Global-level population genomics reveals differential effects of geography and phylogeny on horizontal gene transfer in soil bacteria.

Although microorganisms are known to dominate Earth's biospheres and drive biogeochemical cycling, little is known about the geographic distributions of microbial populations or the environmental factors that pattern those distributions. We used a global-level hierarchical sampling scheme to comprehensively characterize the evolutionary relationships and distributional limitations of the nitrogen-fixing bacterial symbionts of the crop chickpea, generating 1,027 draft whole-genome sequences at the level of bacterial populations, including 14 high-quality PacBio genomes from a phylogenetically representative subset. We find that diverse Mesorhizobium taxa perform symbiosis with chickpea and have largely overlapping global distributions. However, sampled locations cluster based on the phylogenetic diversity of Mesorhizobium populations, and diversity clusters correspond to edaphic and environmental factors, primarily soil type and latitude. Despite long-standing evolutionary divergence and geographic isolation, the diverse taxa observed to nodulate chickpea share a set of integrative conjugative elements (ICEs) that encode the major functions of the symbiosis. This symbiosis ICE takes 2 forms in the bacterial chromosome-tripartite and monopartite-with tripartite ICEs confined to a broadly distributed superspecies clade. The pairwise evolutionary relatedness of these elements is controlled as much by geographic distance as by the evolutionary relatedness of the background genome. In contrast, diversity in the broader gene content of Mesorhizobium genomes follows a tight linear relationship with core genome phylogenetic distance, with little detectable effect of geography. These results illustrate how geography and demography can operate differentially on the evolution of bacterial genomes and offer useful insights for the development of improved technologies for sustainable agriculture.

Biocontrol agents-mediated suppression of oxalic acid induced cell death during Sclerotinia sclerotiorum-pea interaction.

Oxalic acid (OA) is an important pathogenic factor during early Sclerotinia sclerotiorum-host interaction and might work by reducing hydrogen peroxide production (H2 O2 ). In the present investigation, oxalic acid-induced cell death in pea was studied. Pea plants treated with biocontrol agents (BCAs) viz., Pseudomonas aeruginosa PJHU15, Bacillus subtilis BHHU100, and Trichoderma harzianum TNHU27 either singly and/or in consortium acted on S. sclerotiorum indirectly by enabling plants to inhibit the OA-mediated suppression of oxidative burst via induction of H2 O2 . Our results showed that BCA treated plants upon treatment with culture filtrate of the pathogen, conferred the resistance via. significantly decreasing relative cell death of pea against S. sclerotiorum compared to control plants without BCA treatment but treated with the culture filtrate of the pathogen. The results obtained from the present study indicate that the microbes especially in consortia play significant role in protection against S. sclerotiorum by modulating oxidative burst and partially enhancing tolerance by increasing the H2 O2 generation, which is otherwise suppressed by OA produced by the pathogen.

Unraveling the efficient applications of secondary metabolites of various Trichoderma spp.

Recent shift in trends of agricultural practices from application of synthetic fertilizers and pesticides to organic farming has brought into focus the use of microorganisms that carryout analogous function. Trichoderma spp. is one of the most popular genera of fungi commercially available as a plant growth promoting fungus (PGPF) and biological control agent. Exploitation of the diverse nature of secondary metabolites produced by different species of Trichoderma augments their extensive utility in agriculture and related industries. As a result, Trichoderma has achieved significant success as a powerful biocontrol agent at global level. The endorsement of Trichoderma spp. by scientific community is based on the understanding of its mechanisms of action against a large set of fungal, bacterial and in certain cases viral infections. However, it is still an agnostic view that there could be any single major mode of operation, although it is argued that all mechanisms operate simultaneously in a synchronized fashion. The central idea behind this review article is to emphasize the potentiality of applications of target specific secondary metabolites of Trichoderma for controlling phytopathogens as a substitute of commercially available whole organism formulations. With the aim to this point, we have compiled an inclusive list of secondary metabolites produced by different species of Trichoderma and their applications in diverse areas with the major emphasis on agriculture. Outlining the importance and diverse activities of secondary metabolites of Trichoderma besides its relevance to agriculture would generate greater understanding of their other important and beneficial applications apart from target specific biopesticides.

Binding of stress-responsive OsWRKY proteins through WRKYGQK heptapeptide residue with the promoter region of two rice blast disease resistance genes Pi2 and Pi54 is important for development of blast resistance.

Molecular docking was done to investigate the interactions between five differentially expressed rice WRKY proteins when challenged with the rice blast disease caused by Magnaporthe oryzae and drought stresses applied either individually or overlapped, with the promoter region of two blast resistance genes (Pi2 and Pi54). Molecular docking was performed using the HDOCK server. Initially, the homology models for each of the five rice WRKY proteins were prepared using I-TASSER server, and then the secondary structure as well as the DNA-binding pockets were predicted using PSIPRED and BindUP servers, respectively. The molecular docking study revealed a differential binding pattern of the rice WRKYs with the two blast resistance genes. The WRKY proteins (OsWRKY88 and OsWRKY102), whose transcript levels decrease when drought and blast stresses are overlapped, interact with the two resistance genes mostly involving the residues of the zinc finger structure. On the other hand, the WRKY proteins (OsWRKY53-1 and OsWRKY113), whose transcript levels did not reduce significantly when challenged by drought and blast overlapped condition compared to individual treatment of blast, interact mostly involving the residues of the conserved WRKYGQK heptapeptide sequence. Interestingly, the protein OsWRKY74 whose transcript levels are unaffected in both individual and overlapped stresses, interacts with both the blast resistance genes involving few residues of both WRKYGQK heptapeptide and the zinc finger structure. The findings thus indicate that the interaction of OsWRKY proteins involving the conserved WRKYGQK heptapeptide sequence with the blast resistance genes Pi2 and Pi54 is important to mitigate the blast challenge in rice even during overlapping challenges of drought. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03711-y.

Metabolomics of early blight (Alternaria solani) susceptible tomato (Solanum lycopersicum) unfolds key biomarker metabolites and involved metabolic pathways.

Tomato (Solanum lycopersicum) is among the most important commercial horticultural crops worldwide. The crop quality and production is largely hampered due to the fungal pathogen Alternaria solani causing necrotrophic foliage early blight disease. Crop plants usually respond to the biotic challenges with altered metabolic composition and physiological perturbations. We have deciphered altered metabolite composition, modulated metabolic pathways and identified metabolite biomarkers in A. solani-challenged susceptible tomato variety Kashi Aman using Liquid Chromatography-Mass Spectrometry (LC-MS) based metabolomics. Alteration in the metabolite feature composition of pathogen-challenged (m/z 9405) and non-challenged (m/z 9667) plant leaves including 8487 infection-exclusive and 8742 non-infection exclusive features was observed. Functional annotation revealed putatively annotated metabolites and pathway mapping indicated their enrichment in metabolic pathways, biosynthesis of secondary metabolites, ubiquinone and terpenoid-quinones, brassinosteroids, steroids, terpenoids, phenylpropanoids, carotenoids, oxy/sphingolipids and metabolism of biotin and porphyrin. PCA, multivariate PLS-DA and OPLS-DA analysis showed sample discrimination. Significantly up regulated 481 and down regulated 548 metabolite features were identified based on the fold change (threshold ≥ 2.0). OPLS-DA model based on variable importance in projection (VIP scores) and FC threshold (> 2.0) revealed 41 up regulated discriminant metabolite features annotated as sphingosine, fecosterol, melatonin, serotonin, glucose 6-phosphate, zeatin, dihydrozeatin and zeatin-ß-D-glucoside. Similarly, 23 down regulated discriminant metabolites included histidinol, 4-aminobutyraldehyde, propanoate, tyramine and linalool. Melatonin and serotonin in the leaves were the two indoleamines being reported for the first time in tomato in response to the early blight pathogen. Receiver operating characteristic (ROC)-based biomarker analysis identified apigenin-7-glucoside, uridine, adenosyl-homocysteine, cGMP, tyrosine, pantothenic acid, riboflavin (as up regulated) and adenosine, homocyctine and azmaline (as down regulated) biomarkers. These results could aid in the development of metabolite-quantitative trait loci (mQTL). Furthermore, stress-induced biosynthetic pathways may be the potential targets for modifications through breeding programs or genetic engineering for improving crop performance in the fields.

Untargeted Metabolomics of Alternaria solani-Challenged Wild Tomato Species Solanum cheesmaniae Revealed Key Metabolite Biomarkers and Insight into Altered Metabolic Pathways.

Untargeted metabolomics of moderately resistant wild tomato species Solanum cheesmaniae revealed an altered metabolite profile in plant leaves in response to Alternaria solani pathogen. Leaf metabolites were significantly differentiated in non-stressed versus stressed plants. The samples were discriminated not only by the presence/absence of specific metabolites as distinguished markers of infection, but also on the basis of their relative abundance as important concluding factors. Annotation of metabolite features using the Arabidopsis thaliana (KEGG) database revealed 3371 compounds with KEGG identifiers belonging to biosynthetic pathways including secondary metabolites, cofactors, steroids, brassinosteroids, terpernoids, and fatty acids. Annotation using the Solanum lycopersicum database in PLANTCYC PMN revealed significantly upregulated (541) and downregulated (485) features distributed in metabolite classes that appeared to play a crucial role in defense, infection prevention, signaling, plant growth, and plant homeostasis to survive under stress conditions. The orthogonal partial least squares discriminant analysis (OPLS-DA), comprising a significant fold change (≥2.0) with VIP score (≥1.0), showed 34 upregulated biomarker metabolites including 5-phosphoribosylamine, kaur-16-en-18-oic acid, pantothenate, and O-acetyl-L-homoserine, along with 41 downregulated biomarkers. Downregulated metabolite biomarkers were mapped with pathways specifically known for plant defense, suggesting their prominent role in pathogen resistance. These results hold promise for identifying key biomarker metabolites that contribute to disease resistive metabolic traits/biosynthetic routes. This approach can assist in mQTL development for the stress breeding program in tomato against pathogen interactions.

Extending the benefits of PGPR to bioremediation of nitrile pollution in crop lands for enhancing crop productivity.

Incessant release of nitrile group of compounds such as cyanides into agricultural land through industrial effluents and excessive use of nitrile pesticides has resulted in increased nitrile pollution. Release of nitrile compounds (NCs) as plant root exudates is also contributing to the problem. The released NCs interact with soil elements and persists for a long time. Persistent higher concentration of NCs in soil cause toxicity to beneficial microflora and affect crop productivity. The NCs can cause more problems to human health if they reach groundwater and enter the food chain. Nitrile degradation by soil bacteria can be a solution to the problem if thoroughly exploited. However, the impact of such bacteria in plant and soil environments is still not properly explored. Plant growth-promoting rhizobacteria (PGPR) with nitrilase activity has recently gained attention as potential solution to address the problem. This paper reviews the core issue of nitrile pollution in soil and the prospects of application of nitrile degrading bacteria for soil remediation, soil health improvement and plant growth promotion in nitrile-polluted soils. The possible mechanisms of PGPR that can be exploited to degrade NCs, converting them into plant useful compounds and synthesis of the phytohormone IAA from degraded NCs are also discussed at length.

Characterization of sucrose non-fermenting-1 (SNF1) homologue gene in Fusarium udum WSP-V2 and its regulation by the biocontrol agent Pseudomonas fluorescens OKC.

Sucrose non-fermenting 1 (SNF1) is a protein kinase and plays an important role in the energy homeostasis of glucose repressible gene transcription. It derepresses glucose repressed genes and associated with pathogenesis and production of cell wall degrading enzymes in fungal species. In the present study, we identified and characterized SNF1 homologue FuSNF1 in the F. udum strain WSP-V2. Transcript analysis of FuSNF1 along with the MAP kinases and some cell wall degrading enzyme (CWDE) genes of F. udum during interaction with pigeonpea revealed that most MAP kinases and CWDE genes was positively correlated with the FuSNF1 gene. Interestingly, transcript accumulation of all these genes was lowered when pigeonpea seeds were bioprimed with a PGPR strain Pseudomonas fluorescens OKC. Transcript accumulation of FuSNF1 was observed from the day of inoculation and reached maximum level on day 7 in OKC non-bioprimed plants. However, transcript accumulation was low (1.5 fold) in F. udum inoculated with pigeonpea plants bioprimed with OKC. Transcript accumulation patterns of the F. udum MAP Kinases genes and CWDE genes also showed a similar trend and their transcript accumulation was lowered in the OKC bioprimed treatment. The results thus indicate a prime role of FuSNF1 in regulating pathogenicity and virulence of F. udum. The results further emphasize the importance of application of effective PGPR strains in regulating virulence of F. udum. In silico analysis of the SNF1 reference proteins from different fungal species showed that their homologue FuSNF1 is likely to be thermostable and acidic in nature.

Plant G-protein signaling cascade and host defense.

The heterotrimeric guanine-nucleotide-binding proteins (G-proteins) play a crucial role in signal transduction and regulate plant responses against biotic and abiotic stresses. Necrotrophic pathogens trigger Gα subunit and, in contrast, sometimes Gßγ dimers. Beneficial microbes play a vital role in the activation of heterotrimeric G-proteins in plants against biotrophic and necrotrophic pathogens. The subunits of G-protein (α, ß, and γ) are activated differentially against different kinds of pathogens which in turn regulates the entry of the pathogen in a plant cell. Defense mediated by G-proteins in plants imparts resistance against several pathogens. Activation of different G-protein subunits depends on the mode of nutrition of the pathogen. The current review discussed the role of the three subunits against various pathogens. It appeared to be specific in the individual host-pathogen system as well as the role of effectors in the induction of G-proteins. We also discussed the G-protein-mediated production of reactive oxygen species (ROS), including H2O2, activation of NADPH oxidases, hypersensitive response (HR), phospholipases, and ion channels in response to microorganisms.

Trichoderma asperellum T42 induces local defense against Xanthomonas oryzae pv. oryzae under nitrate and ammonium nutrients in tobacco.

Trichoderma has been explored and found to play a vital role in the defense mechanism of plants. However, its effects on host disease management in the presence of N nutrients remains elusive. The present study aimed to assess the latent effects of Trichoderma asperellum T42 on oxidative burst-mediated defense mechanisms against Xanthomonas oryzae pv. oryzae (Xoo) in tobacco plants fed 10 mM NO3 - and 3 mM NH4 + nutrients. The nitrate-fed tobacco plants displayed an increased HR when Xoo infected, which was enhanced in the Trichoderma-treated plants. This mechanism was enhanced by the involvement of Trichoderma, which elicited NO production and enhanced the expression pattern of NO-modulating genes (NR, NOA and ARC). The real-time NO fluorescence intensity was alleviated in the NH4 +-fed tobacco plants compared to that fed NO3 - nutrient, suggesting the significant role of Trichoderma-elicited NO. The nitrite content and NR activity demonstration further confirmed that nitrate metabolism led to NO generation. The production of ROS (H2O2) in the plant leaves well-corroborated that the disease resistance was mediated through the oxidative burst mechanism. Nitrate application resulted in greater ROS production compared to NH4 + nutrient after Xoo infection at 12 h post-infection (hpi). Additionally, the mechanism of enhanced plant defense under NO3 - and NH4 + nutrients mediated by Trichoderma involved NO, ROS production and induction of PR1a MEK3 and antioxidant enzyme transcription level. Moreover, the use of sodium nitroprusside (100 µM) with Xoo suspension in the leaves matched the disease resistance mediated via NO burst. Altogether, this study provides novel insights into the fundamental mechanism behind the role of Trichoderma in the activation of plant defense against non-host pathogens under N nutrients.

A novel function of N-signaling in plants with special reference to Trichoderma interaction influencing plant growth, nitrogen use efficiency, and cross talk with plant hormones.

Trichoderma spp. is considered as a plant growth promoter and biocontrol fungal agents. They colonize on the surface of root in most of the agriculture crops. They secrete different secondary metabolites and enzymes which promote different physiological processes as well as protect plants from various environmental stresses. This is part of their vital functions. They are widely exploited as a biocontrol agent and plant growth promoter in agricultural fields. Colonization of Trichoderma with roots can enhance nutrient acquisition from surrounding soil to root and can substantially increase nitrogen use efficiency (NUE) in crops and linked with activation of plant signaling cascade. Among Trichoderma species, only some Trichoderma species were well characterized which help in the uptake of nitrogen-containing compound (especially nitrate form) and induced nitric oxide (NO) in plants. Both nitrate and NO are known as a signaling agent, involved in plant growth and development and disease resistance. Activation of these signaling molecules may crosstalk with other signaling molecule (Ca2+) and phytohormone (auxin, gibberellins, cytokinin and ethylene). This ability of Trichoderma is important to agriculture not only for increased plant growth but also to control plant diseases. Recently, Trichoderma strains have been shown to encompass the ability to regulate transcripts level of high-affinity nitrate transporters and probably it was positively regulated by NO. This review aims to focus the usage of Trichoderma strains on crops by their abilities to regulate transcript levels, probably through activation of plant N signaling transduction that improve plant health.

Designing and experimental evaluation of gene-specific primers of pea (Pisum sativum) defense proteins.

Real-time gene expression analysis by semi-quantitative and quantitative RT-PCR requires a set of gene-specific primers which should have the ability to amplify the gene of interest specifically. In the present study, we have standardized certain parameters for primer design using the freely available Primer3 software. We have designed the primers for defense genes such as ICS (isochorismate synthase), CCoAOMT (caffeoyl CoA O-methyltransferase), C4H (cinnamate 4-hydroxylase), and G-alpha in pea. We have also discussed, the way of sequence retrieval, when the sequence is not reported in the organism of interest. We have evaluated the designed primers using cDNA prepared from mRNA isolated from the pea leaves. By analyzing the results, we have found that primers are perfectly binding with the target and giving single sharp band on a DNA electrophoresis gel. It can be concluded that the parameters used for primer designing by Primer3 play a critical role in the experimental results and parameters defined in the present study resulting in a very good amplification during PCR.

Modulation in phenolic root exudate profile of Abelmoschus esculentus expressing activation of defense pathway.

Phenolics play a key role in communication between plants and microbes in the rhizosphere. In this study, shikimic, gallic, fumaric, ferulic, vanillic acid and quercetin in root exudates of Abelmoschus esculentus act as chemoattractants of endophytic Alcaligenes faecalis strains, BHU 12, BHU 16 and BHU M7. In vitro chemotaxis assay showed that BHU 12 expressed highest chemotactic movement (CFU ∼50×1012) towards A.esculentus root exudates followed by BHU 16 and BHU M7 (CFU∼ 9×1012), thereby confirming their ability to colonize the host rhizoplane region. However, BHU 16 expressed highest biofilm formation ability followed by BHU 12 and BHU M7. Assessment of chemotactic and biofilm formation potential towards individual phenolic acids revealed BHU 12 to be maximally attracted towards 1µM shikimic acid (2×1015) while BHU 16 towards 1mM vanillic acid (6.5×1012) and BHU M7 towards 1mM ferulic acid (3.5×1012), thereby confirming the phenolic acid components responsible for particularly attracting the endophytic isolates. Upon colonization, the endophytic isolates modified the phenolic profiles of root exudates in planta in a manner so as to plausibly attract more of the beneficial rhizospheric microbiota as well as self-fortification against pathogenic microbes. This hypothesis was verified by monitoring the changes in phenolic components of A. esculentus root exudate owing to S. rolfsii infection, a disastrous soil-borne pathogen. Thus, on the whole, the work provides intricate details of plant-endophyte interactions for biotic stress management through careful manipulation of root exudates, thereby aiding in sustainable agriculture.

Trichoderma asperellum spore dose depended modulation of plant growth in vegetable crops.

In the present study we bioprimed seeds of six vegetable crops (tomato, brinjal, chilli, okra, ridge gourd and guar) with different spore doses of Trichoderma asperellum BHUT8 ranging from 102 to 108 spores ml-1 and the effect of biopriming was seen on seed germination and development. The most effective spore dose for enhancement in seed germination and radicle length was found to be 103 spores ml-1 in tomato and ridge gourd, 104 spores ml-1 in brinjal and okra while 106 spores ml-1 in chilli and guar. At the most effective spore dose, the increase in germination percentage was 5, 146.15, 112.5, 5.4, 28.13 and 0% while increase in radicle length was 73.17, 50.83, 171.6, 107.35, 247.19 and 90.79% in tomato, brinjal, chilli, okra, ridge gourd and guar, respectively, compared to their controls. Higher spore dose i.e. 107-108 spores ml-1 and 106-108 spores ml-1 in tomato and brinjal, respectively, reduced seed germination percentage and radicle growth compared to their controls. Biopriming with T. asperellum BHUT8 also triggered various defense like responses such as high phenylpropanoid activities and lignifications in bioprimed tomato seedlings compared to the non-bioprimed tomato seedlings demonstrating possible use of BHUT8 against phytopathogens.

Biochemical and histochemical analyses revealing endophytic Alcaligenes faecalis mediated suppression of oxidative stress in Abelmoschus esculentus challenged with Sclerotium rolfsii.

Sclerotium rolfsii is a highly aggressive pathogen that causes huge economic losses, especially in temperate climates. Alcaligenes faecalis, particularly in endophytic form, has rarely been used to control this fungus. In this study, endophytic Alcaligenes sp. strain BHU 12, BHU 16 (isolated from Abelmoschus esculentus leaf) and BHU M7 (isolated from Andrographis paniculata leaf) were reported to trigger a wide range of host defenses in Okra plant against the collar-rot pathogen S. rolfsii. Endophytic colonization of the strains in ten days old plants was assessed through re-isolation of the rif-tagged strains on rifampicin augmented nutrient agar media. The ability of the endophytic strains to induce systemic defense responses in above-ground organs was assessed by collecting leaf tissues of the Okra plants grown under non-gnotobiotic conditions at different time intervals post seedling bacterization with the endophytic biocontrol agents. The pathogen challenged unprimed plants exhibited flaccidity of the stem and leaves at 48 h post infection (hpi) in contrast to the bioprimed and challenged plants. Biochemical and histochemical analyses explained the above phenomenon as activation of phyto-peroxidases leading to an increased metabolism of the reactive oxygen species (ROS), accompanied by activation of the phenylpropanoid network and a subsequent enhancement in plant phenolics. Interestingly, though the maximum increase in the defense pathways was observed in treatments with native endophytes of Okra plant, yet the enhancement in antioxidant pathway due to A. paniculata borne endophytes was also quite significant. Thus, this work clearly demonstrates how Okra plants respond to the "non-hostile" colonization of bacterial endophytes and how induced defense response can contribute to the biocontrol activity of the endophytic strains.

Enhancing Plant Disease Resistance without R Genes.

Crop plants encounter constant biotic challenges, and these challenges have historically been best managed with resistance (R) genes. However, the rapid evolution of new pathogenic strains along with the nonavailability or nonidentification of R genes in cultivated crop species against a large number of plant pathogens have led researchers to think beyond R genes. Biotechnological tools have shown promise in dealing with such challenges. Technologies such as transgenerational plant immunity, interspecies transfer of pattern recognition receptors (PRRs), pathogen-derived resistance (PDR), gene regulation, and expression of antimicrobial peptides (AMPs) in host plants from other plant species have led to enhanced disease resistance and increased food security.

Rhizosphere competent microbial consortium mediates rapid changes in phenolic profiles in chickpea during Sclerotium rolfsii infection.

The present study was carried out with the aim of evaluating the effectiveness and potentiality of three compatible rhizosphere microbes, viz., fluorescent Pseudomonas aeruginosa (PHU094), Trichoderma harzianum (THU0816) and Mesorhizobium sp. (RL091), in promoting plant growth and mobilizing phenolic acid biosynthesis in chickpea under challenge of Sclerotium rolfsii. The microbes were applied as seed coating in different combinations in two experimental sets and the pathogen was inoculated after 25 days of sowing in one set. Results revealed that microbe application led to higher growth in chickpea particularly in the triple microbe combination compared to their individual treatments and control. Similarly, pathogen challenged plants accumulated higher amount of phenolic compounds both at the site of attack of the pathogen i.e. collar region as well as leaves compared to unchallenged plants. All the bioagents were found to trigger the level of phenolic compounds at collar region in varying degrees as compared to the healthy control (A). However, the most effective treatment was D7 (combined application of PHU094, THU0816 and RL091 with pathogen challenge) among all the treatments. Shikimic acid was maximally induced amongst all the phenolic compounds. In leaves also, the most effective treatment was D7 where shikimic acid, t-chlorogenic acid, ferulic acid, myricetin, quercetin and syringic acid were produced in higher amounts as compared to treatment B where the plants were challenged only with the pathogen.

Compatible rhizosphere microbes mediated alleviation of biotic stress in chickpea through enhanced antioxidant and phenylpropanoid activities.

The study was conducted to examine efficacy of a rhizospheric microbial consortium comprising of a fluorescent Pseudomonas (PHU094), Trichoderma (THU0816) and Rhizobium (RL091) strain on activation of physiological defense responses in chickpea against biotic stress caused by the collar rot pathogen Sclerotium rolfsii. Results of individual microbes were compared with dual and triple strain mixture treatments with reduced microbial load (1/2 and 1/3rd, respectively, of individual microbial load compared to single microbe application) in the mixtures. Periodical studies revealed maximum activities of phenylalanine ammonia lyase [E.C. 4.1.3.5] and polyphenol oxidase [E.C. 1.14.18.1] and accumulation of total phenol content in chickpea in the triple microbe consortium treated plants challenged with the pathogen compared to the single microbe and dual microbial consortia. Similarly, the expression of the antioxidant enzymes superoxide dismutase [E.C.1.15.1.1] and peroxidase [E.C.1.11.1.7] was also highest in the triple microbial consortium which was correlated with lesser lipid peroxidation in chickpea under the biotic stress. Histochemical staining clearly showed maximum and uniform lignification in vascular bundles of chickpea stem sections treated with the triple microbes. The physiological responses were directly correlated with the mortality rate as least plant mortality was recorded in the triple microbe consortium treated plants. The results thus suggest an augmented elicitation of stress response in chickpea under S. rolfsii stress by the triple microbial consortium in a synergistic manner under reduced microbial load.