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 production 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://darjeeling.gov.in/agriculture.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 complicated 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 unavailable22. Previous studies 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 mechanisms2. Tea rhizosphere harbors diverse plant growth-promoting rhizobacteria, which offer potential applications as biofertilizers in sustainable agricultural practice within a similar agroclimatic setup12,13,14,15,16,18,49. 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 rhizobacterial 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.

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 available 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 genera12,14,15,49. Moreover, Bacillus genera are a dominant cultivable member in the rhizosphere soil of different plants, e.g., rice, wheat, tobacco, Panax notoginseng (Chinese ginseng), etc.50,51,52,53.

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

Figure 2

figure2

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.

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 different PGPR strains54. Previous studies have shown that tea rhizobacteria are efficient in IAA production in the presence of tryptophan15,16. 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).

Table 3 Functional screening of selected tea rhizobacteria for in vitro PGP and antifungal activities.

Microorganisms contribute to the natural phosphorous cycling by solubilizing precipitated and fixed phosphorous present in various soil types in a pH-dependent manner. In the acidic soil of the tea plantation, phosphorus 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 solubilizers 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 medium15,16. 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 phosphate56.

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 microenvironment57. 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 biomass58. 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 proteases 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 activity, 8 (26.6%) showed cellulase activity, and 12 (40%) exhibited ACC deaminase activity (Table 3). To this end, 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.

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, antimicrobial 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 agents15,63. In this study besides Bacillus, members of the genus such as Arthobacter (Arthrobacter sp. AB200), Exiguobacterium (Exiguobacterium mexicanum AB201), Leifsonia (Leifsonia lichenia AB203), Lysinibacillus (Lysinibacillus fusiformis AB332), Micrococcus (Micrococcus luteus AB321), Ochrobactrum (Ochrobactrum anthropi AB285, Ochrobactrum haematophilum AB286, Ochrobactrum haematophilum AB345), Pseudomonas (Pseudomonas stutzeri AB266), and Staphylococcus (Staphylococcus pasteuri AB212, Staphylococcus cohnii AB312, Staphylococcus gallinarum AB328, Staphylococcus 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. 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 comparison to uninoculated control plants (Fig. 4 and Fig. S2).

Figure 3

figure3

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).

Figure 4

figure4

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).

Effect of PGPR treatment on the defense-related enzymes in rice

Plant growth-promoting rhizobacteria (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) scavengers67. In the present study, we examined the status of APX, CAT, Chitinase, and PAL activities in the PGPR treated rice plants (Fig. 5 and Fig. S3).

Figure 5

figure5

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).

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 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 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 plants17,71. 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 metabolites 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 scavenging in the plants73. 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 proteins 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.

Figure 6

figure6

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).

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 tolerance75. In the present study, we measured the total accumulated 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.

Figure 7

figure7

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).

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 resistance 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 rhizobacterial 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. 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 phytopathogens79,80,81,82. Group VI members are comparatively less characterized rhizobacterial species (Table S1) and members like B. paralicheniformis, Lysinibacillus fusiformis, and Ochrobactrum sp. were recently reported to have biocontrol activities83,84,85.

Figure 8

figure8

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.

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