Microbiological Research 239 (2020) 126518

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Microbiological Research
journal homepage: www.elsevier.com/locate/micres

Plant Growth-Promoting Bacillus sp. Cahoots Moisture Stress Alleviation in T

Rice Genotypes by Triggering Antioxidant Defense System
Shobana Narayanasamy, Sugitha Thangappan, Sivakumar Uthandi*
Biocatalysts lab., Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India

A R T I C LE I N FO A B S T R A C T

Keywords: Drought is considered one of the major obstacles for agricultural productivity worldwide such that greater efforts
Moisture stress are required to boost crop production under this stress. One of the methods to overcome this obstacle is to
plant growth-promoting bacteria (PGPB) harness the potential of microbe-induced systemic tolerance against moisture stress. The present work evaluated
Bacillus the potential role of two bacterial strains, namely Bacillus altitudinis FD48 and Bacillus methylotrophicus RABA6
antioxidant defense system
and their combination as a co-inoculant for promoting plant growth and moisture stress resilience in two con-
rice
trast cultivars of Oryza sativa L: CO51 (moderately drought tolerant) and IR64 (drought susceptible) under
conditions of terminal moisture stress. B. altitudinis FD48- and B. methylotrophicus-primed rice seeds (CO51 and
IR64) significantly influenced the source-sink relationship and reduced the relative water content (RWC). While
photosynthetic pigments and proline showed a steady increase owing to the co-inoculant priming, the activity of
reactive oxygen species (ROS)-quenching enzymes, such as catalase, superoxide dismutase, ascorbate perox-
idase, and peroxidase constitutively increased in plants treated with co-inoculant besides,reducing the trend
during the recovery phase. The productive tillers and grain weight were further augmented by the co-inoculant
under induced moisture stress. Moreover, the results revealed a 14% and 19% increase in the harvest index (HI)
in CO51 and IR64, respectively, attenuated with Bacillus sp. as a co-inoculant. The key mechanism in augmenting
energy metabolism by B. altitudinis FD48 and B. methylotrophicus RABA6 could be attributed to the regulation of
ROS-quenching enzymes that aid in moisture stress resilience. The results of the present study conclude that
these strains may be used as a novel bioinoculant for enhancing the drought tolerance in rice grown under
moisture stress regimes.

1. Introduction
Rice serves as a source of food and calories to more than half a
billion people worldwide, predominantly in Asian countries. The world
population in 2050 is projected to be 9 billion, entailing an incessant
increase in food production to ensure food security (Foley et al., 2011).
However, rice productivity remains poor in several parts of the world
owing to numerous constraints, including biotic and abiotic stresses.
Among the abiotic stresses, drought and salinity are highly intricate,
severely affecting rice production. In general, drought stress is the most
critical constraint on rice production and it is estimated to affect ap-
proximately 19 to 23 million hectares (Kumar, 2017). Drought stress
alters the cell’s water potential, stimulates stomata closure, and reduces
several anabolic enzymes implicated in photosynthesis and nitrate as-
similation (Zlatev et al., 2006). Besides, it triggers the production of
reactive oxygen species (ROS) that cause membrane injury, lipid per-
oxidation, DNA damage, and protein degradation (Pompelli et al.,
2010). According to Shukla et al. (2012), the plant system enhances its
physiologic and biochemical activities as a tolerance mechanism to
these stresses. The plants start actively accumulating compatible solutes
including ammonium compounds such as glycine betaine, sugars (e.g.,
sucrose), polyols (e.g., mannitol), organic acids (e.g., malate), inorganic
ions (e.g., calcium), and non-protein amino acids (e.g., proline). These
mechanisms allow plants to lower the water potential; indeed, it is
known to cause a significant reduction in the yield owing to the dis-
rupted metabolic processes during moisture stress.
Therefore, there is increasing attention in finding a solution to these
drought-related issues and its impact on food security. Yang et al.
(2009) determined that plant-associated bacteria could help withstand
the drought stress more effectively through a mechanism known as
microbe-induced systemic tolerance (MIST). It triggers bacterial pro-
duction of phytohormones (indole acetic acid [IAA], cytokinin, and
abscisic acid [ABA]), antioxidants, and 1-aminocyclopropane-1-car-
boxylate deaminase (ACCD) (Glick, 2012). Su et al. (2017) exhibited

⁎
Corresponding author at: Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore, 641 003, Tamil Nadu, India.
E-mail address: usivakumartnau@gmail.com (S. Uthandi).

https://doi.org/10.1016/j.micres.2020.126518
Received 22 January 2020; Received in revised form 19 February 2020; Accepted 23 May 2020
Available online 02 June 2020
0944-5013/ © 2020 Elsevier GmbH. All rights reserved.
S. Narayanasamy, et al.
the role of Bacillus amyloliquefaciens GB03 in mitigating the water stress
by upregulating drought-responsive genes, such as dehydrin, mal-
ondialdehyde, and aquaporins along with several physiological altera-
tions including changes in hormonal homeostasis, antioxidants, and
exopolysaccharides (EPS) and enhanced production of antioxidant en-
zymes (Huang and Guo, 2005; Simova-Stoilova et al., 2008). Elevated
deposition of antioxidant enzymes, including peroxidase (POX), cata-
lase, and nitrate reductase (NR), acts as a critical factor in reducing
oxidative damage, thereby increasing the drought tolerance (Gururani
et al., 2013).
Apoplastic space is the continuum of cell walls of adjacent cells as
well as the extracellular spaces that facilitates the transport of water
and solutes including photosynthates, proteins, amino acids, and nu-
trients from several metabolic pathways (Su et al., 2018). Apoplastic
space is the first battle area to combat plant biotrophic pathogens and
provide a hostile environment to the microbial intruder. A few defense
compounds, such as reactive oxygen species (ROS) and phytoalexins,
which bestow plant immunity toward pathogens, have been found in
the apoplast. This is evident in the adopted decoy strategy of host plants
against biotic and abiotic stresses (Zipfel and Oldroyd, 2017). Fur-
thermore, PGPR (plant growth promoting rhizhobacteria) activates
ABA concentrations and triggers stomatal closure. In response to
drought, the ABA levels increase and enhance several enzyme activities
because of activated ABA receptors (Watkins et al., 2017). Activation of
respiratory burst oxidases in the plasma membrane results in a transient
elevation in ROS. This, in turn, increases the calcium ion influx chan-
nels (Blatt et al., 2007; Minguet-Parramona et al., 2016), and further
activates ion efflux channels. Hence, guard cells become flaccid re-
sulting in stomatal closure owing to change in the ion concentration
and water efflux.
Other mechanisms include the up-regulation of aquaporins and root
hydraulic conductivity (Ngumbi and Kloepper, 2016). According to
Kumar et al. (2017), bacterial priming in plants modulates their mor-
phology and physiologic structure by changing the activity and levels of
phytohormones and enzymes. Furthermore, Bacilli can mitigate the
moisture/oxidative stress in crop plants and inhabit intercellularly,
perhaps as an endophyte or as a phyllospheric or rhizospheric organism
(Timmusk and Wagner, 1999; Vardharajula et al., 2011). Moreover,
microbial volatile organic compounds (mVOCs) can induce systemic
tolerance to soil salinization and drought stress, which pose significant
threats to crop production. The volatile 2,3-butanediol protects plants
from abiotic stress. The plant hormone, salicylic acid and hormonal
signal, nitric oxide, are required for the plant response to 2,3-butane-
diol under abiotic stress.
B. altitudinis FD48 is a multifaceted phyllospheric PGPB obtained
from rice (var.ADT 43). Its role in promoting plant growth and inducing
moisture stress tolerance in rice plants has been investigated in our
previous studies (Kumar et al., 2017). Furthermore, FD48 has been
found to produce phytohormones, including IAA (indole acetic acid),
cytokinin, and gibberellic acid (GA). Besides, FD48 can regulate the
production of plant ethylene through the formation of ACCD. It also
produces 192.5 n moles α-ketobutyrate mg–1. h–1, which may serve as
the sole nitrogen source (Kumar et al., 2017). FD48 has been reported
to solubilize P and Zn and produce siderophores; moreover, it can
survive at –10 bars of osmotic potential (–1.0 MPa) (Kumar et al.,
2017). Additionally, FD48 regulates auxin-responsive genes in the rice
root upon colonization under gnotobiotic conditions, thereby altering
the root system architecture by enhancing the accumulation of IAA in
the roots (Ambreetha et al., 2018). Complete genome sequencing of the
strain FD48 reveals the genes encoding for stress tolerance and plant
growth promoting (PGP)) traits, including those involved in trehalose
synthesis, cysteine synthesis, acetoin synthesis, ACCD, mineral solubi-
lization, osmolytes production, and heat and cold shock chaperones
(Kumaravel et al., 2018). Furthermore, B. methylotrophicus RABA6 iso-
lated from rice apoplast showed enhanced PGP attributes including IAA
production (1.95 μg mL–1) and ACCD activity (146.92 n moles α-
2
Microbiological Research 239 (2020) 126518
ketobutyrate mg–1. h–1) under in vitro induced moisture stress (Punitha,
2019).
The present work evaluated the performance of both B. altitudinis
FD48 and B. methylotrophicus RABA6 on moisture stress tolerance in
rice. In this study, we adopted field water limitation in rice by with-
holding irrigation at the panicle initiation stage to understand the ef-
fects of FD48 and RABA6 on promoting plant growth along with
moisture stress adaptive mechanisms. The experiments focused on the
responses of antioxidant enzymes, proline, and alterations in the
morpho-physiological structure of the plant under induced moisture
stress in commercial rice genotypes.
2. Materials and methods
2.1. Bacterial inoculants
Plant growth-promoting, drought-tolerant strains B. altitudinis FD48
(Kumar et al., 2017) and B. methylotrophicus RABA6 (Punitha, 2019)
were obtained from the Department of Agricultural Microbiology,
Tamil Nadu Agricultural University (TNAU), Coimbatore. In this in-
vestigation, FD48 and RABA6 were used as a co-inoculum. Both strains
were grown in the LB broth, incubated at 28 °C at 120 rpm. Seeds of two
rice genotypes, namely commercial, high-yielding moderately drought-
tolerant variety (CO51) and drought susceptible (IR64) variety, were
obtained from Paddy Breeding Station, Coimbatore and Rice Research
Station, Aaduthurai, Tanjore, Tamil Nadu, respectively.
2.2. Abscisic acid production
FT-IR spectroscopy was used to gain information about the func-
tional groups of ABA and investigate the presence of ABA in the culture
filtrate of FD48 and RABA6 in comparison with the ABA standard
(SIGMA, catalog no. A4906). The cell-free culture filtrate was analyzed
by the Fourier Transform Infrared Spectrophotometer-6800 type A (M/
s. Jasco, Japan) with an ATR sampling accessory. The minimal quantity
of the sample (50 μL) was placed between the infrared transparent
plates. The analyses were run using the TGS detector and the percent
transmittance was recorded against the wavenumbers (cm–1). The
spectral resolution was 4 cm–1 and the scanning was performed in the
mid-IR range from 400 cm–1 to 4000 cm–1. The same procedure was
repeated for the standard ABA at various concentrations, ranging from
10 to 100 ppm.
2.3. Design of greenhouse experiment, inoculants, and moisture stress
Pot culture experiments were conducted in a greenhouse at the
Department of Agricultural Microbiology, TNAU, Coimbatore, India
(situated at an altitude of 426.7 m above mean sea level, 11 °N latitude
and 77 °E longitude), to explore the mechanism of moisture stress mi-
tigation and the interaction of these strains with rice under induced
moisture stress. Wetland soil (soil texture: clay loam, pH 8.0; EC ds/m
0.75; organic carbon (%), 0.68; clay%, 37.7; silt%, 21.4; sand%, 40.5)
collected from D’ Block, TNAU, Coimbatore, was autoclaved prior to
use. The bioinoculant-primed plants were subjected to moisture stress,
and observations were recorded on different days of moisture stress,
namely 0, 7, and 10 days and recovery. In order to recover the plants
from water deficit, irrigation was scheduled for five days after 10 days
of moisture stress imposement.
Criteria Description
Variety CO51, IR64
Soil type Clay loam
S. Narayanasamy, et al.
Treatments T1 : Uninoculated Control (UNI)
T2 : B. altitudinis FD48
T3 : B. methylotrophicus RABA6
T4 : B. altitudinis FD48+ B. methylotrophicus RABA6
2.4. Plant growth and induction of drought stress
Influence of plant growth-promoting bacteria on growth as well as
physiologic and biochemical responses of two rice genotypes, namely
CO51 and IR64, under moisture stress were investigated. The surface-
sterilized seeds of CO51 and IR64 biotized with bacteria (1010cells
mL–1) were placed in a sterile Petri dish for germination. Uniformly
germinated seeds were sown in pots containing sterile soil and main-
tained as two seedlings per pot. Four replicates were maintained for
each treatment. Approximately, 10 mL of Hoagland nutrient solution
(Hoagland and Arnon, 1950) was added to each pot at 10-day intervals
to maintain the nutrients after 30 days after sowing (DAS). Foliar spray
of PGPB at 1010 CFU. mL–1 was administered at 40 DAS. Mock plants
were sprayed with the same volume of water and the plants were wa-
tered up to 45 days. After 50 DAS, the plants were exposed to moisture
stress by withholding irrigation at the panicle initiation stage. Tissues
(shoot and root) were collected from both treated and control plants
(DS) at the same time to avoid variations. All morphologic, physiologic,
and biochemical analyses were performed on each day of stress (0, 7,
and 10 days and recovery). The experiment was repeated thrice to
check the data integrity.
2.5. Assessment of photosynthetic pigments
Chlorophyll a andchlorophyll b were estimated using the following
procedure described by Sumanta et al. (2014). Approximately 500 mg
of leaf samples was homogenized with 80% acetone, and the homo-
genate was centrifuged at 6000 × g for 10 min; the supernatant ob-
tained was used for the analysis of pigments. The absorbance was
measured at, 645 nm, and 663 nm using a spectrophotometer (Spec-
tramax® i3X). The photosynthetic pigment was represented as mg g–1
fresh weight (FW).
2.6. Relative water content
The relative water content (RWC) was determined by the method
described by Barrs and Weatherley (1962). The third leaf of the plants
was collected early in the morning. Leaf samples of 1.5 cm diameter
were soaked in water for 4 h, and turgid weight was determined from
these leaves. The samples were then kept in a hot air oven at 60 °C. The
RWC was worked out after recording dry weight and expressed in
percentage.
2.7. Estimation of proline content
Accumulation of total proline in the leaf sample produced under
abiotic stress was assessed as per the protocol described by Bates et al.
(1973). Leaf samples (500 mg) were homogenized in 3% sulfosalicylic
acid. The mixture was filtered through Whatman no.1 filter paper. For
the estimation of proline, 2 mL of homogenate mixture was mixed with
2 mL of acid ninhydrin and 2 mL of glacial acetic acid. Next, the re-
action mixture was incubated at 100 °C in a boiling water bath for 1 h
and immediately, the tubes were cooled in an ice bath to terminate the
reaction. Later, the total proline was separated with 4 mL toluene and
vigorously mixed. The chromophore layer was separated and the ab-
sorbance was measured at 520 nm against a blank. The total proline
was calculated using the standard graph prepared using proline, and it
was expressed in mg g–1 FW.
3
Microbiological Research 239 (2020) 126518
2.8. Antioxidant enzyme assay
A quantity of 500 mg leaf sample was homogenized for super oxide
dismutase (SOD), catalase (CAT) and peroxidase (POD) assays, using an
ice-cold pestle and mortar in a 5 mL ice-cold buffer containing 50 mM
potassium phosphate buffer (pH 7.0), 1 mM EDTA (ethylene diamine
tetraacetic acid), and 1% (w/v) PVP (polyvinyl pyrrolidone). The
homogenate was centrifuged at 10,000 g for 30 min at 4 °C. The su-
pernatant obtained was used for the enzyme assay.
The ascorbate peroxidase (APX)) activity was determined according
to the protocol described by Nakano and Asada (1987). About 600 μL
enzyme extract was added to 1.5 mL of 10 mM phosphate buffer, 300 μL
ascorbate, and 600 μL H2O2. The decrease in the absorbance was read at
290 nm. The enzyme activity was calculated using the extinction
coefficient 2.8 mM−1 cm−1 at 290 nm and expressed as units min–1 g–1
of FW.
The CAT activity was estimated by the method described by
Azevedo et al. (1998). The assay mixture contained 50 mM phosphate
buffer (pH 7.0), 20 mM H2O2, and 0.1 mL enzyme extract. Reduction in
the absorbance of H2O2 was observed for 1 min at 240 nm on a mi-
croplate reader (Spectramax® i3X). The CAT activity was expressed as
units g–1 FW min–1.
The SOD activity was assessed by the nitroblue tetrazolium (NBT)
method described by Beauchamp and Fridovich (1971). About 100 μL
of the enzyme mixture was added to the reaction mixture containing 3
mL of sodium phosphate buffer (50 mM; pH 7.8) with 13 mM me-
thionine, 75 μM NBT, 2 μM riboflavin, and 0.1 mM EDTA. Then, the
reaction mixture was incubated for 15 to 30 min at 28 ± 2 °C. After
incubation, the absorbance was measured at 560 nm and the activity
was expressed as units g–1 of FW min–1.
For POD activity, the reaction mixture contained 100 μL of the
homogenate, 2.9 mL of 0.01 M sodium phosphate buffer (pH 6.0) with
0.25% v/v guaiacol and 0.1 M H2O2. The absorbance of the colored
product was read at 470 nm, and the activity was represented as units
g–1 FW min–1 (Hammerschmidt et al., 1982).
2.9. Yield components
The panicle length was determined using a centimeter scale.
Measurements were performed from the panicle base to the tip of the
panicle, and the number of grains per panicle was recorded. One
thousand filled grains were sampled from each plant and weighed at
14% moisture content and expressed in grams. The grain yield was
determined at harvest. The yield was extrapolated in grams per plant (g.
plant–1). The straw obtained from each plant was sun-dried and
weighed and the straw yield was expressed in grams per plant (g.
plant–1).
2.9.1. Harvest index
The harvest index was considered as the ratio of economic yield to
biological yield (Yoshida, 1972) and calculated as follows. Harvest
index (HI) = (economic yield)/(biological yield) × 100
2.10. Statistical analysis
All data were statistically analyzed in Microsoft Excel and add-in
with XLSTAT Version 2016.04.325250 (XLSTAT 2016). Significant
differences among the treatments were statistically analyzed using
analysis of variance (ANOVA) and Duncan’s Multiple Range Test
(DMRT) at p < 0.05 significance level. Each treatment was performed
with at least four replicates and the standard deviation was calculated
and expressed in mean ± SD of four replicates. The principal compo-
nent analysis (PCA) was performed with antioxidant enzymes as in-
fluenced by the inoculation of moisture-tolerant microbes and different
levels of stress in rice genotypes in R platform.
S. Narayanasamy, et al. Microbiological Research 239 (2020) 126518

bacterial strains RABA6 and FD48, which further authenticated the

production of ABA by the mentioned cultures. Besides, few wave-
numbers, namely 1714.41, 1590.99, 1195.65, 1068.37, 749.20, and
613.25 cm–1 were observed only for RABA6 and FD48 and represented
the ketone group owing to the C = O stretch, amine group owing to the
NH2 scissoring, C-N stretching, C-O stretching, O-H bending, C-H de-
formation, respectively.
The whole-genome sequencing data of FD48 (NCBI accession no.
CP025643) implied the presence of genes responsible for ABA bio-
synthesis. More specifically, the genes (YerE) encoding geranylgeranyl
glycerol phosphate synthase involved in the carotene biosynthesis were
identified, which, in turn, acts as a precursor for ABA biosynthesis
(Kumaravel et al., 2018). The entire pathway is depicted in Fig.S1.

Fig. 1. ABA production of different bacterial strains determined by FT-IR
spectroscopy. Cell free culture filtrate analyzed in Fourier Transform Infrared
Spectroscopy with the IR range of 400 cm-1 and 4000 cm-1. i) Line in black
represents standard ABA (SIGMA, catalog no. A4906), at 100 ppm concentra-
tion ii) Line in blue represent s B. altitudinis FD48 iii) Red line represents B.
methylotropicus RABA6.
3.2. Impact of Bacillus spp. in rice genotypes under induced moisture stress
Rice plants were collected on 0th, 7th, 10th days after stress and
recovery stages for assessing the physiologic and biochemical para-
meters. The soil moisture contents of pots on these days were recorded
as 830 ± 16.74, 62 ± 3.03, 45 ± 2.63, and 417 ± 14.37%, respec-
tively.

3. Results 3.3. Plant biomass

3.1. ABA production of different bacterial cultures by FT-IR spectroscopy
The comparative FT-IR absorption spectra of abscisic acid with
bacterial strains FD48 and RABA6 are presented in Fig. 1. The FT-IR
spectra of standard ABA (100 ppm) revealed distinct biomolecules.
Major absorption peaks were observed in the ranges between 3200 and
2800 cm–1 and 1500 and 1350 cm–1, 1700 and 1500 cm–1, 1250 and
1000 cm–1, 1000 and 500 cm–1. The wavenumbers 3309.25, 2942.84,
2830.99, 1448.28, 1022.09, 792.6, 628.68, and 421.37 cm-1 corre-
sponded to standard ABA. The observed standard ABA wavenumbers
were compared with bacterial strains, RABA6, and FD48 for their
capability to produce ABA and influence ABA modulation in plants.
Similar wavenumbers of standard ABA were recorded in both the
strains RABA6 and FD48.
The wavenumbers of standard ABA corresponding to 2924.84 cm–1,
2829.06 cm–1, 1441.53 cm–1, and 1025.94 cm–1 represented the alkane
group owing to CH3/CH2/CH stretching, C-H stretch of the aldehyde
group, CH2/CH3 bending deformation, and C-O stretch of the alcohol
group, respectively. Similar functional groups were observed in
Influence of Bacillus spp. (B. altitudinis FD48 and B. methylotrophicus
RABA6) on the physiologic and biochemical activities of rice genotypes
exposed to moisture stress was studied. Our experimental results ex-
hibited a significant decrease in the shoot and root dry weight on the 7th
day after moisture stress. However, the plants inoculated with PGP
bacterial strains FD48 and RABA6 significantly increased the shoot and
root dry weight under stressed (DS) conditions (Table 1). The in-
oculated plants (INO) showed enhanced biomass over un-inoculated
control (UNI). Among the treatments, the co-inoculant showed sig-
nificant biomass in both the genotypes IR64 (7.65 ± 0.098) and CO51
(8.85 ± 0.020).
Moreover, on the 10th day of DS, FD48 + RABA6 (co-inoculant)-
treated plants showed maximum shoot dry weight (43% increase over
UNI control), followed by either FD48 (29%) or RABA6 (21%) as single
inoculum in moderately drought tolerant genotype CO51. Similarly,
IR64 followed the same trend in shoot dry weight, registering 62%
increase over control in co-inoculated plants followed by FD48 (50%)
and RABA6 (31%). In the recovery phase, the co-inoculant (FD48 +
RABA6)treated plants showed a significant effect on recouping of shoot

Table 1
Effect of moisture stress on the growth parameters of rice genotypes influenced by PGPB
Days of stress Treatments Shoot dry weight (g) Root dry weight (g) Relative water content (%)

CO51 IR64 CO51 IR64 CO51 IR64

e e e e a
0 DAS T1 7.730 ± 0.033 8.12 ± 0.058 4.62 ± 0.073 4.03 ± 0.032 73.27 ± 3.82 64.16 ± 0.78a
T2 8.520 ± 0.055b 10.10 ± 0.130b 5.50 ± 0.046b 4.27 ± 0.113b 72.84 ± 1.27a 64.23 ± 1.13a
T3 8.120 ± 0.045d 8.82 ± 0.019d 4.89 ± 0.079d 4.03 ± 0.056d 71.68 ± 1.55a 62.56 ± 2.43a
T4 8.710 ± 0.047a 10.94 ± 0.096a 5.89 ± 0.097a 5.23 ± 0.127a 73.45 ± 1.30a 63.98 ± 3.21a
7 DAS T1 7.78 ± 0.050e 6.45 ± 0.034d 4.54 ± 0.043d 3.85 ± 0.049d 61.32 ± 4.32c 56.76 ± 4.11b
T2 8.12 ± 0.115b 7.53 ± 0.022b 5.38 ± 0.081a 4.87 ± 0.023a 67.24 ± 3.76a 60.12 ± 5.01a
T3 7.68 ± 0.083d 7.26 ± 0.125c 4.59 ± 0.072c 4.36 ± 0.028c 68.15 ± 3.43a 57.13 ± 2.23b
T4 8.85 ± 0.020a 7.65 ± 0.098a 5.79 ± 0.046a 4.96 ± 0.066a 65.13 ± 1.45b 61.23 ± 0.003a
10 DAS T1 5.14 ± 0.061d 4.06 ± 0.076c 2.81 ± 0.063d 2.71 ± 0.065d 49.87 ± 1.62c 45.63 ± 0.23c
T2 4.89 ± 0.052b 5.16 ± 0.065b 3.64 ± 0.005b 3.46 ± 0.073b 57.09 ± 3.76a 54.12 ± 3.21a
T3 4.61 ± 0.069c 4.86 ± 0.039d 3.18 ± 0.037c 3.17 ± 0.026c 54.76 ± 3.12b 50.19 ± 2.89b
T4 5.42 ± 0.006a 5.12 ± 0.058a 4.08 ± 0.076a 3.62 ± 0.020a 57.12 ± 1.89a 53.21 ± 3.98a
Recovery T1 5.76 ± 0.073d 4.86 ± 0.068d 3.16 ± 0.035d 3.16 ± 0.058d 58.21 ± 2.34c 50.89 ± 1.23c
T2 6.84 ± 0.105b 6.12 ± 0.062b 3.28 ± 0.021a 3.07 ± 0.047a 63.23 ± 2.65a 56.32 ± 0.23b
T3 6.56 ± 0.071c 5.41 ± 0.074c 3.46 ± 0.020c 2.98 ± 0.043c 60.56 ± 3.09b 52.14 ± 2.12c
T4 7.21 ± 0.012a 6.88 ± 0.005a 3.31 ± 0.076a 3.31 ± 0.026a 62.31 ± 1.76a 58.12 ± 2.67a

T1 - Uninoculated control, T2 - B. altitudinis FD48, T3 - B. methylotrophicus RABA6, and T4- FD48 + RABA6. Values are mean ( ± standard error) (n = 4) and values
followed by the same letter in each column are not significantly different from each other within the observation day as determined by DMRT (p ≤ 0.05).

4
S. Narayanasamy, et al.
dry weight over control with 25 and 41%, respectively, irrespective of
genotypes followed by FD48 (25%). The strain RABA6 as a single in-
oculum showed the least value of only an 11% increase over the con-
trol. As analogue to the shoot dry weight, the bacterial inoculation
considerably increased the root dry weight in both the genotypes under
induced moisture stress (Table 1).
On 7th and 10th day of DS, FD48 + RABA6 (co-inoculant)-treated
plants showed maximum shoot dry weight (5.79 ± 0.046 and
4.08 ± 0.076 gram respectively), followed by either FD48
(5.38 ± 0.081 and 3.64 ± 0.005 respectively) or RABA6 (4.59 ± 0.072
and 3.18 ± 0.037 gram) as single inoculum in moderately drought
tolerant genotype CO51. Similarly, IR64 followed the same trend in
root dry weight, co-inoculated plants showed maximum root dry weight
over uninoculated control followed by single inoculants FD48 and
RABA6 (Table 1).
The present investigations indicated significant variations among
different genotypes in their responses to PGP strains under induced
moisture stress, which is evident from their root/shoot ratio. The R/S
values ranged from 0.6 to 0.7 and 0.5 to 0.6 for CO51 and IR64, re-
spectively, under DS. The results on R/S showed an increasing trend
with an increase in moisture stress in both genotypes. However, on the
7th and 10th days of stress, FD48 (0.66 and 0.74, respectively) recorded
the maximum R/S ratio, followed by FD48 + RABA6 (0.65 and 0.75) in
the genotype CO51 over the UNI control. On the contrary, IR64 regis-
tered an increase in the R/S ratio over control on the 7th and 10th days
of DS with FD48 + RABA6 (0.64 and 0.70, respectively), followed by
FD48 (0.64 and 0.68), respectively. In general, in comparison with UNI
control, plants inoculated with both the PGPB exhibited a higher R/S
ratio under drought stress. Besides, during recovery phase, the R/S ratio
showed a decreasing trend in plants primed with bacteria. While
comparing both genotypes, recovery of R/S ratio in the susceptible
cultivar IR64 is very slow. (Fig. 2).
3.4. Relative water content
The tissue-water balance of both UNI and INO plants was evaluated
by determining the leaf RWC for both CO51 and IR64 throughout the
period of induced moisture stress and recovery. The results suggested a
dramatic reduction in RWC, irrespective of rice genotypes for induced
stress (Table 1). However, after ten days of moisture stress, FD48 alone
and FD48 + RABA6-treated plants exhibited a 14.5% and 18% re-
duction in RWC, respectively, over UNI control.
5
Microbiological Research 239 (2020) 126518
3.5. Photosynthetic pigments
Our investigation showed a positive influence on the photosynthetic
activity of both CO51 and IR64, inoculated with bacterial strains that
were measured in terms of levels of Chlorophyll a and Chlorophyll b .
However, the result implies a gradual decrease in chlorophyll content
with an increase in the intensity of moisture stress (Table 2). FD48 and
FD48 + RABA6, treated plants registered significantly higher levels of
chla and chlb levels on the 7th and 10th days after induced moisture
stress. The uninoculated control and RABA6 were almost on par with
each other.
3.6. Total proline
Accumulation of proline in both rice genotypes augmented sig-
nificantly with the progression of moisture stress and restored after
rewatering (Fig. 3). These results implied that proline content was
significantly influenced by genotypic nature, PGPB, and stress duration
(F = 3.54, p < 0.02). PGPB-inoculated plants showed a significant in-
crease in the proline content over the uninoculated control plants. On
initial day of stress, no significant difference was found between the
treatments in both the genotypes. However, bacterial strains, FD48,
RABA6, and FD48 + RABA6primed CO51 plants showed increased
proline content of 53%, 24%, and 41%, respectively on 7th day of
moisture stress.
Similarly, on 7th day of moisture stress IR64 attenuated with FD48,
RABA6, and FD48 + RABA6 showed 37%, 24%, and 58% increase over
control plants respectively. On the contrary, FD48-primed CO51 plants
showed a 42% increase in the proline content on the 10th day of
moisture stress, whereas the co-inoculant showed a drastic reduction as
compared to that on the 7th day. Besides, in the recovery phase, the
proline content was further reduced over the control, irrespective of
genotypes (Fig. 3). Furthermore, it was fascinating to observe that IR64
showed increased proline content than CO51 at all levels of stress in
severe moisture stress.
3.7. ROS scavenging enzymes
The osmotic stress response in rice plants was determined by esti-
mating the activity of several ROS scavenging enzymes, such as SOD,
CAT, POD, and APX, in plants over a time interval. The activity of these
enzymes increased with a simultaneous increase in the moisture stress
Fig. 2. Effect of PGPB induced Root/Shoot
ratio on rice genotypes under moisture stress
conditions. T1 - Uninoculated control, T2 - B.
altitudinis FD48, T3 - B. methylotrophicus RABA6
and T4 - FD48 + RABA6. Values are mean
( ± standard error) (n = 4) and values fol-
lowed by the same letter in each column are
not significantly different from each other on
the observation day as determined by DMRT (p
≤ 0.05).
S. Narayanasamy, et al. Microbiological Research 239 (2020) 126518

Table 2
Effect of PGPB inoculation on chlorophyll content of the two genotypes of rice under moisture stress condition
Rice Genotypes Bacterial strains Chlorophyll a (mg g-1 FW) Chlorophyll b (mg g-1 FW)

0day 7days 10 days Recovery 0days 7days 10 days Recovery

d d c d e e c
CO51 T1 2.66 ± 0.035 1.94 ± 0.020 1.15 ± 0.003 1.42 ± 0.014 2.52 ± 0.016 1.78 ± 0.013 0.97 ± 0.047 1.17 ± 0.019c
T2 3.29 ± 0.011ab 2.77 ± 0.047b 1.40 ± 0.008a 1.99 ± 0.016b 3.30 ± 0.055c 2.25 ± 0.028b 1.22 ± 0.016b 1.74 ± 0.024b
T3 2.94 ± 0.024c 2.61 ± 0.003c 1.28 ± 0.016b 1.71 ± 0.003c 2.68 ± 0.014d 2.08 ± 0.023d 1.03 ± 0.014c 1.75 ± 0.024b
T4 3.56 ± 0.007a 2.94 ± 0.036a 1.46 ± 0.024a 2.04 ± 0.002a 3.79 ± 0.041a 2.40 ± 0.025a 1.56 ± 0.027a 2.05 ± 0.015a
IR-64 T1 2.44 ± 0.036d 1.70 ± 0.015d 1.06 ± 0.021d 1.33 ± 0.010d 2.65 ± 0.047c 1.65 ± 0.050d 0.86 ± 0.030d 1.15 ± 0.010c
T2 3.04 ± 0.029b 2.66 ± 0.016b 1.37 ± 0.007b 1.68 ± 0.023b 3.24 ± 0.034b 2.18 ± 0.015b 1.10 ± 0.003b 1.61 ± 0.019b
T3 2.74 ± 0.006c 2.49 ± 0.035c 1.25 ± 0.016c 1.41 ± 0.004c 2.70 ± 0.119c 2.07 ± 0.023c 1.02 ± 0.014c 1.64 ± 0.004b
T4 3.10 ± 0.040a 3.03 ± 0.048a 1.41 ± 0.023a 1.85 ± 0.028a 3.85 ± 0.030a 2.37 ± 0.015a 1.31 ± 0.029a 1.87 ± 0.052a

T1 - Uninoculated control, T2 - B. altitudinis FD48, T3 - B. methylotrophicus RABA6, and T4 - FD48 + RABA6. Values are mean ( ± standard error) (n = 4) and values
followed by the same letter in each column are not significantly different from each other on the observation day as determined by DMRT (p ≤ 0.05).

Fig. 3. Proline content of rice genotypes in-

oculated with PGPB under moisture stress. T1 -
Uninoculated control, T2 - B. altitudinis FD48,
T3 - B. methylotrophicus RABA6 and, T4 - FD48
+ RABA6. Values are mean ( ± standard error)
(n = 4) and values followed by the same letter
in each column are not significantly different
from each other within the observation day as
determined by DMRT (p ≤ 0.05).

intensity in response to bacterial inoculation of both CO51 and IR64 activity (F = 7.93; p < 0.02*) at all time intervals. The SOD activity
genotypes. showed a steady increase up to ten days of exposure to moisture stress.
The co-inoculant treatment registered 34% (0.38 U min–1 g–1 of FW)
and 34% (1.12 U min–1 g–1 of FW) increase as compared to control,
3.7.1. Catalase activity
followed by strain RABA6 in moderately tolerant rice genotype CO51
PGPB-primed CO51 and IR64 plants showed a remarkable CAT
on 7th and 10th days (33% and 19%, respectively) of stress. However,
enzyme activity (F = 3.673, p < 0.01**). The catalase activity gradu-
the SOD activity of the susceptible genotype IR64 was more affected by
ally increased on 0th, 7th, and 10th days of induced moisture stress.
the co-inoculum with a 28% increase over the mock, followed by FD48
Irrespective of the treatment, no significant difference was noticed on
on the 7th and 10th days after the stress (Fig. 5). During the re-watering
the 0th day after stress in both the rice genotypes. However, on the 7th
phase, all PGPB-inoculated plants showed 15% increased recovery over
and 10th days after exposure to moisture stress, the CAT activity sig-
the control and the rate was observed more in CO51 than in IR64.
nificantly increased in PGPB-inoculated plants, irrespective of geno-
typic variation (Fig. 4). Among the treatments, FD48inoculated CO51
exhibited a significant increase in the CAT activity on the 7th day and
3.7.3. Ascorbate peroxidase activity
10th day of induced moisture stress (26% and 11%, respectively), fol-
The APX activity gradually increased on the 7th and 10th days of
lowed by combined inoculation of FD48 and RABA6. Similarly, the
exposure to moisture stress (F = 2.88; p < 0.01** Table 3). No sig-
drought-susceptible genotype IR64 displayed 37% and 29% increase,
nificant difference was observed among the genotypes and treatments
respectively, over the mock plants and followed the same trend as that
on the initial stress. However, as time passed, the APX activity in-
of CO51. On the contrary, the apoplastic bacterial strain RABA6 showed
creased significantly in co-inoculantsprimed CO51 plants (68% and
a similar pattern of CAT activity with no significant variation in both
39% on 7th and 10th days, respectively), followed by FD48 (Fig. 6).
genotypes. Furthermore, during the recovery phase, the FD48inocu-
Moreover, the susceptible IR64 genotype showed a similar response to
lated plants exhibited increased recovery over control plants as com-
APX (44% and 34%) on the 7th and 10th days of the stress, respectively.
pared to RABA6.
In both the genotypes, the co-inoculant was followed by a single in-
oculant FD48. It was interesting to note that during the recovery phase,
3.7.2. Superoxide dismutase activity priming the susceptible genotype (IR64) resulted in rapid recovery
The PGPB-biotized plants showed a significant impact on SOD when compared to its moderately resilient counter genotype CO51.

6
S. Narayanasamy, et al. Microbiological Research 239 (2020) 126518

Fig. 4. Catalase (CAT) activity induced in CO51 and IR64 under moisture stress condition.
T1 - Uninoculated control, T2 - B. altitudinis FD48, T3 - B. methylotrophicus RABA6 and T4 - FD48 + RABA6. Box and whisker plots represent minimum, lower
quartile, median, upper quartile, and maximum trait values.

3.7.4. Peroxidase activity
Bio-inoculants profoundly influenced the peroxidase activity with
the progression of drought stress, irrespective of the treatment (F =
3.25; p = 0.01**). As explained for other ROS enzymes, all treatments
performed more analogous during initial stress. Peroxidase activity
showed an increasing trend toward stress on the 7th and 10th days after
the exposure (Fig. 7). The maximal production of POD activity was
observed in co-inoculum FD48 + RABA6augmented CO51 (28.4 U
min–1 g–1 FW and 41 U min–1 g–1, respectively on 7th and 10th days of
the stress) and IR64 (24.45 U min–1 g–1 FW and 34.1 U min–1 g–1 FW).
Both genotypes followed a similar pattern of POD, indicating that the
co-inoculant played a significant role in maintaining plant integrity
during the unprecedented moisture stress. Furthermore, during re-
watering, the performance of the co-inoculant exceeded with maximum
recovery per se of 22% and 29% in CO5I and IR64, respectively.
3.8. Principle component analysis for ROS scavenging enzymes in rice
genotypes as influenced by moisture stress-tolerant PGP strains
The cumulative variance for genotypes CO51 (93.18%) and IR64
(93.34%) in the stressed environment by the first three axes with
Eigenvalue of more than 1.0 evidenced that the identified attributes
within the axes exhibited significant influence between the genotypes.
Principal component analysis (PCA) of biochemical attributes, such as
proline content and antioxidant enzyme activity, was not significantly
influenced on the 0th day of moisture stress in CO51 and IR64 geno-
types (Fig. S2). However, proline and antioxidant enzyme activity were
significantly influenced by the 7th and 10th days of stress influenced by
the plants primed with FD48 and a combination of FD48 + RABA6. A
similar pattern of ROS activity under moisture stress was followed in
both genotypes. Conversely, it is interesting to note that the recovery
percentage was higher in IR64 as compared with CO51. The PGPB in-
oculation in both genotypes increased the osmolyte proline production
and ROS enzyme activities during moisture stress and aided in drought
alleviation. During the recovery phase, lessening of ROS scavenging
enzymes in PGPBattenuated plants displayed its potential in the resi-
lience of moisture stress (Fig. S2).
From the heat map, it could be concluded that in CO51, the Row Z-
score (> 1.5) is maximum for all antioxidant enzymes and proline in
FD48 and FD48 + RABA6augmented plants on 7th and 10th days of
stress and showed enhanced moisture stress resilience. On the contrary,
the SOD activity with maximum Row Z-score (1.5) was found in
RABA6. The uninoculated control showed lower CAT, APX, and POD
levels (Fig. 8). In the recovery phase, it was evidenced that all PGPB-
inoculated plants exhibited reduced ROS enzymes and proline content
over the control.

Fig. 5. Superoxide Dismutase (SOD) activity induced in CO51 and IR64 under moisture stress condition.
T1 - Uninoculated control, T2 - B. altitudinis FD48, T3 - B. methylotrophicus RABA6 and T4 - FD48 + RABA6. Box and whisker plots represent minimum, lower
quartile, median, upper quartile, and maximum trait values.

7
S. Narayanasamy, et al. Microbiological Research 239 (2020) 126518

Table 3
Effect of PGPB strains at different levels of stress on induction of ROS enzymes in rice genotypes
Factors CAT SOD POD APX Proline

df F P F P F P F P F P

T 4 110.96 0.001 2.93 0.021 92.53 0.01 42.90 0.001 246.20 0.001
DAS 3 3376.45 0.002 1122.48 0.001 2906.03 0.01 798.86 0.001 8428.8 0.001
V 1 536.08 0.001 990.69 0.005 1006.86 0.02 25.07 0.001 2937.38 0.002
T x DAS 12 18.6 0.001 6.07 0.001 25.13 0.01 10.3 0.002 55.41 0.001
TxV 4 2.55 0.042 11.87 0.005 6.524 0.11 2.743 0.001 12.87 0.001
DAS x V 3 178.18 0.001 417.71 0.002 83.29 0.01 13.70 0.070 190.74 0.001
T x DAS x V 12 3.673 0.001 7.93 0.002 3.25 0.01 2.88 0.001 3.54 0.002
Error 159

T - Treatments, DAS - Days after Stress, V - Varieties. P - values indicate statistical significance (p < .05).

In the drought-susceptible genotype IR64, the Row Z-score (> 1)
was the maximum for the proline content. The SOD and APX activities
influenced the inoculation of FD48 and of the combination FD48 +
RABA6 priming in plants on 7th and 10th days of moisture stress,
whereas the priming of RABA6 in IR64 showed lesser production of
ROS enzymes than other PGP strains. The APX activity increased on the
7th and 10th days of stress in RABA6inoculated plants. During recovery,
the proline content and ROS enzymes activity reduced in all PGPB-
treated plants with the minimum Row Z-score (–1.5), indicating that
PGPB inoculation provided drought resilience by reducing the pro-
duction of ROS. Moreover, the inoculation of PGPB greatly influenced
the recovery phase (Fig. 9).
3.9. Influence of moisture stress-tolerant PGP strains on yield performance
3.9.1. Productive tillers and Panicle length
PGPB significantly increased the number of productive tillers in the
range of 10 to 15 hill–1 over the uninoculated control (Fig. S3). The co-
inoculants infused moderately tolerant CO51 recorded the maximum
productive tillers (16.5 hill–1), which was a 69% increase over the
control followed by FD48. Conversely, drought-sensitive IR64 showed
less productive tillers, 13 hill–1 when compared with CO51. Moreover,
the influence of the co-inoculant was significantly followed by FD48
(Fig. S3). The performance of RABA6 was the least.
Similarly, the panicle length was affected due to the induction of
moisture stress at the panicle initiation stage. However, the bacterial
inoculation increased the panicle length over the control. Co-inocula-
tion of FD48 + RABA6 recorded 24% and 30% increase in the panicle
length in CO51 and IR64, respectively, followed by axenic culture of
FD48 (13% and 24% in genotypes CO51 and IR64, respectively). The
results suggest that the effect of PGPB priming was more pronounced in
drought-sensitive IR64.
3.9.2. Grain weight
The total grain weight per plant differed significantly in all treat-
ments (Table 4). The plants exposed to moisture stress during the pa-
nicle initiation stage showed a steady decline in the grain weight. The
co-inoculants-treated plant recorded the highest grain weight of 27.46 g
plant–1 and 25.4 g plant–1 in CO51 and IR64, respectively, followed by
FD48. The uninoculated control registered the lowest grain weight
(18.31 g plant–1 and 16.52 g plant,–1 respectively). A similar trend was
observed with the number of grains per panicle and the co-inoculant
tended to increase the grain number under moisture stress. Moreover,
the co-inoculant significantly influenced 1000 grain weight (27.01 g
and 23.05 g for CO51 and IR64, respectively), followed by axenic FD48
over the mock (19 g and 16 g for CO51 and IR64, respectively). The co-
inoculation in CO51 (38%) and IR64 (39%) increased the grain yield
under induced stress.
3.9.3. Biological yield and harvest index
The biological yield of rice genotypes is represented in Table 4. The
data recorded for straw yield showed significant differences among
treatments and genotypes. The plants exposed to FD48 + RABA6 co-
inoculant showed 14% and 19% increase over the control, followed by
FD48 (11% and 8%) in CO51 and IR64, respectively. In general, the co-
inoculant significantly influenced HI, followed by FD48, as observed for
other yield parameters.

Fig. 6. Ascorbate peroxidase (APX) activity induced in CO51 and IR64 under moisture stress condition.
T1 - Uninoculated control, T2 - B. altitudinis FD48, T3 - B. methylotrophicus RABA6 and T4 - FD48 + RABA6. Box and whisker plots represent minimum, lower
quartile, median, upper quartile, and maximum trait values.

8
S. Narayanasamy, et al. Microbiological Research 239 (2020) 126518

Fig. 7. Peroxidase (POD) activity induced in CO51 and IR64 under moisture stress condition.
T1 - Uninoculated control, T2 - B. altitudinis FD48, T3 - B. methylotrophicus RABA6 and T4 - FD48 + RABA6. Box and whisker plots represent minimum, lower
quartile, median, upper quartile, and maximum trait values.

4. Discussion
Plants are continuously subjected to abiotic stress, such as salinity,
drought, and other environmental cues. Drought is one of the most
severe conditions that affect plant growth and development. Recently,
plant-associated microbial communities have received increased at-
tention for enhancing crop productivity and improving tolerance
against abiotic stress (Yang et al., 2009; Ilangumaran and Smith, 2017).
The underlying mechanisms include the production of plant growth
regulators (IAA, GA, ABA, cytokinin), ACC deaminase activity (Glick,
2012), ROS scavenging enzymes (Kaushal, 2019), and other PGP traits.
The presence of a unique microbiome and their metabolites in the
apoplastic space and inside plant cells highly decoys the abiotic stress
(Punitha, 2019). Furthermore, ABA alters the ion transport in guard
cells and mediates stomatal closure under moisture deficit conditions
(Kim et al., 2010). ABA alleviates stress by stimulating the production
of antioxidants, thereby enhancing protein transport (Li et al., 2014;
Zhou et al., 2014). Thus far, our investigation focused on the impact of

Fig. 8. Heat map for biochemical analysis of rice genotype CO51 under the droughtstressed condition T1 - Uninoculated control, T2 - B. altitudinis FD48, T3 - B.
methylotrophicus RABA6 and T4 - FD48+ RABA6.

9
S. Narayanasamy, et al. Microbiological Research 239 (2020) 126518

Fig. 9. Heat map for biochemical analysis of rice genotype CO51 under the drought-stressed condition.
T1- Uninoculated control, T2 - B. altitudinis FD48, T3 - B. methylotrophicus RABA6 and T4 - FD48+ RABA6.

two Bacillus spp. (Kumar et al., 2017; Punitha, 2019) obtained from rice
phyllosphere (B. altitudinis FD48) and apoplast (B. methylotrophicus
RABA6) on two contrary rice genotypes both as axenic culture and co-
inoculant under induced moisture stress. It is evident from the whole-
genome sequence of FD48 that genes responsible for drought tolerance,
including those encoding for acetoin and butanediol biosynthesis, ACC
deaminase, osmolytes production, heat and cold shock, chaperones, and
ABA biosynthesis, are well conserved (Kumaravel et al., 2018). The FT-
IR spectroscopic results unveiled that both RABA6 and FD48 strains
could produce ABA with a spectral match for standard ABA. Hence, we
assumed that priming these PGP bioinoculants cahoots moisture stress
alleviation via ABA modulation and triggers a well-coordinated ROS
scavenging system.
The experimental results suggested that attenuating two contrary
rice genotypes CO51 (moderately drought tolerant) and IR64 (drought-
sensitive) with the co-inoculant (FD48 + RABA6) increased the root
and shoot growth. The positive influence of the co-inoculant on growth
patterns could be attributed to the synergistic action of both strains
(Gusain et al., 2015). Furthermore, plants generally exhibit phenotypic
elasticity to reduce the harmful effects of the adverse environment by
reallocating assimilates from shoot to root under moisture stress con-
ditions (Rich and Watt, 2013). Similarly, in the present study, plants
inoculated with B. altitudinis FD48 showed more phenotypic plasticity
over the uninoculated control throughout the drought period.
R/S is often found to be increased under harsh environmental
conditions and has been reported as an essential trait for drought re-
silience (Xu et al., 2015; Govindaraj et al., 2010). Cultivars with a
higher R/S ratio signify a good source-sink relationship and are the
most preferred cultivars to screen resilience to moisture stress. The
present results revealed that the co-inoculant priming effect was much
pronounced with a higher R/S ratio in both moderately drought-tol-
erant and sensitive genotypes of CO51 and IR64, respectively. Such an
increase in the R/S ratio in the present investigation suggests that the
diversification of nutrients is well balanced in bio-inoculant- aug-
mented plants. Similarly, in the present study, the synergistic effect of
both FD48 and RABA6 in co-inoculant-treated plants contributed to a
better source-sink relationship, leading to the production of biomass in
both shoots and roots.
Similarly, RWC is the most vital marker to assess the water balance
in crop plants (Lata et al., 2011). In the present study, RWC of leaves

Table 4
Effect of PGP bacterial strains on yield components under moisture stress
Treatments Grain Yield plant-1 (g) Biological yield plant-1 (g) Harvest Index (HI)

CO51 IR64 CO51 IR64 CO51 IR64

T1 18.31 ± 0.163d 16.52 ± 0.435c 55.7 ± 0.390d 60.28 ± 0.433c 32.89 ± 0.368c 27.42 ± 0.850b
T2 25.57 ± 0.289b 22.50 ± 0.375a 62.64 ± 0.622b 65.28 ± 1.441b 40.84 ± 0.611ab 34.49 ± 0.410a
T3 22.61 ± 0.575c 20.86 ± 0.763b 58.59 ± 0.541c 65.34 ± 0.443b 38.60 ± 1.108b 31.16 ± 0.438ab
T4 27.46 ± 0.260a 23.70 ± 0.529a 64.88 ± 0.677a 72.26 ± 1.053a 42.33 ± 0.285a 35.22 ± 0.414a

T1 - Uninoculated control, T2 - B. altitudinis FD48, T3 - B. methylotrophicus RABA6, and T4 - FD48 + RABA6. Values are mean ( ± standard error) (n = 4) and values
followed by the same letter in each column are not significantly different from each other on the observation day as determined by DMRT (p ≤ 0.05).

10
S. Narayanasamy, et al.
decreased in both inoculated and uninoculated plants of CO51 and IR64
under moisture stress. Nevertheless, the inoculation of FD48 could
protect the plants by stabilizing their RWC during water deficit periods.
Similarly, inoculation of Bacillus sp. in Zea mays L. maintained their
RWC content and leaf water potential over mock plants under drought
stress (Vardharajula et al., 2011). Furthermore, PGPB profoundly in-
fluenced the chlorophyll and carotenoid levels over mock plants under
all levels of stress evaluated. The enzyme ACC deaminase in bio-in-
oculants suppressed the synthesis of ethylene under moisture stress,
thereby reducing chlorophyll decay. This, in turn, prevented senescence
and increased photosynthetic efficiency. For example, Arshad et al.
(2008) reported that ACCD-producing bacteria extensively suppressed
the ethylene synthesis and prevented leaf senescence owing to en-
hanced ethylene production and reduced chlorophyll degradation.
Moreover, previous studies claimed that PGPB-inoculated plants in-
creased the chlorophyll content and led to the accumulation of ascorbic
acid, which, in turn, acted as an antioxidant in plants under moisture
deficit (Patel and Saraf, 2013; Nadeem et al., 2007).
Osmolytes prevent cell membrane disruption and maintain mem-
brane stability under drought stress. Accumulation of proline stabilizes
membrane proteins and cellular contents (Yadav et al., 2004). In the
present investigation, FD48 and RABA6 contributed to significant ac-
cretion of proline in both the rice genotypes and subsequent restoration
was observed during recovery. We propose that PGPB mediate the
upregulation of the proline biosynthetic pathway, which, in turn,
maintains the cellular water content and protects membrane proteins
(Yoshiba et al., 1997).
Furthermore, drought stress is related to osmotic and oxidative
damage, which is related to the overproduction of ROS, including OH−,
O2− and H2O2. Scavenging these free radicals by low molecular weight
enzymatic antioxidants ensures plant survival (You and Chan, 2015).
Under induced moisture stress, H2O2 induces POD, CAT, and APX,
which, in turn, converts H2O2 into H2O and O2. The PGP bacterial in-
oculation significantly affected CAT, APX, and POD activities, irre-
spective of the drought-sensitive nature of test genotypes. However, it
varied with bacterial strains employed, namely FD48 and RABA6. The
complementary effect of both strains as co-inoculant pronounced the
positive influence. Similar results on ROS production by plant-asso-
ciated microbes (Pseudomonas sp.) were previously reported in rice
(Gusain et al., 2015). Compared with all H2O2− scavenging enzymes,
CAT was highly triggered in both tolerant and sensitive rice genotypes.
Another enzyme, SOD, plays an integral role in catalyzing the dis-
mutation of O2−. An increase observed in SOD activity following PGP
co-inoculation agrees with the results of previous studies under induced
moisture stress (Punitha, 2019). The overall results suggest that
priming with a combination of FD48 and RABA6 triggers ROS-scaven-
ging cascade, thereby assuaging uncontrolled oxidation under moisture
stress.
Seed priming with PGPB inoculants improved the yield perfor-
mance. Moisture stress affected the rice yield by reducing the grain
filling period, disrupting the leaf gas exchange, and reducing the size of
the source and sink tissues (Farooq et al., 2009). All seed priming
treatments improved the panicle length, number of grains, and 1000-
grain weight as compared with the control under all moisture regimes.
However, the performance of co-inoculant-primed plants far exceeded
that of the control, with 24% and 30% increase in the panicle length
over the control for CO51 and IR64, respectively. Our present in-
vestigation was concomitant with the previous reports where the en-
hanced plant growth and yield performance of treated plants might be
associated with the enhanced uptake of plant nutrients (Islam and
Hossain, 2012) and alterations in the endogenous phytohormone levels
in plants (Kurepin et al., 2015). Water stress at the reproductive stage
greatly affects the plant growth than the water stress at the vegetative
stage (Liu et al., 2006). Moisture stress during the reproductive phase of
rice genotypes profoundly reduced the grain yield. However, Punitha
et al. (2019) reported that co-inoculant priming increased the grain
11
Microbiological Research 239 (2020) 126518
yield per plant (26.58% and 46%, respectively) in CO51 and IR64 as
compared to the uninoculated control; the PGPB-treated plants dis-
played increased grain weight under drought stress as compared with
the control.
Similarly, PGPB inoculation demonstrated a direct impact on 1000
grain weight in rice under moisture stress (Vasudevan et al., 2002) as
observed in the present study that 1000-grain weight increased in
plants augmented with the co-inoculant (43% and 39% over control) in
CO51 and IR64 genotypes, respectively. Furthermore, HI revealed the
proportion of assimilate distribution between economic yield and total
biomass (Donald and Hamblin, 1976). According to Zhang et al. (2008),
HI is correlated with water use efficiency (WUE) and grain yield in rice.
Barnabas et al. (2008) reported that rice plants inoculated with a
Pseudomonas sp. registered the highest HI over the control. Similarly, in
the present investigation, co-inoculantsprimed plants recorded the
highest HI.
Furthermore, the present investigation revealed that PGP priming in
IR64, generally considered as a drought-sensitive genotype, manifested
moisture stress tolerance and performed better in terms of biochemical,
physiological, and yield attributes. Moreover, the results vividly sug-
gested that the seedlings from primed seeds coped with harsh en-
vironmental stresses by cross-tolerance or induced tolerance. Initial
imbibition of PGP bacteria might have promoted mitochondrial devel-
opment during priming. At induced moisture stress, cellular processes,
such as de-novo synthesis of nucleic acid and protein, ATP production,
DNA repair, and antioxidant formation, are triggered in PGP-primed
seeds and seedlings (Chen and Arora, 2013). Another key mechanism of
augmenting energy metabolism by PGP B. altitudinis FD48 and B. me-
thylotrophicus RABA6 might be ABA-modulated ROS regulation that
prevented oxidative stress (Watkins et al., 2017).
5. Conclusion
The present study provides evidence that PGP moisture stress-tol-
erant B. altitudinis FD48 and B. methylotrophicus RABA6 primed in two
contrary rice seeds (CO51 and IR64) modulated the ROS regulation
under induced moisture stress. As a co-inoculant, the two strains sig-
nificantly influenced the R/S (20% increase) and HI (28%) under
conditions of terminal moisture stress at the panicle initiation stage.
Moreover, seeds primed with the co-inoculant significantly influenced
the antioxidant enzymes, namely CAT, POD, SOD, and APX, irrespec-
tive of the genotypic variation. Furthermore, complementary ABA sig-
naling influenced the source-sink relationship by enhancing photo-
synthetic efficiency. Seed priming with these PGP Bacillus strains could
potentially assuage an unprecedented moisture deficit. The results also
provided significant insights into the impact of microbe-induced sys-
temic tolerance (MIST) in contrast to rice genotypes including moder-
ately tolerant CO51 and susceptible IR64. It may be adopted as “smart
agricultural practices” if integrated with other sustainable options for
clima rice farming. However, further field trials in different drought-
prone environments are required.
Acknowledgement
This study was supported by the Ministry of Human Resource
Development, Government of India through MHRD-FAST-CoE
(F.No.5–6/2013-TSVII) sanctioned to SU.
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