which is
essential to create plant adaptation strategies in response to drought stress.
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Table 3. Chlorophyll, Carotenoid and Proline Content of rice (Oryza sativa L ‘IR64’ and ‘Situ Bagendit’) at week 12 under
100%, 50%, and 25% field capacity.
‘IR64’
‘Situ Bagendit’
Without
With
Without
With
Rhizobacteria
Rhizobacteria
Rhizobacteria
Rhizobacteria
100%
3.55 cba
3.40 cba
2.56 c
2.67 cb
Chlorophyll (mg.g-1 FW)
50%
3.34 cba
3.98 a
3.00 cba
3.86 ba
25%
3.15 cba
3.13 cba
3.15 cba
3.42 cba
100%
0.85 i
0.16 kj
0.14 k
0.32 kj
Carotenoid (mg.g-1 FW)
50%
0.59 kji
0.23 kj
0.52 kji
0.33 kj
kji
kj
ji
25%
0.41
0.31
0.62
0.33 kj
r
r
rq
100%
0.105
0.109
0.137
0.107 r
Proline (µmol g-1 FW)
50%
0.117 r
0.123 r
0.147 rq
0.161 rq
r
rq
q
25%
0.125
0.199
0.223
0.344 p
Values having same letter (s) in a row and column of each parameter was not significantly different at (p ≤ 0.05) level of
significant by DMRT.
Parameter
Field
Capacity
The highest carotenoid content was found in ‘IR64’ with 100% field capacity
treatment without rhizobacterial osmotolerant inoculation. Rhizobacterial
osmotolerant inoculation treatment resulted in lower carotenoid content
compared to the treatment without rhizobacteria osmotolerant inoculation,
both in ‘IR64’ and ‘Situ Bagendit in under the three field capacity treatments
(Table 3).
Drought has also been linked to changes the metabolism of soluble
carbohydrates, a group of molecules that can act as suitable solutes and antioxidants. When there is a lack of water, there are chemicals which are tend
to rise. Free amino acids are another type of molecules that may be impacted
by lack of water. Water stressed leaves had higher levels of proline and total
free amino acids (Pinheiro et al. 2004; Van Heerden 2002). A distinctive
plant response to environmental challenges, notably drought stress, is the
accumulation of defensive solutes like proline and soluble sugar in the leaf
(Sakamoto 2002). During stress, proline and soluble sugar, both operate as
osmoprotectans (HongBo et al. 2005; Reddy et al. 2004). Proline may also
operate as an antioxidant. Proline content in 'Situ Bagendit' rice at 25% field
capacity treatment resulted in the highest proline content. The inoculation
using rhizobacteria also increases the proline content produced. The increase
of the proline content in stressed plants is an adaptation to deal with stressful
conditions (Table 3). Under severe situations, proline builds up and provides
energy for growth and survival (Chandrashekar & Sandhyarani 1996). Proline, sucrose, glycinebetaine, and other substances accumulation in the cytoplasm have a function in osmotic adjustment, which can improve the rate of
water absorption (Shehab et al. 2010; Usman et al. 2013). Proline biosynthesis is stimulated directly during stress as a drought resistance strategy. Proline
has been shown to scavenge ROS and other free radicals in studies. However, exogenous proline at high concentrations (40-50 mM) has little effect on
rice plants under abiotic stress (Hayat et al. 2012). As a result of the induced
drought stress in the roots, plants’ proline levels rise. Drought tolerant rice
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(such as Padi Hitam Mumere, Shintara, Padi Merah Noemuti, and Gogo Sikka) showed the highest levels of proline from control to severe drought curcumtances (Salsinha et al. 2021).
Biochemical properties
Drought stress causes a plant’s reaction to be complex, involving the synthesis of polyamines and the emergence of a new group of proteins whose function is unknown. Abscisic acid is crucial to the reaction because it causes
closing stomata, limiting water loss while also reducing CO2 available for
photosynthesis, which can lead to electron production in the photosystem
(Arora et al. 2002). Plants have highly efficient scavenging systems for reactive oxygen species, which protect them from oxidative processes that harmful to them. These defenses are not only prevalent within the cell, but also in
the apoplast to a lesser extent. Plants evolved cellular adaptive responses as a
result, such as up-regulation of oxidative stress protectors and the accumulation of protective solutes. Superoxide dismutase (SOD), ascorbate peroxidase
(APX), and peroxidase (POX) are antioxidant defense enzymes that help to
reduce superoxide and hydrogen peroxide concentrations. The dismutation
of superoxide into oxygen and hydrogen peroxide is catalyzed by superoxidedismutase (SOD). Peroxidases which comprise both enzymic and nonenzymic H2O2 degradation, remove H2O2 (Peltzer et al. 2002). The activity of
ascorbate peroxidase has mostly been found in chloroplasts and cytosols.
SOD and Ascorbate Peroxidase (APX) enzymes are found in both soluble
and thylakoid-bound forms in chloroplasts. The activity of APX had higher
activity drought stress at 25% field capacity and 50% field capacity compared
to 100% field capacity in both rice cultivars. Ascorbate peroxidase is another
key antioxidant enzyme. APX participates in the oxidative chain reaction that
transforms H2O2 into O2 and H2O with absorbic acid as one of the electron
suppliers (Refli & Yekti 2016).
The catalytic alterations in the detoxification of peroxide radicals into
water and oxygen are catalyzed by peroxidase (POX) enzymes (Hiraga et al.
2000). This enzyme is also involved in plant adaptations such as lignification,
suberization, and auxin metabolism simulation (Lagrimini et al. 1997). POX
activity in rice ‘IR64’ and ‘Situ Bagendit’ experienced a decrease in rice
drought rate of 50% and 25% in field capacity compared to 100% (Table 4).
Peroxidase are important for scavenging H2O2 toxicity. Under adverse situation such as drought stress, the combined activity CAT and SOD transforms
the deadly superoxide radical (O2) and hydrogen peroxide (H2O2) to water
and molecular oxygen, preventing cellular damage (Noctor et al. 2000;
Chaitanya et al. 2002). Drought-tolerant wheat, coffee, rice, and caper cultivars had higher antioxidant system than susceptible cultivars, according to
(Guo et al. 2006; Lascano et al., 2001; Lima et al. 2002; Ozkur et al. 2009).
The results of this study indicate that drought stress can simulate
changes in antioxidant enzymes activity (SOD, APX, and POX) of rice leaves
in both rice cultivars, ‘IR64’ and ‘Situ Bagendit’. The change in enzyme activ-11-
J. Tropical Biodiversity Biotechnology, vol. 07 (2022), jtbb67359
Table 4. Oxidative Enzyme Activity of rice plant (Oryza sativa L ‘IR64’ and ‘Situ Bagendit’) at 12 weeks under 100%,
50%, and 25% field capacity.
‘IR64’
‘Situ Bagendit’
Without
With
Without
With
Rhizobacteria
Rhizobacteria
Rhizobacteria
Rhizobacteria
100%
0.640 a
0.914 a
1.145 a
1.527 a
50%
0.868 a
1.295 a
1.091 a
1.517 a
SOD (U/L)
a
a
a
25%
0.985
1.100
0.997
0.969 a
100%
2.336 j
4.403 ji
2.166 j
3.182 ji
ji
ji
ji
50%
3.734
5.041
3.760
3.272 ji
APX (U/L)
25%
3.801 ji
5.764 i
5.092 ji
4.990 ji
100%
0.210 p
0.361 p
0.318 p
0.300 p
p
p
p
POX (A420)
50%
0.202
0.239
0.283
0.298 p
25%
0.164 p
0.228 p
0.228 p
0.174 p
Values having same letter (s) in a row and column of each parameter was not significantly different at (p ≤ 0.05) level of
significant by DMRT.
Parameter
Field Capacity
ity plays a role in suppressing the destructive activity of free radicals. The activity of these oxidative enzymes tends to be more active in ‘Situ Bagendit’ (drought resistant) rice plants than in ‘IR64’ (Table 4). The high activity of oxidative enzymes indicates the development of a better oxidative defense system compared to drought rice.
CONCLUSION
Under stress condition, the mechanism plants defense was increased to ensure the tolerance of plant in responding to stress. The rice plants (Oryza sativa L.) ‘IR64’ and ‘Situ Bagendit’ are becoming increasingly stunted as drought
stress increases. Inoculating osmotolerant rhizobacteria help accelerate plant
growth, as indicated by increased plant height, number of leaves, tillers, and
panicles in both rice cultivars, as well as a higher percentage of filled-grain in
‘IR64’. The biochemical and physiological response of ‘IR64’ and ‘Situ Bagendit’ to drought were enhanced by inoculation with osmotolerant rhizobacteria, as evidenced by an increase in proline content, SOD and APX enzyme activity, while the carotenoid level reduced. The rhizobacteria osmotolerant inoculation showed the increased POX activity at IR64 cultivar. Inoculation of osmotolerant rhizobacteria can be used to increase the cultivation of
rice plants under drought stress.
AUTHORS CONTRIBUTION
TY and DR designed the research and supervised all the process, HDK collected and analyzed the data and prepared the publication. All authors read
and approved the final version of the manuscript.
ACKNOWLEDGMENTS
The author thanked the Faculty of Biology and Faculty of Agriculture, Universitas Gadjah Mada (UGM), Yogyakarta, Indonesia as the provider of the
Research Station and research facilities.
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J. Tropical Biodiversity Biotechnology, vol. 07 (2022), jtbb67359
CONFLICT OF INTEREST
There are no conflicts of interest declared by the authors.
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