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OPEN Evaluation of plant growth

promotion properties and induction
of antioxidative defense
mechanism by tea rhizobacteria
of Darjeeling, India
Chandrima Bhattacharyya1, Srimoyee Banerjee2, Udita Acharya2, Aroni Mitra2, Ivy Mallick1,
Anwesha Haldar3, Shyamalina Haldar4, Anupama Ghosh 2* & Abhrajyoti Ghosh 1*

A total of 120 rhizobacteria were isolated from seven different tea estates of Darjeeling, West
Bengal, India. Based on a functional screening of in vitro plant growth-promoting (PGP) activities,
thirty potential rhizobacterial isolates were selected for in-planta evaluation of PGP activities in rice
and maize crops. All the thirty rhizobacterial isolates were identified using partial 16S rRNA gene
sequencing. Out of thirty rhizobacteria, sixteen (53.3%) isolates belong to genus Bacillus, five (16.6%)
represent genus Staphylococcus, three (10%) represent genus Ochrobactrum, and one (3.3%) isolate
each belongs to genera Pseudomonas, Lysinibacillus, Micrococcus, Leifsonia, Exiguobacterium, and
Arthrobacter. Treatment of rice and maize seedlings with these thirty rhizobacterial isolates resulted
in growth promotion. Besides, rhizobacterial treatment in rice triggered enzymatic [ascorbate
peroxidase (APX), catalase (CAT), chitinase, and phenylalanine ammonia-lyase (PAL)], and non-
enzymatic [proline and polyphenolics] antioxidative defense reactions indicating their possible role in
the reduction of reactive oxygen species (ROS) burden and thereby priming of plants towards stress
mitigation. To understand such a possibility, we tested the effect of rhizobacterial consortia on biotic
stress tolerance of rice against necrotrophic fungi, Rhizoctonia solani AG1-IA. Our results indicated
that the pretreatment with rhizobacterial consortia increased resistance of the rice plants towards the
common foliar pathogen like R. solani AG1-IA. This study supports the idea of the application of plant
growth-promoting rhizobacterial consortia in sustainable crop practice through the management of
biotic stress under field conditions.

A diverse population of soil bacteria usually inhabits plant rhizospheres. A subset of these is beneficial to the
plants in terms of promoting their growth. These bacteria, therefore, are very commonly referred to as plant
growth-promoting rhizobacteria (PGPR) and serve as potential environment-friendly substitutes of chemi-
cal ­fertilizers1,2. Phytostimulating rhizobacteria, in general, do not show host specificity and can exhibit their
growth-promoting features when associated with a broad range of ­hosts3. This makes them very suitable as
biofertilizers as their application can be extended to multiple plants that might not serve as their natural hosts.
In the case of biocontrol, for endophytic and mycorrhiza helper PGPR, however, genotype-dependent specificity
in PGPR- plant cooperation has been r­ eported3,4. PGPR contributes to the general well-being of the associated
plants either by direct or indirect ­manner1. In the direct mechanism, PGPR stimulates plant growth by fixing
atmospheric nitrogen, solubilizing soil insoluble phosphates and potassium, making iron available for the host
plant, and finally, by producing different phytohormones to support plant g­ rowth1. The indirect mechanism of

1
Department of Biochemistry, Bose Institute, Centenary Campus, P1/12, C.I.T. Road, Scheme VIIM,
Kolkata 700054, India. 2Division of Plant Biology, Bose Institute, Centenary Campus, P1/12, C.I.T. Road, Scheme
VIIM, Kolkata 700054, India. 3Department of Geography, University of Calcutta, 35 Ballygunge Circular Road,
Kolkata 700019, India. 4Department of Biochemistry, Asutosh College, University of Calcutta, Kolkata 700026,
India. *email: ghosh.anupama1982@gmail.com; aghosh78@gmail.com

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PGPR involves the protection of plants from biotic and abiotic stresses. The major indirect mechanisms adopted
by PGPR are hydrolytic enzyme production, exo-polysaccharide production, bioremediation of heavy metals, and
stimulation of induced systemic resistance (ISR)1. In plants, PGPR activates the immune response by stimulating
an induced systemic resistance (ISR) through strengthening the physical and biochemical responses of the plant
cell towards environmental ­stresses5,6. Association of PGPR with host plants has often been found to enhance the
biosynthesis of defense-related molecules in the later. The elevated levels of the defense proteins thus provide the
host plant a better chance of survival under stress ­conditions5 6. Both biotic, as well as abiotic stresses, impose
a number of different physiological changes in plant cells, including the generation of reactive oxygen species
(ROS)7. Accumulation of a high concentration of ROS in plant cells leads to oxidative damage and results in
the disruption of cellular ­homeostasis7. Plant cells are equipped with sophisticated antioxidative mechanisms
involving antioxidative defense enzymes like Ascorbate peroxidase (APX), Catalase (CAT), peroxidase (PO),
superoxide dismutase (SOD), glutathione reductase, glutathione S-transferase and guaiacol p ­ eroxidase8. These
enzymes are involved in scavenging and transforming ROS into non-toxic end products and thereby protect
cells from oxidative ­damage8. Besides, plant cells also produce various antioxidant molecules such as carotenoids
and phenylpropanoids to overcome oxidative ­damage9. PGPR mediated ISR primes host plants towards resisting
pathogen invasion through the production of defense-related antioxidative enzymes and m ­ olecules5.
Tea (Camellia sinensis (L.) O. Kuntze.) is an economically important perennial crop plant mostly cultivated
in the North-Eastern part in India. Extensive use of agrochemicals to meet the global requirement of tea resulted
in an alteration of the microbial community associated with the tea ­plants10,11. Interestingly, little is known
regarding the tea rhizosphere microbiome of Indian tea. Several culture-dependent analyses have shown that
the tea rhizosphere is constituted of a variety of metabolically versatile PGPR that has the potential to be used
as ­biofertilizer12–16. Moreover, a few of these rhizobacteria were also found to act as biocontrol ­agents12,13,17,18.
However, a lack of systematic analysis combining different rhizobacterial isolates rendered only a little progress
in the development of consortia-based biofertilizer formulations.
In the present study, we report isolation, characterization, and plant growth-promoting (PGP) activities
of 30 rhizobacteria isolated from the rhizospheric soils of seven tea estates in Darjeeling, West Bengal. All the
rhizobacterial isolates were tested for their PGP activities in two major crop plants, rice, and maize. After tea,
rice and maize together cover the majority of the cultivable crop area in Darjeeling ­region19 (Source: https:​ //darje​
eling​.gov.in/agric​ultur​e.html). However, their yields are significantly affected due to several factors, including
local agro-climatic conditions and conventional agricultural ­practices19,20. The soil in this region is mostly acidic,
leading to low availability of essential base cations and ­phosphate21,22. Moreover, the use of local low yielding
seed varieties contributes towards the reduced total production of the food grains. Decreasing productivity of
these crops raises further concern about food security and the social livelihood of the local farming population.
Application of PGPR might come to a great rescue under such a circumstance. PGPR have well-established
properties of restoring the bioavailability of nutrients in addition to promoting crop growth. Besides, we also
evaluated the effect of rhizobacterial treatment on the production of antioxidative defense enzymes and mol-
ecules in rice plants. Furthermore, we took a consortia based approach to evaluate the effect of rhizobacterial
treatment on rice plants in inducing resistance towards infection with necrotrophic fungi Rhizoctonia solani
AG1-IA leading to sheath blight disease.

Materials and methods

Geography of the terrain of the study sites. The Darjeeling District is mostly set over the Himalayan
hill region in the north, and Terai plains in the south with the local relief varying from 100 m up to 3,636 m
in Sandakphu peak intersected largely by tributary streams of the Teesta, Mahanadi and Jaldhaka rivers. The
amount of precipitation influences the slope stability and vegetation of the region. The southern slopes receive
4,000 to 5,000 mm rainfall annually while the northern leeward slopes 2,000–2,500 mm, concentrating over 218
rainy days annually. Temperature isolines have a wider range both seasonally as well as over the district. The
vegetation sharply progresses from the moist tropical deciduous forests from 300–1,000 m to evergreen montane
forests to 3,000 m, and temperate forests above 3,000 m. Geologically, the district comprises of relatively recent
rock structures. The sub-Himalayas are made up of Siwalik systems composed of mudstones, sandstones, shale,
and conglomerates, followed by lower Godowana systems or Damuda series that comprise the outer region. In
contrast, the Daling series group of crystalline rocks composed of hard-grained sandstone chlorite shales, phyl-
lites and schists associated with quartzites form towards the inner H­ imalayas23,24. Of the 2,417 km2 area of the
Darjeeling hills, 18% is covered under the tea plantations. The tea gardens across the district are mostly situated
on steep slopes where well-drained podzolic soils are in abundance.

Sampling of tea rhizosphere soil. The rhizospheric soil samples of tea plants were collected in May 2016
from seven different tea estates of Darjeeling, India. Detailed site characteristics are provided in Table 1 and
Fig. 1. Soil samples were collected from the rhizosphere area at 20 ± 2 cm depth. The area of the tea estates was
divided into three zones, from each zone samples were collected from four randomly selected healthy tea plants
containing roots and root-adhered soil. Finally, all four rhizosphere soil samples were pooled, resulting in a total
of three composite rhizosphere soil samples from each tea estate. All the samples were immediately stored at 4 °C
and subsequently either directly used for chemical analysis and microbiological analysis or stored at − 80 °C for
molecular biological studies.

Isolation of tea rhizobacteria. The tea rhizobacterial strains were isolated using enrichment media fol-
lowing methods described ­previously25. Further functional screening of the isolated rhizobacteria was per-
formed in Pikovskaya Agar (PA), Burks Agar (BA), Jensen’s Agar (JA), Azotobacter Agar (AA) (HiMedia Labo-

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Coordinates (latitude and Total soil bacterial count (CFU ­g−1 Number of isolates (number used
Sampling sites (tea estates) longitude) Soil type (USDA) Soil pH range) for downstream analysis)
Tukvar tea estate 27° 5′42.01ʺ N; 88°15′30.24ʺ E Sandy loam, Silty 4.7 3 × 104 − 3 × 106 15 (3)
Barnesbeg tea estate 27° 6′17.23ʺ N; 88°15′49.92ʺ E Sandy loam 4.6 5 × 104 − 6 × 106 17 (5)
Happy Valley Tea estate 27° 3′3.81ʺ N; 88°15′36.12ʺ E Sandy loam, Silty 4.2 2 × 104 − 3 × 106 13 (3)
Maharani tea estate 26°55′48.36ʺ N; 88°17′55.66ʺ E Sandy loam and clay 4.6 5 × 104 − 6 × 106 16 (6)
Rohini Tea estate 26°50′22.42ʺ N; 88°17′23.98ʺ E Sandy loam and clay 4.5 6 × 104 − 7 × 106 17 (4)
Makai Bari Tea estate 26°50′37.67ʺ N; 88°16′0.85ʺ E Sandy loam 5.0 1 × 105 − 2 × 107 22 (5)
Long view tea estate 26°49′1.75ʺ N; 88°15′39.05ʺ E Sandy loam 5.2 2 × 105 − 2 × 107 20 (4)

Table 1.  The location, soil type and number of total bacteria isolated from tea rhizosphere soils collected from
the sampling sites in the tea estate of Darjeeling.

Figure 1.  The geographical location of the sampling tea estates in the Darjeeling district of West Bengal, India.
Coordinates of the sampling tea estates and description of the stations are presented in Table 1.

ratories, India) to screen their ability to show in vitro plant-growth promotion activities. Upon inoculation, the
agar plates were incubated either at 30 °C or 37 °C for 24–48 h. The rhizobacterial colonies appeared on the plates
were sub-cultured and store at − 80 °C in minimal medium containing 20% glycerol. The morphology and gram-
characteristics of the selected rhizobacteria were ascertained using a light microscope (40X, Euromex Oxion
trinocular Microscope, Netherlands).

Molecular identification of the isolates. To identify rhizobacterial isolates, partial amplification of the
16S rRNA gene was carried out using universal primer pair 27F (5′-AGA​GTT​TGATCMTGG​CTC​AG-3′) and
1492R (5′-TAC​GGY​TAC​CTT​GTT​ACG​ACTT-3′) and genomic DNA as template following protocols described
­previously26,27. The amplified partial rRNA gene was sequenced in ABI 3500 XL Genetic Analyzer with primers
27F, 515F, or 1492R, as described ­previously28.
The evolutionary history of rhizobacterial isolates was inferred using Neighbor-Joining m ­ ethod29. The per-
centages of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates)
are shown next to the b ­ ranches30. The evolutionary distances were computed using the Maximum Composite
Likelihood ­method31 and are in the units of the number of base substitutions per site. Evolutionary analyses
were conducted in MEGA ­X32. 16S rRNA gene sequence of Sulfolobus solfataricus P2 was used to assign an out-
group species.

Phosphate solubilization. For screening of phosphate solubilizing activity of the rhizobacterial isolates,
Pikovskaya’s agar plates were used with certain ­modifications33. The reaction was considered positive when a
clear halo surrounding the spot inoculated bacterial colonies were observed after seven days of incubation at
30 °C. Furthermore, the ability of the strains to solubilize inorganic tri-calcium phosphate was quantitatively
assessed using the malachite green method as described p ­ reviously14.

Siderophore production. Chrome azurol sulfonate (CAS) agar solid medium was used to screen sidero-
phore production by the rhizobacterial ­isolates34. The reaction was considered positive when an orange halo
surrounding the bacterial colony appeared due to the removal of iron from CAS by the siderophore. For the

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quantitative estimation of siderophore, CAS-shuttle assay was performed following the protocol described
­previously28. Briefly, the quantification of siderophore production was estimated by following the formula: %
siderophore unit = [(Ar−As)/Ar] × 100, where, Ar = absorbance of reference at 630 nm (minimal medium + CAS
assay solution), and As = absorbance of the sample at 630 nm.

IAA production. Production of indole-3-acetic acid (IAA) by the rhizobacterial isolates was estimated by
growing the isolates in M9 medium, supplemented with or without L-tryptophan (100 µg ml−1), and incubated
for 48 h at 30 °C. After incubation, 1 ml of supernatant was mixed vigorously with 4 ml of Salkowski’s reagent
(150 ml conc. ­H2SO4, 250 ml ­H2O, 7.5 ml 0.5 M ­FeCl3. ­6H2O), incubated for 30 min and the absorbance was
measured at 520 nm with the help of a spectrophotometer (Halo XB-10 by Dynamica Scientific Ltd., United
Kingdom). The concentration of IAA produced by the rhizobacterial isolates was determined from a standard
curve generated using a standard solution of commercial IAA (Sigma–Aldrich, Germany).

Ammonia production. To detect the production of ammonia, the rhizobacterial isolates were grown in
4% peptone broth and incubated for seven days at 30 °C. Following incubation, 0.5 ml of Nessler’s reagent was
added to the bacterial suspension. The development of brown to yellow color indicates ammonia production.
The absorbance was measured at 450 nm using a spectrophotometer. Furthermore, quantitative estimation of
the amount of ammonia production by the rhizobacterial isolates was carried out and compared with a standard
curve generated using a standard ammonium sulphate solution.

Effect of the isolates on the growth of fungal cultures. To assess whether the isolates exhibit any
antifungal activity, their effects on the growth of different fungal cultures were measured using dual-culture
­technique35. On a single potato dextrose agar (PDA) plate, both the fungal strain and an individual isolate were
inoculated on two halves. These PDA plates were then incubated at 30 °C for three days. The effect of each isolate
on the growth of the fungal culture was then assessed by measuring the spread of the culture towards the bacte-
rial colony. The less the spread is, the more the antifungal effect of the isolate is. Two different fungal cultures,
Rhizoctonia solani AG1-IA and Ustilago maydis SG200, a necrotrophic and a biotrophic phytopathogen respec-
tively, were used in this assay.

Protease activity. The protease activity of the bacterial isolates was determined using the skim milk agar
medium as described ­previously25. The isolates were spot inoculated on skim milk agar medium, and after two
days of incubation at 30 °C, proteolytic activity was assessed by clear zone around the colonies.

Cellulase activity. The rhizobacterial isolates were screened for cellulase production by plating them onto
M9 agar media supplemented with 10 g l−1 carboxymethyl cellulose (CMC) (Sigma-Aldrich, Germany). After
48 h incubation at 30 °C, plates were flooded with congo red dye, and the clear halos formed surrounding the
colonies indicated their cellulolytic ­activity36.

ACC deaminase activity. To detect whether the rhizobacterial isolates were able to produce the ACC
deaminase enzyme, the organisms were streaked onto M9 minimal media containing 1-aminocyclopropane-
1-carboxylic acid (ACC) as the sole source of nitrogen. The plates were incubated at 30 °C for 48–72 h. An abun-
dant growth on the medium indicated the positive result for a corresponding rhizobacterial isolate.

Effect of rhizobacterial isolates on the growth of rice and maize seedlings. To assess the effect
of rhizobacterial isolates on rice seedlings, surface-sterilized rice seeds (Variety: IR64) were sown in sterile pots
(20–25 seeds/pot) [Pot size (L × W × H): 20 cm × 20 cm × 16 cm] filled with sterilized soilrite (Keltech Energies
Limited, Bangalore, India) and allowed to germinate in the dark for about five days. Following germination, the
pots were transferred to light, and the rhizosphere of the rice seedlings was treated with freshly grown cultures
of about ­108–109 cfu ml−1 of individual rhizobacterial i­solates14,37. Rice seedlings treated with the sterile growth
media for the culture of the rhizobacterial isolates were used as a negative control. One rhizobacterial isolate
was tested in 16 different pots. Four of these 16 pots were used for the experiment of assessing the effect of indi-
vidual rhizobacterial isolates on the growth of rice seedlings. Different growth parameters like the root length,
shoot length, dry weight, and wet weights of 4 randomly selected rice seedlings from each of the four pots were
measured at 21 days post-treatment and compared with that of the control seedlings. These 16 rice seedlings
in total (four from each of the four pots) represented 16 replicates of the experiment. For the measurement of
chlorophyll content following 21 days post-treatment, 16 rice seedlings were randomly selected from the same
four pots, as mentioned in the case of growth parameter assessment experiment. The seedlings in the remaining
12 pots were used at 14 days post-treatment for assaying the antioxidative enzymatic activities, the total contents
of proline and phenols in three sets of four pots each. The plants were regularly irrigated. Both the control and
treated seedlings were maintained in a phytochamber in a 16 h/8 h light/dark cycle. Day and night temperatures
were maintained at 30 °C and 24 °C, respectively, with average humidity 70% throughout the experiment.
To assess the effect of rhizobacterial treatment on the growth parameters of maize seedlings (Variety: Early
Golden Bantam), a previously described methodology was adopted with certain m ­ odifications14. Briefly, after ger-
mination, rhizospheres of 7-day-old seedlings were treated with freshly grown cultures of about ­108–109 cfu ml−1
of individual rhizobacterial isolates. Similar to rice, each of the rhizobacterial isolates were tested for growth
promotion in 16 replicates. In total, 16 pots were used for the treatment of each of the rhizobacterial isolate,
each pot containing two maize seedlings. While one of the treated seedlings in each pot was used for assessing

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different growth parameters like shoot length, root length, dry weight, and wet weight, the other seedling was
used to assess the total chlorophyll content following 15 days post-treatment. Control and treated maize plants
were grown in a phytochamber in a 15 h/9 h light/dark cycle, and the temperature was maintained at 28 °C and
20 °C with relative humidity 40% and 60% during light and dark periods, respectively.

Measurement of chlorophyll. Chlorophyll a, and chlorophyll b contents were measured in leaves of soil
rite grown, and PGPR inoculated rice and maize plants. Chlorophyll pigments were extracted from fresh leaf
­ reviously38, and the quantification was carried out
samples in 80% acetone following the procedures described p
following the method described by Porra et al.39.

Assessment of antioxidative enzymes, proline, and total phenol contents in treated rice
plants. To evaluate the triggering of antioxidative defense response in rice plants upon PGPR treatment, a
series of plant defense-related enzymatic assays and estimation of non-enzymatic molecules were performed.
Plant samples either treated with growth media only or individual rhizobacterial isolates were collected 14 days
post-treatment and washed adequately to remove traces of soil rite. Following this, the plant samples were
crushed in liquid nitrogen and stored at − 80 °C till further use.

Plant defense‑related enzyme assay. Root and shoot proteins were extracted from frozen crushed
samples in 3 mL of extraction buffer containing 100 mM K-phosphate buffer (pH 7.0), 2 mM EDTA, 20 mM
ascorbate, and 0.1% (v/v) Triton X-100 for catalase (CAT) and ascorbate peroxidase (APX) activities or 100 mM
Citrate–phosphate buffer (pH 5.5) for chitinase or 100 mM Na-Borate buffer (pH 8.8) containing 5 mM β- mer-
captoethanol, 2 mM EDTA and 10% acid-washed polyvinylpolypyrrolidone (PVP) for Phenylalanine ammonia-
lyase (PAL). The homogenate was then centrifuged at 12,000 × g for 20 min at 4 °C. The supernatant was used for
total protein and enzymatic assays.
Root and shoot ascorbate peroxidase (APX) activities were measured at 290 nm following a method described
­previously40. 20 μL enzyme extract was added to a 3 mL APX assay mixture containing 50 mM K-phosphate
buffer (pH 7.0), 0.1 mM H ­ 2O2, and 0.5 mM ascorbate. The amount of ascorbate oxidized was calculated using
the extinction coefficient = 2.8 mM−1 cm−1. All assays were performed in triplicate.
The total root and shoot catalase (CAT) activities were estimated based on the rate of ­H2O2 consumption at
240 nm41. The assay mixture of 3 mL contained 100 mM K-phosphate buffer (pH7.0), 0.1 mM EDTA, 0.1% H ­ 2O2,
and 20 μL enzyme extract. After addition of the enzyme extract to the reaction mixture, a decrease in H ­ 2O2 levels
was measured at 240 nm with a Halo XB-10 spectrophotometer and quantified by using the extinction coefficient
(0.036 ­cm2 µmol−1). All assays were performed in triplicate.
Root and shoot chitinase activities were measured following the protocol described previously with slight
­modifications42. Briefly, 200 µl of crude extract was incubated with 400 µl of 100 mM citrate–phosphate buffer
(pH 5.5) and 200 µl of 1% colloidal chitin at 37 °C for 1 h in a water bath. The reaction was then arrested by add-
ing 1.5 ml 3, 5-dinitrosalicylic acid (DNS) reagent, followed by heating at 100 °C for 10 min in a boiling water
bath. After cooling the reaction mixture to room temperature, the colored solution was centrifuged at 10,000 × g
for 10 min, and the absorbance of the supernatant was measured at 540 nm using a UV- visible spectrophotom-
eter (Halo XB-10). One unit of the chitinase activity was defined as the amount of enzyme that catalyzed the
release of 1 µmol/ml/min of reducing sugar under the assay conditions. All assays were performed in triplicate.
Phenylalanine ammonia-lyase (PAL) activity of the root and shoot extracts were measured following the
method described p ­ reviously43. 200 µl of the enzyme extract was preincubated with 30 mM Na-Borate buffer (pH
8.8) at 30 °C for 10 min, and then 100 µM phenylalanine was added as substrate making the total reaction volume
of 3 ml. The reaction mixture was then incubated at 37 °C for 70 min. The reaction was stopped with 100 µl of
6 N HCl, and the absorbance was measured at 290 nm using a Halo XB-10 UV–visible spectrophotometer. The
specific activity of PAL was defined as the amount of enzyme required for the formation of 1 mol of product in
1 s per mg of protein under the assay conditions.

Estimation of total proline. Proline was estimated using a method described previously with slight
­modifications44. Briefly, 500 µl of 3% sulfosalicylic acid was added to 100 mg of crushed samples prepared from
the PGPR-treated and non-treated control rice plants. The mixture was then centrifuged for 10 min at room
temperature at 10,000 × g, and the supernatant was used for further analysis. 100 µl of supernatant was mixed
with 100 µl of 3% sulfosalicylic acid, 200 µl acid-ninhydrin, and 200 µl of glacial acetic acid in a microfuge tube.
The reaction mixture was mixed thoroughly and was further incubated for 1 h at 100 °C. The reaction was termi-
nated by placing the microfuge tube on ice followed by proline extraction with 1 ml toluene. The chromophore-
containing layer was collected and kept at room temperature for 15 min before measuring the absorbance at
520 nm using a spectrophotometer. The proline concentration was determined using a standard curve, and the
results were expressed as µg/gram fresh weight.

Total phenolic contents. The total phenolic content (TPC) was estimated using a modified Folin–Ciocal-
teu ­method45. Briefly, the total phenolics were extracted twice (each for 10 min at 70 °C with occasional mixing)
with 50 ml of hot demineralized water from 1.5–2 g of air-dried leaves. 500 μl of extracts in demineralized water
was mixed with 2.5 ml of Folin- Ciocalteu reagent (0.2 N). After 5 min, 2 ml of Na2CO3 solution (75 g/l) was
added and kept in the dark for 120 min, before measuring the absorbance at 760 nm against a blank using a
Halo XB-10 UV–visible spectrophotometer. The total phenolic contents were calculated based on the calibration
curve of gallic acid and expressed as gallic acid equivalents (GAE) in milligrams per gram of the sample.

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Evaluation of the effect of multispecies rhizobacterial consortia on the susceptibility of rice

plants towards R. solani AG1‑IA infection. To assess the effect of rhizobacterial consortia, if any, in the
disease resistance of host plants, rice seedlings pretreated with rhizobacterial consortia were infected with foliar
pathogen Rhizoctonia solani AG1-IA that causes sheath blight infection in rice. To develop multispecies rhizo-
bacterial consortia, 30 rhizobacterial isolates were randomly distributed in six different groups, each consisting
of five isolates representing different genera/species (Table S1). For the pretreatment of rice seedlings with the
rhizobacterial consortia, each of the individual isolates of the consortia is inoculated in 5 ml Luria Bartoni (LB)
broth and grown at 30 °C with constant agitation at 160 rpm till an O ­ D600 of the culture reached 0.6. Following
this, equal volumes of each of the rhizobacterial suspension cultures were mixed to achieve a final concentration
of ­108–109 cfu ml−1. 1 ml of the resulting culture was used to treat the rhizospheric region of the five days old
IR64 variety of rice seedlings. The treated plants were grown at 24 °C under 16 h/8 h day/night cycle for 3 days.
Following this, the seedlings were inoculated with R. solani AG1-IA. The inoculation was done by incubating
about 1 cm length of the rice seedlings with potato dextrose agar plaques containing both sclerotia and mycelia
of R. solani AG1-IA. The rice seedlings thus infected with the fungus were grown further for two days following
which the infection symptoms were scored as the length of the chlorotic lesions in the infected area of the leaves.
The severity of the infection was then accessed as the disease index (DI). The DI is the measure of the spread of
infection beyond 1 cm inoculation area. The average spread of infection in the case of rice seedlings that were
untreated with any rhizobacterial consortia (control) was considered as the standard. In the case of all of the
rhizobacterial consortia treated rice seedlings (treated), the spread of the infection was presented as the fraction
of that of the control. The DI, therefore, in case of the treated R. solani AG1-IA infected seedlings was calculated
as below:
   
DItreated = Total lesion length in cm−1 treated /Average of Total lesion length in cm−1 control .

The DI of the control is considered as 1. The DI of all the treated samples thus calculated was then compared
with that of the control to evaluate the effect of the respective consortia on inducing resistance to disease sus-
ceptibility in rice.

Statistical analysis. For each of the investigated biochemical parameters from control and treated samples,
three separate replication sets were conducted. All the experimental measurement values were expressed as
means of three measurements [± standard deviation (SD)]. The significance of the differences between the mean
values of control and treated plant samples were statistically evaluated by two-tailed t-test at P ≤ 0.05 using the
Windows 2016/Microsoft Excel 2013 computer package.

Nucleotide sequence accession numbers. The partial 16S rRNA gene sequences of the rhizobacterial
isolates reported in this study were submitted to GenBank under the accession numbers: MT436081–MT436110.

Results and discussion

Isolation and identification of rhizobacteria from tea rhizosphere. Soil microorganisms play a
crucial role in plant health and development. Moreover, they contribute immensely to the agricultural produc-
tion of different crops. In the district of Darjeeling, tea is cultivated as the major cash crop. Besides tea, a number
of other crops such as rice, maize, wheat, mustard, millet, ginger, orange, large cardamom, and vegetable crops
are ­cultivated19 (Source: https​://darje​eling​.gov.in/agric​ultur​e.html). Rice and maize are the most important food
grain crops grown in this region. However, because of the acidic nature of the soil of this region, crop cultivation
becomes increasingly difficult. Agrochemicals, including N fertilizers, make the situation even more compli-
cated as they further assist soil acidification. In the slightly acidic soils of Darjeeling district (4.2 < pH < 7.0), base
cations such as calcium (Ca), magnesium (Mg), potassium (K) and sodium (Na) are replaced by protons and
subsequently leached from the rhizosphere zone of the crops and thereby become u ­ navailable22. Previous stud-
ies have shown that the lower soil availability of Ca, Mg, K, and P can be detrimental towards crop growth and
development in acidic ­soils46,47. Moreover, crop production is shown to be already limiting at pH values below
5.5–6.548. Under these agroclimatic conditions, plant growth-promoting rhizobacteria offer useful alternatives to
agrochemicals for better growth and development of crop plants by direct as well as indirect m ­ echanisms2. Tea
rhizosphere harbors diverse plant growth-promoting rhizobacteria, which offer potential applications as biofer-
tilizers in sustainable agricultural practice within a similar agroclimatic ­setup12–16,18,49. We, therefore, sought to
isolate, characterize, and explore tea PGPR for their potential in plant growth promotion and biocontrol using
the two most important food grain crops in the Darjeeling region.
In the present study, a total of 120 unique rhizobacteria were isolated from the soil samples collected from
seven tea estates of Darjeeling, West Bengal, India (Fig. 1) using different enrichment media. All the rhizobac-
terial isolates were then functionally screened on specific media for assessing their plant-growth-promoting
(PGP) potential, and eventually, thirty pure cultures of rhizobacteria were selected for further analysis. At the
time of biochemical and microbiological studies, sixteen isolates were found to be Bacillus (16 different strains)-
like. All of them were found to be spore-forming, Gram-positive, catalase-positive, and rod-shaped bacteria
(data not shown). Also, several gram-positive, spherical (grape-like appearance under the microscope) shaped
Staphylococcus-like bacterial isolates were evident from microbiological analyses (data not shown). In general,
the microbiological assessment of the tea rhizosphere soil revealed a bacterial count ranged between 2 × 104 to
2 × 107. The number of rhizobacterial strains isolated from rhizosphere soils of each tea estates is summarized in
Table 1 along with soil pH and texture as measured following USDA methodologies.

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To determine the identity of the tea rhizobacterial isolates, the 16S rRNA gene was amplified partially and
sequencing was performed as described in the method section. DNA sequencing and analysis using the BLAST
tool revealed that all the 30 rhizobacterial isolates show an identity ranging from 97.88% to 100% with the avail-
able 16S rRNA gene sequences in GenBank (Table 2). Furthermore, the phylogenetic analysis revealed that these
30 rhizobacterial isolates represent nine different genera (Fig. 2). Out of these 30 isolates, 16 (53.3%) isolates
belong to genus Bacillus, 5 (16.6%) represent genus Staphylococcus, 3 (10%) represent genus Ochrobactrum, and
1 (3.3%) isolates each belongs to genera Pseudomonas, Lysinibacillus, Micrococcus, Leifsonia, Exiguobacterium,
and Arthrobacter (Table 2 and Fig. 2). Previous studies have shown that the cultivable rhizobacterial population
in the tea rhizosphere is dominated by Bacillus g­ enera12,14,15,49. Moreover, Bacillus genera are a dominant culti-
vable member in the rhizosphere soil of different plants, e.g., rice, wheat, tobacco, Panax notoginseng (Chinese
ginseng), etc.50–53.

In vitro plant‑growth promoting activities. Thirty selected rhizobacterial isolates were evaluated
in vitro for properties that are known to be essential for plant growth-promoting activities of bacteria, such as
the IAA production, phosphate solubilization, siderophore production, and ammonia production.
Plant growth-promoting rhizobacteria often produce indole acetic acid (IAA) and thereby assist plant growth.
The biosynthesis of bacterial IAA takes place either by tryptophan-dependent or independent manners. Out of
30 rhizobacterial isolates, 21 (70%) showed IAA production of the level of 20 µg ml−1 or more when grown in
the presence of 100 µg ml−1 L-tryptophan (Table 3). Some of the rhizobacterial isolates (AB304, AB312, AB328,
and AB331) were found to synthesize 90 µg ml−1 or more of IAA with AB331 synthesizing the maximum amount
(134.67 ± 3.59 µg ml−1) (Table 3). Biosynthesis of IAA utilizing tryptophan has been reported previously in dif-
ferent PGPR ­strains54. Previous studies have shown that tea rhizobacteria are efficient in IAA production in the
presence of t­ ryptophan15,16. However, in the present study, we also determined rhizobacterial IAA production
in the absence of tryptophan. In the absence of tryptophan, 8 (26.6%) isolates showed 20 µg ml−1 or more IAA
production with AB304 being the highest (55.17 ± 5.42 µg ml−1) (Table 3).
Microorganisms contribute to the natural phosphorous cycling by solubilizing precipitated and fixed phos-
phorous present in various soil types in a pH-dependent manner. In the acidic soil of the tea plantation, phos-
phorus is fixed by free oxides and hydroxides of aluminum and iron, resulting in the low availability of soluble
­phosphate55. In the present study, most of the rhizobacterial isolates were found to be efficient phosphate solubi-
lizers and therefore exhibit potentials to be used as plant growth promoters. About 11 (36.6%) isolates exhibited
phosphate solubilization of 400 µg ml−1 or more with AB345 being the most efficient phosphate solubilizer
(841.42 ± 10.89 µg ml−1) (Table 3). Previous studies have shown that phosphate solubilization by tea PGPR is
accompanied by a lowering of pH in the m ­ edium15,16. In the present study, we observed a significant decrease in
the pH of the medium associated with an in vitro solubilization of phosphate by the selected PGPR. We believe
that the production of organic acids by PGPR facilitated the solubilization of the insoluble phosphates. Previously
it has been shown that organic acids such as gluconic acid, lactic acid, malic acid, succinic acid, formic acid, citric
acid, malonic acid, and tartaric acid are often involved in effective solubilization of the inorganic p­ hosphate56.
Selected rhizobacterial isolates were found to be highly efficient producers of siderophore. The siderophore
production by the isolates was found to be between 52 to 99% siderophore units (Table 3). About 70% (21 isolates)
of rhizobacterial isolates were found to show siderophore production of more than 90% siderophore units or
more (Table 3). In general, rhizobacteria require strategies to survive in the highly competitive micro-ecological
zone of the rhizosphere. Amongst many strategies adopted by rhizobacteria, biosynthesis of siderophore is one
of the critical strategies, where rhizobacteria inhibit the growth of phytopathogenic bacteria or fungi or non-
rhizospheric bacteria by depriving them of the essential iron in the rhizosphere m ­ icroenvironment57. Previous
studies have reported that tea PGPR from Assam and Darjeeling are efficient in siderophore production, and
our results corroborate well with previous ­findings15,16.
Ammonia production by PGPR is one of the essential traits linked to plant growth promotion. In general,
ammonia produced by PGPR has been shown to supply nitrogen to their host plants and thereby promote root
and shoot elongation and their b ­ iomass58. In the present study, the ammonia production by the rhizobacterial
isolates was observed in the range of 2.5 μmol ml−1 to 7.54 μmol ml−1 (Table 3). About 67% (20 isolates) showed
ammonia production of more than 4 μmol ml−1, and isolate AB331 was found to produce the highest amount
of ammonia (7.54 μmol ml−1).
Together, the selected tea PGPR isolates were found to be highly efficient in various in vitro PGP activities
and showed possibilities in in-planta growth promotion activities.

Anti‑fungal (antagonistic) activities. The application of PGPR to mitigate biotic stress has attracted
considerable attention in recent years. Such an application can help both in growth promotion as well as in
disease control within the host plant, thereby increasing crop productivity to meet the global demand. All the
selected rhizobacterial isolates were screened for antifungal activity against two fungal pathogens, viz. rice
necrotrophic pathogen R. solani AG1-IA and maize biotrophic pathogen U. maydis SG200, respectively. Out
of 30 selected rhizobacteria, 18 (60%) isolates were found to be active against R. solani AG1-IA (Table 3), and
21 (70%) isolates were active against U. maydis SG200 (Data not shown). Seventeen rhizobacterial isolates were
found to show activity against both the fungal pathogen tested in the present study. All 30 isolates were further
evaluated for protease, cellulase, and ACC deaminase activities. While different hydrolytic enzymes such as pro-
teases and cellulases help in biocontrol by promoting fungal cell wall degradation, ACC deaminase produced by
rhizobacteria promotes plant growth by sequestering and cleaving 1-aminocyclopropane-1-carboxylate (ACC)
produced in plants under biotic and abiotic ­stresses59,60. Among the isolates, 21 (70%) showed protease activ-
ity, 8 (26.6%) showed cellulase activity, and 12 (40%) exhibited ACC deaminase activity (Table 3). To this end,

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Nearest neighbor and % identity

from NCBI with Accession
Serial number Strain name Accession number Base pair length number Isolation source
Arthrobacter sp. 690 (99.7%)
1 AB200 MT436081 1,321 Long view tea estate
(EU086821)
Exiguobacterium mexicanum
2 AB201 MT436082 1,427 Maharani tea estate
(100%) (CP040676)
Leifsonia lichenia (98.37%)
3 AB203 MT436083 1,353 Happy Valley Tea estate
(H432650)
Bacillus niacini (98.23%)
4 AB209 MT436084 1,415 Maharani tea estate
(MF177863)
Staphylococcus pasteuri (99.79%)
5 AB212 MT436085 1,401 Long view tea estate
(MT072161)
Bacillus nakamurai (99.93%)
6 AB214 MT436086 1,414 Barnesbeg tea estate
(MH057388)
Bacillus atrophaeus (98.45%)
7 AB228 MT436087 1,416 Makai Bari Tea estate
(KP209406)
Bacillus velezensis (99.93%)
8 AB230 MT436088 1,399 Maharani tea estate
(MT378136)
9 AB233 MT436089 1,417 Bacillus sp. (99.72%) (KP217809) Rohini Tea estate
Bacillus cereus (99.65%)
10 AB236 MT436090 1,413 Tukvar tea estate
(KU922345)
Bacillus velezensis (100%)
11 AB237 MT436091 1,349 Makai Bari Tea estate
(MH157241)
Bacillus altitudinis (100%)
12 AB242 MT436092 1,410 Barnesbeg tea estate
(KX344022)
Bacillus wiedmannii (99.86%)
13 AB246 MT436093 1,405 Maharani tea estate
(LC515603)
14 AB255 MT436094 1,413 Bacillus flexus (100%) (MH542283) Happy Valley Tea estate
Pseudomonas stutzeri (99.33%)
15 AB266 MT436095 1,349 Barnesbeg tea estate
(MN932299)
Bacillus subtilis (99.86%)
16 AB267 MT436096 1,411 Tukvar tea estate
(LC040931)
Bacillus pumilus (100%)
17 AB276 MT436097 1,359 Long view tea estate
(MK603127)
Ochrobactrum anthropi (100%)
18 AB285 MT436098 1,255 Rohini Tea estate
(MT093466)
Ochrobactrum haematophilum
19 AB286 MT436099 1,346 Makai Bari Tea estate
(100%) (MH236269)
Bacillus nitratireducens (100%)
20 AB304 MT436100 1,355 Long view tea estate
(MT341781)
Staphylococcus cohnii (97.88%)
21 AB312 MT436101 1,390 Makai Bari Tea estate
(MN581170)
Bacillus megaterium (99.92%)
22 AB320 MT436102 1,332 Rohini Tea estate
(CP010586)
Micrococcus luteus (99.93%)
23 AB321 MT436103 1,366 Barnesbeg tea estate
(AB539843)
Staphylococcus gallinarum (100%)
24 AB328 MT436104 1,419 Barnesbeg tea estate
(MK015788)
Bacillus paralicheniformis (100%)
25 AB330 MT436105 1,401 Maharani tea estate
(MG780252)
Staphylococcus edaphicus (100%)
26 AB331 MT436106 1,255 Tukvar tea estate
(MT269536)
Lysinibacillus fusiformis (98.07%)
27 AB332 MT436107 1,362 Happy Valley Tea estate
(LT547806)
Staphylococcus haemolyticus
28 AB336 MT436108 1,400 Rohini Tea estate
(100%) (MN581168)
Bacillus thuringiensis (99.56%)
29 AB341 MT436109 1,373 Makai Bari Tea estate
(MN911366)
Ochrobactrum sp. (98.84%)
30 AB345 MT436110 1,296 Maharani tea estate
(KY678891)

Table 2.  16S rRNA gene based molecular identity of isolated tea rhizobacteria, their sequence accession
numbers and their isolation sites.

most of the selected rhizobacterial isolates possess the necessary arsenal to act as possible biocontrol agents. We,
therefore, sought to test their ability to act as biocontrol agents in our subsequent experiments.

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Figure 2.  Neighbor-joining phylogenetic tree showing phylogenetic relationship between rhizobacterial isolates
based on the 16S rRNA gene sequences. The 16S rRNA sequence of Sulfolobus solfataricus P2 was used to assign
an outgroup species.

Assessment of plant‑growth promotion activity under laboratory conditions. The Plant growth
promotion experiments using rhizobacterial isolates revealed that the application of almost all the selected
rhizobacteria had increased plant biometric parameters (viz. wet weight, dry weight, shoot length and root
length) of both rice and maize seedlings in a statistically significant manner (Fig. 3 and Fig. S1). In the present
study, Bacillus was the most abundant member of the cultivable tea rhizobacterial isolates. All the 16 (53.33%)
isolates of Bacillus genera were found to induce a significant increase in wet weight, dry weight, shoot length
and root length in both rice and maize seedlings (Fig. 3 and Fig. S1). Several species of the genus Bacillus such
as B. amyloliquefaciens, B. aryabhattai, B. circulans, B. coagulans, B. licheniformis, B. megaterium, B. subtilis,
B. thuringiensis and B. velezensis have been previously identified and characterized as PGPR and biocontrol
­agents1,12,14,61,62. They promote plant growth using various direct and indirect mechanisms, including nitrogen
fixation, phosphate and potassium solubilization, phytohormone production, siderophores production, antimi-
crobial and hydrolytic enzymes biosynthesis, stimulation of induced systemic resistance (ISR) and antioxidative
defense system in ­plants62. In the present study, we evaluated plant growth-promoting activities of several known
PGPR of the genus Bacillus, i.e., B. atrophaeus AB228, B. velezensis AB230, B. velezensis AB237, B. cereus AB236,
B. altitudinis AB242, B. wiedmannii AB246, B. flexus AB255, B. subtilis AB267, B. nitratireducens AB304, B.
magatarium AB320, B. thuringiensis AB341 (Fig. 3 and Fig. S1)61. Besides, we also found several new members
that fall under the genus Bacillus, e.g., B. niacini AB209, B. nakamurai AB214, Bacillus sp. AB233, B. pumilus
AB276, and B. paralicheniformis AB330, as potential plant growth stimulating rhizobacterial isolates (Fig. 3 and
Fig. S1). Previous studies have shown that the genus Bacillus dominates the cultivable portion of the Darjeeling
tea rhizobacterial population and are useful as PGPR and biocontrol a­ gents15,63. In this study besides Bacillus,
members of the genus such as Arthobacter (Arthrobacter sp. AB200), Exiguobacterium (Exiguobacterium mexi-
canum AB201), Leifsonia (Leifsonia lichenia AB203), Lysinibacillus (Lysinibacillus fusiformis AB332), Micrococ-
cus (Micrococcus luteus AB321), Ochrobactrum (Ochrobactrum anthropi AB285, Ochrobactrum haematophilum
AB286, Ochrobactrum haematophilum AB345), Pseudomonas (Pseudomonas stutzeri AB266), and Staphylococ-
cus (Staphylococcus pasteuri AB212, Staphylococcus cohnii AB312, Staphylococcus gallinarum AB328, Staphylo-
coccus saprophyticus AB331, Staphylococcus haemolyticus AB336) were also found to increase plant biometric
parameters (viz. wet weight, dry weight, shoot length and root length) in both rice and maize seedlings (Fig. 3
and Fig. S1). To our knowledge, among these isolates, members of the genus Arthrobacter (Arthrobacter sp.

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Plant growth promoting traits Antifungal traits

IAA
production
when
supplemented
IAA with Siderophore Phosphate Protease Cellulase ACC Anti fungal
production (µg Tryptophan (µg production solubilization Ammonia production production deaminase activity against
Sl No Sample ­ml−1)* ­ml−1)* (%)* (µg ­ml−1)* ­production# (qualitative) (qualitative) (qualitative) R. solani
1 AB200 6.67 ± 2.86 15 ± 2.36 97.21 ± 0.56 30 ± 2.52 Moderate − − − +
2 AB201 1.95 ± .034 7.97 ± 1.03 95.05 ± 1.15 230.52 ± 8.75 Small + + + −
3 AB203 0.76 ± .19 20.34 ± 1.26 98.88 ± 0.42 175.88 ± 8.42 Small − + − −
4 AB209 3.67 ± 0.47 14.83 ± 2.63 52.31 ± 4.46 280.18 ± 69.09 Large + − − +
5 AB212 0.68 ± .024 30 ± 2.71 92.71 ± 2.16 275.58 ± 43.63 Moderate − − − +
6 AB214 3.33 ± 0.36 16.67 ± 1.43 72.07 ± 3.67 572.62 ± 23.59 Moderate + + − +
7 AB228 1.36 ± 0.72 9.92 ± 6.11 98.55 ± 1.50 51.99 ± 2.04 Small − + − +
8 AB230 25.33 ± 2.83 37.33 ± 2.83 97.69 ± 1.49 794.86 ± 12.87 Moderate + − + −
9 AB233 10.33 ± 1.89 20 ± 2.84 94.77 ± 3.22 187.19 ± 37.26 Small + + − +
10 AB236 25.33 ± 2.83 56.33 ± 3.33 94.43 ± 1.04 22 ± 2.05 Moderate + − + −
11 AB237 22 ± 0.47 25.33 ± 1.89 75.05 ± 1.64 635.75 ± 18.89 Large + − − +
12 AB242 4 ± 1.41 38 ± 3.33 72.56 ± 2.46 12 ± 3.0 Large − − + −
13 AB246 16.5 ± 0.71 20 ± 0.47 80.25 ± 4.60 591.28 ± 53.44 Moderate + − − +
14 AB255 7 ± .94 15.33 ± 2.83 99.26 ± 1.21 191.51 ± 8.31 Small + − + −
15 AB266 9.67 ± 1.41 39.33 ± 1.86 88.18 ± 2.77 70.12 ± 5.92 Small − − + +
16 AB267 7 ± 2.83 12 ± 2.83 68.77 ± 3.69 335.62 ± 23.59 Small + − − +
17 AB276 15 ± 2.36 16.67 ± 2.86 97.21 ± 0.56 21.78 ± 5.75 Moderate − − + +
18 AB285 34.67 ± 4.24 27 ± 3.29 93.25 ± 1.25 229.40 ± 24.67 Small − − + +
19 AB286 4 ± 1.42 15.67 ± 1.89 82.71 ± 1.25 33.52 ± 4.25 Moderate + − − −
20 AB304 55.17 ± 5.42 90 ± 4.33 83.87 ± 2.26 137.52 ± 14.41 Large + + − +
21 AB312 9.17 ± 3.06 113 ± 2.36 99.01 ± 0.21 252.83 ± 9.35 Large + − − −
22 AB320 28.17 ± 2.12 74.83 ± 5.89 98.21 ± 0.24 452.65 ± 1.28 Moderate + + − −
23 AB321 13.83 ± 3.06 58.33 ± 2.36 98.92 ± 0.49 446.76 ± 3.84 Moderate + − + +
24 AB328 15.33 ± 0.47 99.83 ± 2.12 98.42 ± 0.42 577.71 ± 53.83 Moderate + − − −
25 AB330 26.17 ± 1.18 53 ± 2.65 97.07 ± 0.11 312.37 ± 6.73 Small + − − +
26 AB331 25.17 ± 3.54 134.67 ± 3.59 93.60 ± 0.07 275.49 ± 4.57 Large + + + +
27 AB332 19.33 ± 2.36 48.83 ± 2.78 96.38 ± 0.31 522.89 ± 26.27 Large − − + −
28 AB336 19.67 ± 3.77 30.67 ± 2.71 97.76 ± 0.45 822.39 ± 41.97 Moderate + − − +
29 AB341 13 ± 4.71 28.50 ± 1.18 93.99 ± 0.41 728.55 ± 71.77 Small + − + +
30 AB345 9 ± 1.41 60.50 ± 2.42 98.60 ± 0.24 841.42 ± 10.89 Moderate + − − −

Table 3.  Functional screening of selected tea rhizobacteria for in vitro PGP and antifungal activities. *Mean
value (all values are triplicate) ± Standard deviation (SD), + means positive activity and—means no activity.
#
Ammonia production attributes are equivalent to Small: 1–3 µmole ­ml−1 , Moderate: 3–6 µmole m ­ l−1, and
−1
Large: > 6 µmole ­ml .

AB200), Exiguobacterium (Exiguobacterium mexicanum AB201), Leifsonia (Leifsonia lichenia AB203), and
Lysinibacillus (Lysinibacillus fusiformis AB332) were isolated from the tea rhizosphere of Darjeeling and tested
for PGP activities for the first time. Out of thirty rhizobacterial isolates, a treatment with 27 (90%) isolates
resulted in a statistically significant increase in total chlorophyll content both in rice and maize seedling in com-
parison to uninoculated control plants (Fig. 4 and Fig. S2).

Effect of PGPR treatment on the defense‑related enzymes in rice. Plant growth-promoting rhizo-
bacteria (PGPR) are known to impart induced systemic resistance (ISR) to bacterial, fungal, and viral diseases in
­plants64. They have been shown to elicit plant defense systemically against foliar and root ­pathogens65,66. Previous
investigations revealed that different PGPR strains protect the plants from various pathogens by activating plant
defense genes encoding chitinase, β-1,3 glucanase, PAL (phenylalanine ammonia-lyase), CAT (Catalase), APX
(Ascorbate peroxidase), POD (peroxidase) and other enzymes, many of which act as primary reactive oxygen
species (ROS) s­ cavengers67. In the present study, we examined the status of APX, CAT, Chitinase, and PAL activi-
ties in the PGPR treated rice plants (Fig. 5 and Fig. S3).
The enzyme ascorbate peroxidase detoxifies ­H2O2 generated as a byproduct of antioxidative mechanisms and
converts it into water within chloroplast, cytoplasm, and ­mitochondria9,68. Increased activity of APX is proposed
to have a contribution in the detoxification of augmented ­H2O2 accumulation in the ­cells63. Plants inoculated
with tea rhizobacterial isolates showed a differential APX activity in the shoots and roots, respectively. In the

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Figure 3.  Evaluation of plant-growth promoting traits upon individual treatments of selected tea rhizobacterial
isolates on IR64 variety of rice seedlings. Five days old seedlings were treated with individual rhizobacterial
isolates, and the growth parameters such as (a) wet weight, (b) dry weight, (c) root length, and (d) shoot length
were measured 21 days post-treatment. A two-sided t-test determined the significance level, and data are
mean ± SD. (a: P-value- 0.05–0.01, b: P-value 0.01–0.001, and c: P-value less than 0.001).

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Figure 4.  Evaluation of Chlorophyll concentration upon individual treatments of selected tea rhizobacterial
isolates on the IR64 variety of rice seedlings. Five days old seedlings were treated with individual rhizobacterial
isolates, and the total chlorophyll concentration was measured 21 days post-treatment. A two-sided t-test
determined the significance level, and data are mean ± SD. (a: P-value- 0.05–0.01, b: P-value 0.01–0.001, and c:
P-value less than 0.001).

shoot lysate, APX activity was significantly increased in plants treated with 12 rhizobacterial isolates (40%)
(P ≤ 0.05–0.001) (Fig. 5a). In cases of isolates AB237, AB242, and AB285, the APX activity in the shoot lysate
were found to be maximum among the treated plants (P ≤ 0.001) (Fig. 5a). In the case of root lysate, significant
enhancement of APX activity was evident for 13 rhizobacterial isolates (43.3%) (P ≤ 0.05–0.001) (Fig. S3a).
Treatment with isolates AB212, AB236, AB246, and AB255 were found to show maximum APX activity in the
root lysates of the treated plants (P ≤ 0.001) (Fig. S3a). Together, APX activity measurements revealed that about
40% and 43.3% rhizobacterial isolates showed statistically significant enhancement of APX activity in the shoot
and/or root of the treated plants.
Catalase (CAT) acts as a cellular sink of ­H2O2 and catalyzes its disproportionation into ­H2O and ­O269. Out of
30 rhizobacterial isolates, 16 isolates (53.3%) showed a statistically significant increase in the elicitation of CAT
activity in plant shoot lysates compared to untreated control (P ≤ 0.05–0.001) (Fig. 5b). Among these isolates,
AB201 and AB237 were found to induce maximum CAT activity in plant shoot (P ≤ 0.001) (Fig. 5b). While in the
root lysates, CAT activity was found to be significantly higher in the plants treated with 14 rhizobacterial isolates
(46.6%) (P ≤ 0.05–0.001) (Fig. S3b). Among these isolates, treatments with AB237 showed a maximum increase
in CAT activity in plant root (P ≤ 0.001) (Fig. S3b). Together, the CAT activity was found to increase significantly
in the shoot and root of treated plants for treatments with about 53.3% and 46.6% of rhizobacterial isolates.
Chitinases are the member of pathogen-related proteins in plants. These enzymes are strongly induced when
a plant is challenged with a fungal pathogen or due to stimulation of induced systemic resistance (ISR) as a result
of PGPR–plant ­interaction5,70. Chitinases act as an essential arsenal to mitigate fungal infection in plants by direct
lytic action on fungal cell walls or by stimulating a variety of plant defenses by releasing oligosaccharide signaling
­molecules70. Among the selected rhizobacteria, 19 isolates (63.3%) showed a significant increase in chitinase
activity in the shoot of treated rice plants (P ≤ 0.05–0.001) (Fig. 5c). Treatment with isolates AB214 and AB285
resulted in statistically most significant increase in the chitinase activity (P ≤ 0.001) (Fig. 5c). Contrary to shoot,
the root lysates of the treated plants showed a substantial escalation of the chitinase activity for treatments with
15 rhizobacterial isolates (50%) (P ≤ 0.05–0.001) (Fig. S3c). Furthermore, treatment with AB228 and AB233
showed maximum induction of chitinase activity in the root of the treated plants (P ≤ 0.001) (Fig. S3c). Overall,
when treated with about 63.3% and 50% rhizobacterial isolates, a significant increase of the chitinase activity in
the shoot and root of treated plants was evident.
Phenylalanine Ammonia Lyase (PAL) is an important enzyme that helps plants to mitigate different stress
­conditions5,67. PAL offers physiological and structural support to the plants by converting L-phenylalanine to
ammonia and trans-cinnamic acid. In rice, it was shown that microbial treatment increase PAL activity and
accumulation of polyphenols in the leaves and thereby help to ameliorate stress conditions (drought, salinity,
etc.)67. In the present study, we observed that in the majority of the cases, rhizobacterial treatment resulted in
enhanced activity of PAL enzyme in rice shoot lysates. In cases of 26 rhizobacterial isolates (86.6%), a statistically
significant increase in the PAL activity was noted for shoot samples of the treated rice plants (P ≤ 0.05–0.001)
(Fig. 5d). The most significant PAL activity was, however, documented in the shoot samples of the plants treated
with isolates AB228, AB267, AB276, and AB336 (P ≤ 0.001) (Fig. 5d). In root samples, PAL activity was found to
increase for treatments with 16 rhizobacterial isolates (53.3%) (P ≤ 0.05–0.001) (Fig. S3d). Out of rhizobacterial
isolates, treatment with AB267, and AB341 showed maximum PAL activity in the root samples of the treated
plants (P ≤ 0.001) (Fig. S3d). Together, our analysis revealed that about 86.6% of rhizobacterial isolates increase
PAL activity in the shoot regions, while only 53.3% isolates could promote PAL activity in the root region of the
treated rice plants.
To the end, increased activity of the defense-related enzymes such as APX, CAT, chitinase, and PAL in the
PGPR treated rice plants led us to propose that (i) rhizobacterial treatment can stimulate induced systemic
resistance (ISR), a state of enhanced defensive capacity in rice plants, and (ii) the rhizobacterial isolates are

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Figure 5.  Effect of rhizobacterial treatment on (a) ascorbate peroxidase (APX), (b) catalase (CAT), (c)
chitinase, and (d) phenylalanine ammonia-lyase (PAL) activity in shoot fraction of IR64 variety of rice seedlings.
Five days old rice seedlings were treated with individual rhizobacterial isolates, and the antioxidative defense
enzymes were measured in the shoot lysate preparation 14 days post-treatment. A two-sided t-test determined
the significance level, and data are mean ± SD. (a: P-value- 0.05–0.01, b: P-value 0.01–0.001, and c: P-value less
than 0.001).

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Figure 6.  Impact of rhizobacterial treatment on (a) shoot proline content, and (b) root proline content in IR64
variety of rice seedlings. Five days old rice seedlings were treated with individual rhizobacterial isolates, and
the antioxidative defense molecules were measured 14 days post-treatment. A two-sided t-test determined the
significance level, and data are mean ± SD. (a: P-value- 0.05–0.01, b: P-value 0.01–0.001, and c: P-value less than
0.001).

capable of modulating defense-related enzyme activity and thereby help the plant to prepare itself for a future
challenge with biotic and abiotic stresses. Limited information is available about how PGPR from tea rhizosphere
modulate defense pathways in host p ­ lants17,71. To our knowledge, this is possibly the first report of modulation
of defense-related enzyme activities by tea PGPR in parallel to their typical plant growth promoting attributes.

Proline and polyphenols accumulation in inoculated rice plants. ROS scavenging small metabo-
lites such as carotenoids, phenolics, proline, and tocopherol maintain redox balance in cells during oxidative
­damage72. PGPR treatment enhance proline and polyphenolics concentrations that usually favors ROS scaveng-
ing in the p ­ lants73. In the present study, we measured proline and polyphenolics concentrations in the treated
rice plants.
Proline is an excellent osmolyte that helps in the stabilization of sub-cellular macromolecules such as pro-
teins and cell membranes. Besides, it is involved in scavenging free radicals, balancing redox homeostasis and
signaling, thereby assisting plants to cope under stress ­conditions74. Eighteen rhizobacterial isolates (60%) were
found to cause a statistically significant increase in the proline concentration in shoot samples in treated rice
compared to untreated control plants (P ≤ 0.05–0.001) (Fig. 6a). The isolates AB321 caused the most significant
increase in proline concentration in the shoot of treated rice plants (P ≤ 0.001) (Fig. 6a). In the case of the same
set of treatments, analysis of the root samples revealed that 14 rhizobacterial isolates (46.6%) caused a statistically
significant increase in proline content (P ≤ 0.05–0.001) with isolate AB228 being the most efficient (P ≤ 0.001)
(Fig. 6b). Together, proline estimates in the rhizobacteria treated rice plants revealed that in the majority of
the treatments proline concentration was increased in shoot samples (60%) and also in root samples (46.6%)
indicating possible PGPR assisted priming (both local and systemic) of the plants to face future challenges of
biotic and abiotic stresses.
Accumulation of polyphenolics in plant leaves is shown to have a protective role against biotic and abiotic
stresses through anti-oxidation and ROS ­deactivation67. Microbial treatment influences the accumulation of
polyphenolics in plant l­eaves75. Being a potent antioxidant, high accumulation of polyphenolics in the leaves
is supposed to strengthen plants’ stress t­ olerance75. In the present study, we measured the total accumulated

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Figure 7.  Impact of rhizobacterial treatment on the accumulation of total polyphenol in IR64 variety of rice
seedlings. Five days old rice seedlings were treated with individual rhizobacterial isolates, and the antioxidative
defense molecules were measured 14 days post-treatment. A two-sided t-test determined the significance level,
and data are mean ± SD. (a: P-value- 0.05–0.01, b: P-value 0.01–0.001, and c: P-value less than 0.001).

Figure 8.  The effect of rhizobacterial treatment on the relative disease indices (DI) of sheath blight infection in
IR64 variety of rice seedlings. Five days old rice seedlings were treated with individual rhizobacterial consortia
(as described in Table S1), and the treated plants were grown at 24 °C under 16–8 h day-night cycle for three
days. Following this, the seedlings were inoculated with R. solani AG1-IA, as described in the method section.
The rice seedlings thus infected with the fungus were grown further for two days, and the infection symptoms
were scored as the disease index (DI). The DI of all the treated samples was calculated and compared with that
of the control to evaluate the effect of the respective consortia on inducing resistance of rice seedlings. Error bars
represent the standard deviation calculated from three independent experiments.

polyphenolics in the treated rice plants. Our analysis revealed that for treatments with 23 rhizobacterial isolates
(76.6%), a statistically significant increase in total polyphenolics was observed in the leaves (P ≤ 0.05–0.001)
(Fig. 7). However, treatment with isolates AB304 caused the most significant effect in terms of total polyphenolics
measurement in the leaves (P ≤ 0.001) (Fig. 7). Together, an accumulation of polyphenolics was observed in the
majority of the treatments (76.6%), indicating possible enhancement of anti-oxidants in the plants.

Increased resistance of PGPR pretreated rice plants towards sheath blight infection. To assess
whether the increased activity of defense-related enzymes in rice due to PGPR pretreatment is indeed involved
in inducing disease resistance, we studied sheath blight infection in rice under PGPR pretreated and untreated
conditions. PGPR were distributed in six consortia (Table S1), and the rice seedlings were pretreated with each
of these six consortia separately before R. solani AG1-IA infection. Figure 8 shows a positive response with
respect to the increased resistance of rice in the case of each of the six consortia. However, the degree of resist-
ance induced within the rice plants varied with the individual consortia used for pretreatment. Like for instance,
groups I, V, and VI showed the maximum effect with about 60 to 70 percent reduction in DI. In all of these cases,
the DI ranged between 0.3 and 0.4. Group III showed a moderate effect with a DI of 0.42, and group II and IV
showed the least impact. While group IV consortia pretreatment resulted in a DI of 0.65, group II treated rice
plants showed a DI of 0.72 upon infection with R. solani AG1-IA. Among the designed multispecies rhizobacte-
rial consortia, group I was found to be the most effective in the mitigation of R. solani AG1-IA infection. Group I
have consisted of Arthrobacter sp. AB200, Staphylococcus pasteuri AB212, Bacillus sp. AB233, Bacillus altitudinis
AB242, and Pseudomonas stutzeri AB266 (Table S1). Out of five rhizobacterial isolates in group I, Arthobacter sp.

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was shown previously to inhibit the in vivo growth of potato pathogen Phytophthora infestans76, P. stutzeri was
found effective against plant root rot Fusarium solani77, and B. altitudinis was found to be effective against the
root rot disease caused by Thanatephorus cucumeris78. Besides, groups V and VI were also found highly efficient
in the mitigation of R. solani infection in rice (Fig. 8). Members of group V, e.g., B. subtilis, B. thuringiensis, B.
pumilus, and S. gallinarum are well-known biocontrol agents against a number of p ­ hytopathogens79–82. Group VI
members are comparatively less characterized rhizobacterial species (Table S1) and members like B. paralicheni-
formis, Lysinibacillus fusiformis, and Ochrobactrum sp. were recently reported to have biocontrol ­activities83–85.

Conclusion
In the present study, thirty plant growth-promoting rhizobacteria were isolated and characterized from the
tea rhizosphere of Darjeeling, West Bengal, India. All the thirty rhizobacterial isolates were found to effec-
tively promote the growth of rice and maize seedlings implying their possible utilization in microbe-based bio-
formulations. Moreover, the treatment of these rhizobacterial isolates was found to activate the antioxidative
defense mechanisms in rice seedlings through the induction of APX, SOD, chitinase, and PAL activities and
accumulation of proline and polyphenols. This, therefore, indicated a significant role of the microbial inoculum
in reducing the stress-induced ROS burden on host plants. We further evaluated the effect of six different rhizo-
bacterial consortia, each of which is composed of five different rhizobacterial isolates on the stress tolerance
of rice towards R. solani AG1-IA infection. Results showed a positive effect on disease tolerance by each of the
six consortia formulations, although to different extents. To summarize, tea rhizobacteria have the potential to
promote plant growth and modulating plant antioxidative defense systems, thereby aiding in biotic stress man-
agement in model plant systems. However, further studies are necessary to evaluate the usefulness of these PGP
rhizobacteria in tea plantation. Finally, it is intriguing to explore the genome of individual rhizobacteria to find
out their physiological robustness to become useful in the field conditions. Also, a study of the molecular cross-
talk between the individual isolates of the different consortia is necessary to account for the relative efficiencies
of the individual consortia in their plant growth promotion abilities.

Received: 25 May 2020; Accepted: 31 August 2020

References
1. Mahanty, T. et al. Biofertilizers: A potential approach for sustainable agriculture development. Environ. Sci. Pollut. Res. Int. 24,
3315–3335. https​://doi.org/10.1007/s1135​6-016-8104-0 (2017).
2. Bhattacharyya, C. R. R., Tribedi, P., Ghosh, A. & Ghosh, A. Biofertilizers as Substitute to Commercial Agrochemicals 263–290
(Elsevier Ltd., Amsterdam, 2020).
3. Drogue, B., Dore, H., Borland, S., Wisniewski-Dye, F. & Prigent-Combaret, C. Which specificity in cooperation between phyto-
stimulating rhizobacteria and plants?. Res. Microbiol. 163, 500–510. https​://doi.org/10.1016/j.resmi​c.2012.08.006 (2012).
4. Berg, G. & Smalla, K. Plant species and soil type cooperatively shape the structure and function of microbial communities in the
rhizosphere. FEMS Microbiol. Ecol. 68, 1–13. https​://doi.org/10.1111/j.1574-6941.2009.00654​.x (2009).
5. Choudhary, D. K., Prakash, A. & Johri, B. N. Induced systemic resistance (ISR) in plants: Mechanism of action. Indian J. Microbiol.
47, 289–297. https​://doi.org/10.1007/s1208​8-007-0054-2 (2007).
6. Kumar, A. & Verma, J. P. Does plant-Microbe interaction confer stress tolerance in plants: A review?. Microbiol. Res. 207, 41–52.
https​://doi.org/10.1016/j.micre​s.2017.11.004 (2018).
7. Gill, S. S. & Tuteja, N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol.
Biochem. PPB 48, 909–930. https​://doi.org/10.1016/j.plaph​y.2010.08.016 (2010).
8. Hussain, S., Khan, F., Cao, W., Wu, L. & Geng, M. Seed priming alters the production and detoxification of reactive oxygen
intermediates in rice seedlings grown under sub-optimal temperature and nutrient supply. Front. Plant Sci. 7, 439. https​://doi.
org/10.3389/fpls.2016.00439​(2016).
9. Sharma, P., Jha, A. B., Dubey, R. S. & Pessarakli, M. Reactive oxygen species, oxidative damage, and antioxidative defense mecha-
nism in plants under stressful conditions. J. Bot. 1–26, 2012. https​://doi.org/10.1155/2012/21703​7 (2012).
10. Cernava, T. et al. The tea leaf microbiome shows specific responses to chemical pesticides and biocontrol applications. Sci. Total
Environ. 667, 33–40. https​://doi.org/10.1016/j.scito​tenv.2019.02.319 (2019).
11. Bishnu, A., Saha, T., Mazumdar, D., Chakrabarti, K. & Chakraborty, A. Assessment of the impact of pesticide residues on microbio-
logical and biochemical parameters of tea garden soils in India. J. Environ. Sci. Health 43, 723–731. https​://doi.org/10.1080/03601​
23080​23888​50 (2008).
12. Chakraborty, A. P. C. & Chakraborty, U. Bacillus megaterium from tea rhizosphere promotes growth and induces systemic resist-
ance in tea against Sclerotium rolfsii. Indian Phytopathol. 68, 237–247 (2015).
13. Chakraborty, U., Chakraborty, B. N., Basnet, M. & Chakraborty, A. P. Evaluation of Ochrobactrum anthropi TRS-2 and its talc
based formulation for enhancement of growth of tea plants and management of brown root rot disease. J. Appl. Microbiol. 107,
625–634. https​://doi.org/10.1111/j.1365-2672.2009.04242​.x (2009).
14. Bhattacharyya, C. et al. Genome-guided insights into the plant growth promotion capabilities of the physiologically versatile
Bacillus aryabhattai strain AB211. Front. Microbiol. 8, 411. https​://doi.org/10.3389/fmicb​.2017.00411​ (2017).
15. Dutta, J. & Thakur, D. Evaluation of multifarious plant growth promoting traits, antagonistic potential and phylogenetic affiliation
of rhizobacteria associated with commercial tea plants grown in Darjeeling, India. PLoS ONE 12, e0182302. https:​ //doi.org/10.1371/
journ​al.pone.01823​02 (2017).
16. Dutta, J., Handique, P. J. & Thakur, D. Assessment of culturable tea rhizobacteria isolated from tea estates of assam, india for growth
promotion in commercial tea cultivars. Front. Microbiol. 6, 1252. https​://doi.org/10.3389/fmicb​.2015.01252​ (2015).
17. Mishra, A. K., Morang, P., Deka, M., Nishanth Kumar, S. & Dileep Kumar, B. S. Plant growth-promoting rhizobacterial strain-
mediated induced systemic resistance in tea (Camellia sinensis (L) O. Kuntze) through defense-related enzymes against brown
root rot and charcoal stump rot. Appl. Biochem. Biotechnol. 174, 506–521. https​://doi.org/10.1007/s1201​0-014-1090-0 (2014).
18. Chakraborty, U. C., Chakraborty, A. P., Sunar, K. & Dey, P. L. Plant growth-promoting rhizobacterial strain-mediated induced
systemic resistance in tea. Indian J. Biotechnol. 12, 20–31 (2013).
19. Majumdar, K., Ray, D. P., Chakraborty, S. & Pandit, T. Change of nutrient status of hilly soil in darjeeling district within five years.
Int. J. Bioresour. Sci. 1, 25–30 (2014).
20. Mukherjee, D. Resource conservation through indigenous farming system in hills of West Bengal. J. Crop Weed 8, 160–164 (2012).

Scientific Reports | (2020) 10:15536 | https://doi.org/10.1038/s41598-020-72439-z 16

Vol:.(1234567890)
www.nature.com/scientificreports/

21. Roy, S. K. & Mukhopadhyay, D. A study on physicochemical properties of soils under different tea growing regions of WEST
BENGAL (INDIA). Int. J. Agric. Sci. 4, 325–329 (2012).
22. Zhu, Q. et al. Cropland acidification increases risk of yield losses and food insecurity in China. Environ. Pollut. 256, 113145. https​
://doi.org/10.1016/j.envpo​l.2019.11314​5 (2020).
23. Gansser, A. Geology of Himalayas. 289 (Inter Science Publishers, 1964).
24. Desai, M. Geological survey on NH 55, Darjeeling—a draft report (Engineering Department, Darjeeling, 2005).
25. Ghosh, A., Maity, B., Chakrabarti, K. & Chattopadhyay, D. Bacterial diversity of East Calcutta Wet land area: possible identifica-
tion of potential bacterial population for different biotechnological uses. Microb. Ecol. 54, 452–459. https​://doi.org/10.1007/s0024​
8-007-9244-z (2007).
26. Frank, J. A. et al. Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Appl. Environ.
Microbiol. 74, 2461–2470. https​://doi.org/10.1128/aem.02272​-07 (2008).
27. Ghosh, A. et al. Culture independent molecular analysis of bacterial communities in the mangrove sediment of Sundarban, India.
Saline Syst. 6, 1. https​://doi.org/10.1186/1746-1448-6-1 (2010).
28. Mallick, I. et al. Effective rhizoinoculation and biofilm formation by arsenic immobilizing halophilic plant growth promoting
bacteria (PGPB) isolated from mangrove rhizosphere: A step towards arsenic rhizoremediation. Sci. Total Environ. 610–611,
1239–1250. https​://doi.org/10.1016/j.scito​tenv.2017.07.234 (2018).
29. Saitou, N. & Nei, M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425.
https​://doi.org/10.1093/oxfor​djour​nals.molbe​v.a0404​54 (1987).
30. Felsenstein, J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39, 783–791. https​://doi.
org/10.1111/j.1558-5646.1985.tb004​20.x (1985).
31. Tamura, K., Nei, M. & Kumar, S. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc. Natl.
Acad. Sci. 101, 11030–11035. https​://doi.org/10.1073/pnas.04042​06101​ (2004).
32. Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. MEGA X: Molecular evolutionary genetics analysis across computing
platforms. Mol. Biol. Evol. 35, 1547–1549. https​://doi.org/10.1093/molbe​v/msy09​6 (2018).
33. Pikovskaya, R. I. Mobilization of phosphorus in soil in connection with the vital activity of some microbial species. Mikrobiologiya
17, 362–370 (1948).
34. Alexander, D. B. & Zuberer, D. A. Use of chrome azurol S reagents to evaluate siderophore production by rhizosphere bacteria.
Biol. Fertil. Soils 12, 39–45. https​://doi.org/10.1007/bf003​69386​ (1991).
35. Ji, S. H. et al. Biocontrol activity of bacillus amyloliquefaciens CNU114001 against fungal plant diseases. Mycobiology 41, 234–242.
https​://doi.org/10.5941/MYCO.2013.41.4.234 (2013).
36. Bera, A., Ghosh, A., Mukhopadhyay, A., Chattopadhyay, D. & Chakrabarti, K. Improvement of degummed ramie fiber proper-
ties upon treatment with cellulase secreting immobilized A. larrymoorei A1. Bioprocess Biosyst. Eng. 38, 341–351. https​://doi.
org/10.1007/s0044​9-014-1274-6 (2014).
37. García de Salamone, I. E. et al. Field response of rice paddy crop to Azospirillum inoculation: Physiology of rhizosphere bacterial
communities and the genetic diversity of endophytic bacteria in different parts of the plants. Plant Soil 336, 351–362. https​://doi.
org/10.1007/s1110​4-010-0487-y (2010).
38. Bharti, N., Pandey, S. S., Barnawal, D., Patel, V. K. & Kalra, A. Plant growth promoting rhizobacteria Dietzia natronolimnaea
modulates the expression of stress responsive genes providing protection of wheat from salinity stress. Sci. Rep. 6, 34768. https​://
doi.org/10.1038/srep3​4768 (2016).
39. Porra, R. J., Thompson, W. A. & Kriedemann, P. E. Determination of accurate extinction coefficients and simultaneous equations
for assaying chlorophylls a and b extracted with four different solvents: Verification of the concentration of chlorophyll standards
by atomic absorption spectroscopy. Biochim. et Biophys. Acta Bioenerg. 975, 384–394. https:​ //doi.org/10.1016/s0005-​ 2728(89)80347​
-0 (1989).
40. Chen, G. & Asada, K. Ascorbate peroxidase in tea leaves: Occurrence of two isozymes and the differences in their enzymatic and
molecular properties. Plant Cell Physiol. 30, 987–998. https​://doi.org/10.1093/oxfor​djour​nals.pcp.a0778​44 (1989).
41. Havir, E. A. & McHale, N. A. Biochemical and developmental characterization of multiple forms of catalase in tobacco leaves.
Plant Physiol. 84, 450–455. https​://doi.org/10.1104/pp.84.2.450 (1987).
42. Karthik, N., Binod, P. & Pandey, A. Purification and characterisation of an acidic and antifungal chitinase produced by a Strepto-
myces sp. Biores. Technol. 188, 195–201. https​://doi.org/10.1016/j.biort​ech.2015.03.006 (2015).
43. Cheng, G. W. & Breen, P. J. Activity of phenylalanine ammonia-lyase (PAL) and concentrations of anthocyanins and phenolics in
developing strawberry fruit. J. Am. Soc. Hortic. Sci. 116, 865–869. https​://doi.org/10.21273​/jashs​.116.5.865 (1991).
44. Bates, L. S., Waldren, R. P. & Teare, I. D. Rapid determination of free proline for water-stress studies. Plant Soil 39, 205–207. https​
://doi.org/10.1007/bf000​18060​ (1973).
45. Singleton, V. L., Orthofer, R. & Lamuela-Raventós, R. M. Analysis of total phenols and other oxidation substrates and antioxidants
by means of folin-ciocalteu reagent. Methods Enzymol. 299, 152–178. https​://doi.org/10.1016/s0076​-6879(99)99017​-1 (1999).
46. Baquy, M.A.-A., Li, J.-Y., Xu, C.-Y., Mehmood, K. & Xu, R.-K. Determination of critical pH and Al concentration of acidic Ultisols
for wheat and canola crops. Solid Earth 8, 149–159. https​://doi.org/10.5194/se-8-149-2017 (2017).
47. Lucas, R. E. & Davis, J. F. Relationships between Ph values of organic soils and availabilities of 12 plant nutrients. Soil Sci. 92,
177–182. https​://doi.org/10.1097/00010​694-19610​9000-00005​ (1961).
48. Holland, J. E., White, P. J., Glendining, M. J., Goulding, K. W. T. & McGrath, S. P. Yield responses of arable crops to liming—An
evaluation of relationships between yields and soil pH from a long-term liming experiment. Eur. J. Agron. 105, 176–188. https​://
doi.org/10.1016/j.eja.2019.02.016 (2019).
49. Chakraborty, B. N., Chakraborty, U., Chakraborty, A. P. & Dey, P. L. Molecular identification and immunological characterization
of plant growth promoting rhizobacteria of Camellia sinensis. Int. J Tea Sci. 10, 41–52 (2014).
50. Upadhyay, S. K., Singh, D. P. & Saikia, R. Genetic diversity of plant growth promoting rhizobacteria isolated from rhizospheric
soil of wheat under saline condition. Curr. Microbiol. 59, 489–496. https​://doi.org/10.1007/s0028​4-009-9464-1 (2009).
51. Beneduzi, A., Peres, D., Vargas, L. K., Bodanese-Zanettini, M. H. & Passaglia, L. M. P. Evaluation of genetic diversity and plant
growth promoting activities of nitrogen-fixing bacilli isolated from rice fields in South Brazil. Appl. Soil. Ecol. 39, 311–320. https​
://doi.org/10.1016/j.apsoi​l.2008.01.006 (2008).
52. Jin, F. et al. Genetic diversity and phylogeny of antagonistic bacteria against phytophthora nicotianae isolated from tobacco rhizo-
sphere. Int. J. Mol. Sci. 12, 3055–3071. https​://doi.org/10.3390/ijms1​20530​55 (2011).
53. Fan, Z.-Y. et al. Diversity, distribution, and antagonistic activities of rhizobacteria of Panax notoginseng. J. Ginseng. Res. 40, 97–104.
https​://doi.org/10.1016/j.jgr.2015.05.003 (2016).
54. Spaepen, S. & Vanderleyden, J. Auxin and plant-microbe interactions. Cold Spring Harbor Perspect. Biol. 3, a001438–a001438.
https​://doi.org/10.1101/cshpe​rspec​t.a0014​38 (2010).
55. Mahdi, S. S., Talat, M. A., Dar, M. H., Hamid, A. & Ahmad, L. Soil phosphorus fixation chemistry and role of phosphate solubiliz-
ing bacteria in enhancing its efficiency for sustainable cropping–a review. J. Pure Appl. Microbiol. 6, 1905–1911 (2012).
56. Johnson, S. E. & Loeppert, R. H. Role of organic acids in phosphate mobilization from iron oxide. Soil Sci. Soc. Am. J. 70, 222–234.
https​://doi.org/10.2136/sssaj​2005.0012 (2006).
57. Berendsen, R. L., Pieterse, C. M. J. & Bakker, P. A. H. M. The rhizosphere microbiome and plant health. Trends Plant Sci. 17,
478–486. https​://doi.org/10.1016/j.tplan​ts.2012.04.001 (2012).

Scientific Reports | (2020) 10:15536 | https://doi.org/10.1038/s41598-020-72439-z 17

Vol.:(0123456789)
 www.nature.com/scientificreports/

58. Marques, A. P. G. C., Pires, C., Moreira, H., Rangel, A. O. S. S. & Castro, P. M. L. Assessment of the plant growth promotion abili-
ties of six bacterial isolates using Zea mays as indicator plant. Soil Biol. Biochem. 42, 1229–1235. https​://doi.org/10.1016/j.soilb​
io.2010.04.014 (2010).
59. Glick, B. R., Cheng, Z., Czarny, J. & Duan, J. Promotion of plant growth by ACC deaminase-producing soil bacteria. Eur. J. Plant
Pathol. 119, 329–339. https​://doi.org/10.1007/s1065​8-007-9162-4 (2007).
60. Lugtenberg, B. & Kamilova, F. Plant-growth-promoting rhizobacteria. Annu. Rev. Microbiol. 63, 541–556. https​://doi.org/10.1146/
annur​ev.micro​.62.08130​7.16291​8 (2009).
61. Saxena, A. K., Kumar, M., Chakdar, H., Anuroopa, N. & Bagyaraj, D. J. Bacillus species in soil as a natural resource for plant health
and nutrition. J. Appl. Microbiol. https​://doi.org/10.1111/jam.14506​ (2019).
62. Goswami, D., Thakker, J. N., Dhandhukia, P. C. & Tejada Moral, M. Portraying mechanics of plant growth promoting rhizobacteria
(PGPR): A review. Cogent Food Agric. https​://doi.org/10.1080/23311​932.2015.11275​00 (2016).
63. Pandey, A. & Palni, L. M. S. Bacillus species: The dominant bacteria of the rhizosphere of established tea bushes. Microbiol. Res.
152, 359–365. https​://doi.org/10.1016/s0944​-5013(97)80052​-3 (1997).
64. Choudhary, D. K., Prakash, A. & Johri, B. N. Induced systemic resistance (ISR) in plants: Mechanism of action. Indian J. Microbiol.
47, 289–297. https​://doi.org/10.1007/s1208​8-007-0054-2 (2008).
65. Liu, K., Newman, M., McInroy, J. A., Hu, C.-H. & Kloepper, J. W. Selection and assessment of plant growth-promoting rhizobacteria
for biological control of multiple plant diseases. Phytopathology 107, 928–936. https:​ //doi.org/10.1094/phyto-​ 02-17-0051-r (2017).
66. Sahni, S. & Prasad, B. D. Management of collar rot disease using vermicompost and a PGPR strain Pseudomonas sp and their effect
on defense-related enzymes in chickpea. Indian Phytopathol. https​://doi.org/10.1007/s4236​0-020-00203​-4 (2020).
67. Singh, D. P. et al. Microbial inoculation in rice regulates antioxidative reactions and defense related genes to mitigate drought
stress. Sci. Rep. https​://doi.org/10.1038/s4159​8-020-61140​-w (2020).
68. Caverzan, A. et al. Plant responses to stresses: role of ascorbate peroxidase in the antioxidant protection. Genet. Mol. Biol. 35,
1011–1019. https​://doi.org/10.1590/s1415​-47572​01200​06000​16 (2012).
69. Mittler, R. ROS are good. Trends Plant Sci. 22, 11–19. https​://doi.org/10.1016/j.tplan​ts.2016.08.002 (2017).
70. Grover, A. Plant chitinases: Genetic diversity and physiological roles. Crit. Rev. Plant Sci. 31, 57–73. https​://doi.org/10.1080/07352​
689.2011.61604​3 (2012).
71. Saravanakumar, D., Vijayakumar, C., Kumar, N. & Samiyappan, R. PGPR-induced defense responses in the tea plant against blister
blight disease. Crop Protect. 26, 556–565. https​://doi.org/10.1016/j.cropr​o.2006.05.007 (2007).
72. Roychoudhury, A., Basu, S. & Sengupta, D. N. Antioxidants and stress-related metabolites in the seedlings of two indica rice varie-
ties exposed to cadmium chloride toxicity. Acta Physiol. Plant. 34, 835–847. https​://doi.org/10.1007/s1173​8-011-0881-y (2011).
73. Shavalikohshori, O., Zalaghi, R., Sorkheh, K. & Enaytizamir, N. The expression of proline production/degradation genes under
salinity and cadmium stresses in Triticum aestivum inoculated with Pseudomonas sp. Int. J. Environ. Sci. Technol. 17, 2233–2242.
https​://doi.org/10.1007/s1376​2-019-02551​-9 (2019).
74. Szabados, L. & Savoure, A. Proline: A multifunctional amino acid. Trends Plant Sci. 15, 89–97. https​://doi.org/10.1016/j.tplan​
ts.2009.11.009 (2010).
75. Singh, R. P. & Jha, P. N. The PGPR stenotrophomonas maltophilia SBP-9 augments resistance against biotic and abiotic stress in
wheat plants. Front. Microbiol. https​://doi.org/10.3389/fmicb​.2017.01945​ (2017).
76. Town, J., Audy, P., Boyetchko, S. M. & Dumonceaux, T. J. High-quality draft genome sequence of Arthrobacter sp. OY3WO11, a
strain that inhibits the growth of phytophthora infestans. Genome Announc. https​://doi.org/10.1128/genom​eA.00585​-16 (2016).
77. Lim, H. S., Kim, Y. S. & Kim, S. D. Pseudomonas stutzeri YPL-1 genetic transformation and antifungal mechanism against fusarium
solani, an agent of plant root rot. Appl. Environ. Microbiol. 57, 510–516 (1991).
78. Sunar, K., Dey, P., Chakraborty, U. & Chakraborty, B. Biocontrol efficacy and plant growth promoting activity of Bacillus altitudinis
isolated from Darjeeling hills, India. J. Basic Microbiol. 55, 91–104. https​://doi.org/10.1002/jobm.20130​0227 (2015).
79. Asaka, O. & Shoda, M. Biocontrol of Rhizoctonia solani damping-off of tomato with Bacillus subtilis RB14. Appl. Environ. Microbiol.
62, 4081–4085 (1996).
80. Yu, X., Ai, C., Xin, L. & Zhou, G. The siderophore-producing bacterium, Bacillus subtilis CAS15, has a biocontrol effect on Fusarium
wilt and promotes the growth of pepper. Eur. J. Soil Biol. 47, 138–145. https​://doi.org/10.1016/j.ejsob​i.2010.11.001 (2011).
81. Agarwal, M. et al. Differential antagonistic responses of Bacillus pumilus MSUA3 against Rhizoctonia solani and Fusarium
oxysporum causing fungal diseases in Fagopyrum esculentum Moench. Microbiol. Res. 205, 40–47. https​://doi.org/10.1016/j.micre​
s.2017.08.012 (2017).
82. Priest, F. G. Biological control of mosquitoes and other biting flies by Bacillus sphaericus and Bacillus thuringiensis. J. Appl. Bacteriol.
72, 357–369. https​://doi.org/10.1111/j.1365-2672.1992.tb018​47.x (1992).
83. Valenzuela-Ruiz, V. et al. Draft genome sequence of Bacillus paralicheniformis TRQ65, a biological control agent and plant growth-
promoting bacterium isolated from wheat (Triticum turgidum subsp. durum) rhizosphere in the Yaqui Valley, Mexico. 3 Biotech
https​://doi.org/10.1007/s1320​5-019-1972-5 (2019).
84. Singh, R. K. et al. Optimization of media components for chitinase production by chickpea rhizosphere associatedLysinibacillus
fusiformisB-CM18. J. Basic Microbiol. 53, 451–460. https​://doi.org/10.1002/jobm.20110​0590 (2013).
85. Zang, C. et al. The biological control of the grapevine downy mildew disease using Ochrobactrum sp. Plant Prot. Sci. 56, 52–61.
https​://doi.org/10.17221​/87/2019-pps (2019).

Acknowledgements
This work was supported by an extramural Grant (No. 38(1475)/19/EMR-II) from the Council of Scientific &
Industrial Research (CSIR), Government of India. CB was supported by INSPIRE-Fellowship (IF150075/2015)
from SERB, India. Authors thank Prof. Gaurab Gangopadhyay, Mr. Vivek Gurung, Mr. Sabyasachi Majee, Mr.
Jadab K Ghosh, and Mr. Bipul Nag for their help in tea rhizosphere soil collection.

Author contributions
Abhrajyoti Ghosh conceived the project and collected the rhizosphere soil samples. C.B. isolated the bacterial
strains, performed biochemical, microbiological, and plant growth promotion experiments with the help of S.B.,
I.M., and U.A in rice and maize. C.B., S.B., U.A., and A.M. were involved in measuring antioxidative defense
molecules and consortia-based biocontrol experiments in rice. Abhrajyoti Ghosh, Anupama Ghosh, S.H., and
C.B. were involved in data analysis. A.H. helped in geographical analysis of the sampling sites. Abhrajyoti Ghosh,
Anupama Ghosh, and C.B. wrote the manuscript. All authors read and approved the final manuscript.

Competing interests
The authors declare no competing interest.

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