Organic amendments modulate the crop yield and rhizospheric bacterial community diversity: a 3-year field study with Cajanus cajan / Las enmiendas orgánicas modulan el rendimiento de los cultivos y la diversidad de la comunidad bacteriana rizosférica: un estudio de campo de 3 años con Cajanus cajan

Excessive use of chemicals to enhance soil nutrient status and crop yield has resulted in a decline in soil health. Organic farming promotes organic amendments, which help to balance the ecosystem. Understanding the dynamic patterns of belowground microbial populations is essential for developing sustainable agricultural systems. Therefore, the study was designed to evaluate the effect of different agri-practices on rhizospheric bacterial diversity and crop yield in an Indian agricultural system. A 3-year field experiment was set up in a randomized block design using Cajanus cajan as a model crop, comparing conventional farming with organic practice (with animal manure and bio-compost as amendments). Plant and rhizospheric soil samples were collected at the harvest stage for assessing various growth attributes, and for characterizing rhizospheric bacterial diversity. Enhanced crop productivity was seen in conventional farming, with a 2.2-fold increase in grain yield over control. However, over the 3 years, an overall positive impact was observed in the bio-compost-based organic amendment, in terms of bacterial abundance, over other treatments. At the harvest stage of the third cropping season, the bacterial diversity in the organic treatments showed little similarity to the initial bacterial community composition of the amendment applied, indicating stabilization along the growth cycles. The study emphasizes the significance of the choice of the amendment for ushering in agricultural sustainability.(AU)

Organic amendments modulate the crop yield and rhizospheric bacterial community diversity: a 3-year field study with Cajanus cajan.

Excessive use of chemicals to enhance soil nutrient status and crop yield has resulted in a decline in soil health. Organic farming promotes organic amendments, which help to balance the ecosystem. Understanding the dynamic patterns of belowground microbial populations is essential for developing sustainable agricultural systems. Therefore, the study was designed to evaluate the effect of different agri-practices on rhizospheric bacterial diversity and crop yield in an Indian agricultural system. A 3-year field experiment was set up in a randomized block design using Cajanus cajan as a model crop, comparing conventional farming with organic practice (with animal manure and bio-compost as amendments). Plant and rhizospheric soil samples were collected at the harvest stage for assessing various growth attributes, and for characterizing rhizospheric bacterial diversity. Enhanced crop productivity was seen in conventional farming, with a 2.2-fold increase in grain yield over control. However, over the 3 years, an overall positive impact was observed in the bio-compost-based organic amendment, in terms of bacterial abundance, over other treatments. At the harvest stage of the third cropping season, the bacterial diversity in the organic treatments showed little similarity to the initial bacterial community composition of the amendment applied, indicating stabilization along the growth cycles. The study emphasizes the significance of the choice of the amendment for ushering in agricultural sustainability.

Antagonistic Activity of Pseudomonas fluorescens Strain X1 Against Different Fusaria and it's In Vivo Analysis Against Fusarium udum Infected Pigeon Pea.

A plant growth-promoting rhizobacterial strain, Pseudomonas fluorescens X1 isolated from the garden soil was employed for antagonistic activity against different species of fusaria. Strain X1 inhibited four different fusaria (Fusarium moniliforme, Fusarium oxysporum, Fusarium semitectum and Fusarium udum) in dual culture plate assay, and in broth culture using cell-free culture filtrate. Scanning electron microscopic (SEM) analysis revealed deformation and shrinkage in mycelia of fusaria after treatment with strain X1. Confocal micrographs showed degeneration of nuclei inside the cells of fusaria for the same effect. Strain X1 exhibited maximum antifungal activity, when it was grown in nutrient broth yeast (NBY) medium amended with 1 mM NH4MoO4 and 1% glucose. The antifungal extracts eluted from thin-layer chromatography (TLC) followed by high performance liquid chromatography (HPLC) showed two fractions active against different fusaria. Liquid chromatography-mass spectrometry (LCMS) analysis of the two fractions 1 and 2 corresponded to molecular ions at m/z 177.16 and m/z 177.09, respectively. Infra-red (IR) analysis showed five similar absorption bands in both the fractions analysed. In vivo analysis of strain X1 alone and along with fungicide inhibited the growth of F. udum and improved the biomass and growth of pigeon pea. These results indicated that strain X1 could be possibly used as a biocontrol agent to inhibit the growth of soil-borne diseases of different fusaria including F. udum that causes wilting in pigeon pea.

Employing mutants to study the role of a three-membered bacterial consortium in growth promotion of Cajanus cajan.

Bacterial consortia, comprising plant growth promoting (PGP) rhizobacteria, are known to outcompete their impacts on plant attributes compared to their individual application. However, tracking of individual bacterial strains post application as consortium, remains challenging. The primary goal of this study was to develop an efficient method of tracking bioinoculants by generating spontaneous mutants of three different bacterial strains in an established consortium, using antibiotic-based screening, followed by their enumeration after application in Cajanus cajan. Mutants were generated for consortium members, viz. Azotobacter chroococcum (A), Priestia megaterium (formerly Bacillus megaterium) (B), and Pseudomonas sp. (P), against streptomycin, kanamycin and rifampicin, respectively. Those mutants having similar growth rates and PGP properties as compared to wild type bacterial strains were selected to test their efficacy in plant growth promotion. Selected mutant strains were applied as mono, dual and triple cultures to C. cajan grown hydroponically. Enumeration of mutant bacterial strains was carried out to check their viability. Bacterial colonization on roots was also analyzed. The application of triple (mutant) inoculants improved plant growth attributes significantly in comparison to mono and dual culture treatments and control. Cell enumeration revealed that the abundance of each bacterial strain increased till the 5th day of treatment. No significant change was observed later in their abundance for any treatment. The triple culture treatment showed greater abundance of bacterial mutant strains in comparison to mono- or dual cultures. To the best of our knowledge, this is the first mutant-based study to have reported the successful tracking and enumeration of bacterial consortium members, post their application in C. cajan.

Plant Growth Promoting Bacterial Consortium Induces Shifts in Indigenous Soil Bacterial Communities and Controls Listeria monocytogenes in Rhizospheres of Cajanus cajan and Festuca arundinacea.

The rhizosphere is a dynamic and complex interface between plant roots and microorganisms. Owing to exudates, a web of interactions establishes among the microbial members of this micro-environment. The present study explored the impact of a bacterial consortium (Azotobacter chroococcum, Bacillus megaterium and Pseudomonas fluorescens, ABP), on the fate of a human pathogen, Listeria monocytogenes EGD-e, in soil and in the rhizospheres of Cajanus cajan and Festuca arundinacea, in addition to its plant growth promoting effect. The study further assessed the impact these bioinoculants exert on the autochthonous soil bacterial communities. Experiments in sterilised soil inoculated with bioinoculants and L. monocytogenes revealed the inhibition of L. monocytogenes by approximately 80-fold compared to that without the consortium. Subsequently, experiments were conducted in non-sterile soil microcosms planted with C. cajan and F. arundinacea, and in bulk soil. The consortium led to a significant increase in plant growth in both plants and prevented growth of L. monocytogenes. However, the presence of resident soil bacterial communities overshadowed this inhibitory effect, and a sharp decline in L. monocytogenes populations (5-6 log reduction) was recorded under non-sterile soil conditions. A shift in the soil resident bacterial communities was observed upon amendment with the bioinoculants. A significant increase of potential Plant Growth Promoting Rhizobacteria (PGPR) and biocontrol agents was observed, while the abundance of potential phytopathogens dropped. The present study opens up new avenues for the application of such a consortium given their dual benefits of plant growth promotion and restricting phytopathogens as well as human pathogen.

GC-MS and LC-MS: an integrated approach towards the phytochemical evaluation of methanolic extract of Pigeon Pea [Cajanus cajan (L.) Millsp] leaves.

Pigeon Pea is a well-received traditional nutritious and medicinal plant in India since ancient times. The plant is identified for its impressive pharmacological activities like antimicrobial, anti-inflammatory, antitubercular, antioxidant, neuroprotective, antihypertensive, antihyperglycemic and anticancer properties. The metabolites from Pigeon Pea leaves methanolic extract were identified using hyphenated instruments namely Gas Chromatography-Mass Spectroscopy (GC-MS) and Liquid Chromatography-Mass Spectroscopic (LC-MS) method. GC and LC chromatogram showed proper separation and presence of several bioactive compounds such as flavonoids, terpenoids, essential oils, alkaloids, and coumarins. LC-MS investigation reported the presence of Selidin (coumarin) and Tamarixetin (monomethoxyflavone) in the Pigeon Pea Leaves. The present investigations have demonstrated that methanolic extract of Pigeon Pea leaves exhibited impressive antibacterial activity against E. coli and antifungal activity against C. albicans with MIC values 50 µg/ml 250 µg/ml respectively. The extracts studied here have demonstrated to be a new remedial source used against diseases caused by these microorganisms.

Comparative transcriptomic profiling of susceptible and resistant cultivars of pigeonpea demonstrates early molecular responses during Fusarium udum infection.

Vascular wilt caused by Fusarium udum Butler is the most important disease of pigeonpea throughout the world. F. udum isolate MTCC 2204 (M1) inoculated pigeonpea plants of susceptible (ICP 2376) and resistant (ICP 8863) cultivars were taken at invasion stage of pathogenesis process for transcriptomic profiling to understand defense signaling reactions that interplay at early stage of this plant-pathogen encounter. Differential transcriptomic profiles were generated through cDNA-AFLP from M1 inoculated resistant and susceptible pigeonpea root tissues. Twenty five percent of transcript derived fragments (TDFs) were found to be pathogen induced. Among them 73 TDFs were re-amplified and sequenced. Homology search of the TDFs in available databases and thorough study of scientific literature identified several pathways, which could play crucial role in defense responses of the F. udum inoculated resistant plants. Some of the defense responsive pathways identified to be active during this interaction are, jasmonic acid and salicylic acid mediated defense responses, cell wall remodeling, vascular development and pattering, abscisic acid mediated responses, effector triggered immunity, and reactive oxygen species mediated signaling. This study identified important wilt responsive regulatory pathways in pigeonpea which will be helpful for further exploration of these resistant components for pigeonpea improvement.

Poor Competitiveness of Bradyrhizobium in Pigeon Pea Root Colonization in Indian Soils.

Pigeon pea, a legume crop native to India, is the primary source of protein for more than a billion people in developing countries. The plant can form symbioses with N2-fixing bacteria; however, reports of poor crop nodulation in agricultural soils abound. We report here a study of the bacterial community associated with pigeon pea, with a special focus on the symbiont population in different soils and vegetative and non-vegetative plant growth. Location with respect to the plant roots was determined to be the main factor controlling the bacterial community, followed by developmental stage and soil type. Plant genotype plays only a minor role. Pigeon pea roots have a reduced microbial diversity compared to the surrounding soil and select for Proteobacteria, especially for Rhizobium spp., during vegetative growth. While Bradyrhizobium, a native symbiont of pigeon pea, can be found associating with roots, its presence is dependent on plant variety and soil conditions. A combination of 16S rRNA gene amplicon survey, strain isolation, and co-inoculation with nodule-forming Bradyrhizobium spp. and non-N2-fixing Rhizobium spp. demonstrated that the latter is a much more successful colonizer of pigeon pea roots. Poor nodulation of pigeon pea in Indian soils may be caused by a poor Bradyrhizobium competitiveness against non-nodulating root colonizers such as Rhizobium. Hence, inoculant strain selection of symbionts for pigeon pea should be based not only on their nitrogen fixation potential but, more importantly, on their competitiveness in agricultural soils. IMPORTANCE Plant symbiosis with N2-fixing bacteria is a key to sustainable, low-input agriculture. While there are ongoing projects aiming to increase the yield of cereals using plant genetics and host-microbiota interaction engineering, the biggest potential lies in legume plants. Pigeon pea is a basic food source for a billion low-income people in India. Improving its interactions with N2-fixing rhizobia could dramatically reduce food poverty in India. Despite the Indian origin of this plant, pigeon pea nodulates only poorly in native soils. While there have been multiple attempts to select the best N2-fixing symbionts, there are no reliable strains available for geographically widespread use. In this article, using 16S rRNA gene amplicon, culturomics, and plant co-inoculation assays, we show that the native pigeon pea symbionts such as Bradyrhizobium spp. are able to nodulate their host, despite being poor competitors for colonizing roots. Hence, in this system, the establishment of effective symbiosis seems decoupled from microbial competition on plant roots. Thus, the effort of finding suitable symbionts should focus not only on their N2-fixing potential but also on their ability to colonize. Increasing pigeon pea yield is a low-hanging fruit to reduce world hunger and degradation of the environment through the overuse of synthetic fertilizers.

Effect of substitution of chemical fertilizer by bioinoculants on plant performance and rhizospheric bacterial community: case study with Cajanus cajan.

Improper nutrient management is one of the major limitations linked with cultivation of Cajanus cajan. This calls for an urgent need for a promising alternative, employing both bioinoculants and chemical fertilizer. Present study attempted to understand the impact of bioinoculants {Azotobacter chroococcum, Bacillus megaterium, and Pseudomonas fluorescens (ABP)} as their mono-inoculations, triple-inoculation, and their combination with different doses of fertilizer on (a) plant parameters, (b) soil nitrogen (N) economy, (c) resident bacterial community, (d) genes and transcripts involved in N cycle, and to evaluate the extent to which fertilizer could be replaced by ABP without compromising on grain yield. Bradyrhizobium sp. was used in all the treatments (as it was recommended for C. cajan). Combined application of bioinoculants and 75% of recommended dose of fertilizer (RDF) led to 1.28-fold enhancement in grain yield as compared to RDF alone. Apart from exerting a positive impact on grain yield, the combined application of ABP and fertilizer led to an improvement in soil fertility, and modified the culturable rhizospheric bacterial community involved in N cycle. Integrated use of bioinoculants and fertilizer led to better N substrate utilization and hence, metabolic diversity when compared with application of fertilizer alone. An increase in the transcripts of nifH gene at the harvest stage in the soil treated with ABP alone and its combination with fertilizer, over individual treatment with fertilizer was observed. The combined use of ABP and fertilizer shaped the resident bacterial community towards a more beneficial community, which helped in increasing soil nitrogen turnover and hence, soil fertility as a whole.

Complete genome sequence of sixteen plant growth promoting Streptomyces strains.

The genome sequences of 16 Streptomyces strains, showing potential for plant growth-promotion (PGP) activities in rice, sorghum, chickpea and pigeonpea, isolated from herbal vermicompost, have been decoded. The genome assemblies of the 16 Streptomyces strains ranged from 6.8 Mb to 8.31 Mb, with a GC content of 72 to 73%. The extent of sequence similarity (in terms of shared ortholog) in 16 Streptomyces strains showed 70 to 85% common genes to the closest publicly available Streptomyces genomes. It was possible to identify ~1,850 molecular functions across these 16 strains, of which close to 50% were conserved across the genomes of Streptomyces strains, whereas, ~10% were strain specific and the rest were present in various combinations. Genome assemblies of the 16 Streptomyces strains have also provided genes involved in key pathways related to PGP and biocontrol traits such as siderophores, auxin, hydrocyanic acid, chitinase and cellulase. Further, the genome assemblies provided better understanding of genetic similarity among target strains and with the publically available Streptomyces strains.

Biochemical, functional and molecular characterization of pigeon pea rhizobia isolated from semi-arid regions of India.

Pigeon pea (Cajanus cajan (L.) Millspaugh) is among the top ten legumes grown globally not only having high tolerance to environmental stresses along, but also has the high biomass and productivity with optimal nutritional profiles. In the present study, 55 isolates of rhizobia were identified from 22 nodule samples of pigeon pea collected from semi-arid regions of India on the basis of morphological, biochemical, plant growth promoting activities and their ability to tolerate the stress conditions viz. pH, salt, temperature and drought stress. Amongst all the 55 isolates, 37 isolates showed effective nodulation under in vitro conditions in pigeon pea. Further, five isolates having multiple PGP activities and high in vitro symbiotic efficiency were subjected to 16S rRNA sequencing and confirmed their identities as Rhizobium, Mesorhizobium, Sinorhizobium sp. Further these 37 isolates were characterized at molecular level using ARDRA and revealed significant molecular diversity. Based on UPGMA clustering analysis, these isolates showed significant molecular diversity. The high degree of molecular diversity is due to mixed cropping of legumes in the region. The assessment of genetic diversity and molecular characterization of novel strains is a very important tool for the replacement of ineffective rhizobial strains with the efficient strains for the improvement in the nodulation and pigeon pea quality. The pigeon pea isolates with multiple PGPR activities could be further used for commercial production.

Rhizodegradation of Petroleum Oily Sludge-contaminated Soil Using Cajanus cajan Increases the Diversity of Soil Microbial Community.

Most components of petroleum oily sludge (POS) are toxic, mutagenic and cancer-causing. Often bioremediation using microorganisms is hindered by the toxicity of POS. Under this circumstance, phytoremediation is the main option as it can overcome the toxicity of POS. Cajanus cajan a legume plant, was evaluated as a phyto-remediating agent for petroleum oily sludge-spiked soil. Culture dependent and independent methods were used to determine the rhizosphere microorganisms' composition. Degradation rates were estimated gravimetrically. The population of total heterotrophic bacteria (THRB) was significantly higher in the uncontaminated soil compared to the contaminated rhizosphere soil with C. cajan, but the population of hydrocarbon-utilizing bacteria (HUB) was higher in the contaminated rhizosphere soil. The results show that for 1 to 3% oily sludge concentrations, an increase in microbial counts for all treatments from day 0 to 90 d was observed with the contaminated rhizosphere CR showing the highest significant increase (p < 0.05) in microbial counts compared to other treatments. The metagenomic study focused on the POS of 3% (w/w) and based on the calculated bacterial community abundance indices showed an increase in the values for Ace, Cho, Shannon (Shannon-Weaver) and the Simpson's (measured as InvSimpson) indices in CR3 compared to CN3. Both the Simpson's and the Shannon values for CR3 were higher than CN3 indicating an increase in diversity upon the introduction of C. cajan into the contaminated soil. The PCoA plot revealed community-level differences between the contaminated non-rhizosphere control and contaminated rhizosphere microbiota. The PCoA differentiated the two treatments based on the presence or absence of plant. The composition and taxonomic analysis of microbiota-amplified sequences were categorized into eight phyla for the contaminated non-rhizosphere and ten phyla for the contaminated rhizosphere. The overall bacterial composition of the two treatments varied, as the distribution shows a similar variation between the two treatments in the phylum distribution. The percentage removal of total petroleum hydrocarbon (TPH) after 90 days of treatments with 1, 2, 3, 4, and 5% (w/w) of POS were 92, 90, 89, 68.3 and 47.3%, respectively, indicating removal inhibition at higher POS concentrations. As the search for more eco-friendly and sustainable remediating green plant continues, C. cajan shows great potential in reclaiming POS contaminated soil. Our findings will provide solutions to POS polluted soils and subsequent re-vegetation.

Bradyrhizobium ivorense sp. nov. as a potential local bioinoculant for Cajanus cajan cultures in Côte d'Ivoire.

For many smallholder farmers of Sub-Saharan Africa, pigeonpea (Cajanus cajan) is an important crop to make ends meet. To ascertain the taxonomic status of pigeonpea isolates of Côte d'Ivoire previously identified as bradyrhizobia, a polyphasic approach was applied to strains CI-1BT, CI-14A, CI-19D and CI-41S. Phylogeny of 16S ribosomal RNA (rRNA) genes placed these nodule isolates in a separate lineage from current species of the B. elkanii super clade. In phylogenetic analyses of single and concatenated partial dnaK, glnII, gyrB, recA and rpoB sequences, the C. cajan isolates again formed a separate lineage, with strain CI-1BT sharing the highest sequence similarity (95.2 %) with B. tropiciagri SEMIA 6148T. Comparative genomic analyses corroborated the novel species status, with 86 % ANIb and 89 % ANIm as the highest average nucleotide identity (ANI) values with B. elkanii USDA 76T. Although CI-1BT, CI-14A, CI-19D and CI-41S shared similar phenotypic and metabolic properties, growth of CI-41S was slower in/on various media. Symbiotic efficacy varied significantly between isolates, with CI-1BT and CI-41S scoring on the C. cajan 'Light-Brown' landrace as the most and least proficient bacteria, respectively. Also proficient on Vigna radiata (mung bean), Vigna unguiculata (cowpea, niébé) and additional C. cajan cultivars, CI-1BT represents a potential bioinoculant adapted to local soil conditions and capable of fostering the growth of diverse legume crops in Côte d'Ivoire. Given the data presented here, we propose the 19 C. cajan isolates to belong to a novel species called Bradyrhizobium ivorense sp. nov., with CI-1BT (=CCOS 1862T=CCMM B1296T) as a type strain.

Bioinoculants play a significant role in shaping the rhizospheric microbial community: a field study with Cajanus cajan.

The present study is an attempt to understand the impact of bioinoculants, Azotobacter chroococcum (A), Bacillus megaterium (B), Pseudomonas fluorescens (P), on (a) soil and plant nutrient status, (b) total resident and active bacterial communities, and (c) genes and transcripts involved in nitrogen cycle, during cultivation of Cajanus cajan. In terms of available macro- and micro-nutrients, triple inoculation of the bioinoculants (ABP) competed well with chemical fertilizer (CF). Their 'non-target' effects were assessed in terms of the abundance and activity of the resident bacterial community by employing denaturing gradient gel electrophoresis (DGGE). The resident bacterial community (16S rRNA gene) was stable, while the active fraction (16S rRNA transcripts) was influenced (in terms of abundance) by the treatments. Quantification of the genes and transcripts involved in N cycle by qPCR revealed an increase in the transcripts of nifH in the soil treated with ABP over CF, with an enhancement of 3.36- and 1.57- fold at flowering and maturity stages of plant growth, respectively. The bioinoculants shaped the resident microflora towards a more beneficial community, which helped in increasing soil N turnover and hence, soil fertility as a whole.

Embryonic Explant and Plumular Meristem Transformation Methods for Development of Transgenic Pigeon Pea.

A reliable pigeon pea transformation system can assist the rapid improvement of this important grain legume through transgenic development. Here we describe two methods of Agrobacterium tumefaciens-mediated pigeon pea transformation. In the tissue culture based embryonic explant transformation method, microshoot grafting was included to obtain rapid root induction, while the other method was culture independent and designated as plumular meristem transformation. Both methods drastically enhanced the transformation frequency and have the potential to provide reasonable solutions for maximum transgenic recovery in biotechnological breeding programs.

Elucidation of quorum sensing components and their role in regulation of symbiotically important traits in Ensifer nodulating pigeon pea.

Quorum sensing (QS) in rhizobia regulates diverse processes determining the success and efficiency of association with the legume host. Despite the notable importance of QS as well as the well-known underlying variability in the genomic and metabolic components thereof, its study in rhizobia is largely restricted to few laboratory strains. In this work, QS phenomenon in the rhizobia nodulating pigeon pea- one of the most important legume crops of the global-south, is characterized. Using 16S rRNA and recombinaseA sequencing analysis, the selected QS-positive and host-beneficial isolates were identified to be taxonomically affiliated to the genus Ensifer. Their QS components, including homologues of QS genes, and the repertoire of N-acyl homoserine lactone (AHL) autoinducers were identified. Sequences of the QS homologues showed significant variabilities ranging from 10 to >20% with the known Ensifer sequences. Autoinducer profiling using LC-MS/MS revealed the production of long and short chain AHLs variably by the isolates, including 3-oxo-C12-homoserine lactone (3-O-C12-HSL) and 3-OH-C16-HSL as their first report in Rhizobiaceae. Motility and attachment- two of the most crucial traits for effective establishment on host roots were discovered to be QS dependent in in vitro analysis and the same was confirmed using expression analysis of their regulatory genes using qRT-PCR; both revealing a QS mediated repression of motility and promotion of attachment. This study highlights that Ensifer nodulating pigeon pea, although with significant variance in the anatomy of their QS components, regulate symbiotically crucial cell-processes via QS in a scheme that is conserved in multiple genera.

Root colonization by heavy metal resistant Enterobacter and its influence on metal induced oxidative stress on Cajanus cajan.

BACKGROUND: Heavy metal resistant bacterium Enterobacter sp. C1D was evaluated for cadmium (Cd) mediated exopolysaccharide production, biofilm formation and legume root colonization ability under Cd stress to alleviate metal induced stress. RESULTS: The plant was sensitive to Cd (IC50 3-4 µg mL-1 ), whereas the bacterium showed high Cd tolerance (MIC99 120 µg mL-1 ). Confocal laser scanning microscopy of the Cajanus cajan roots showed heavy loads of green fluorescence protein labelled Enterobacter sp. C1D on the surface of plant root, specifically at the point of root hair/lateral root formation along with cortex, even under metal stress. The root colonizing ability of Enterobacter sp. C1D was not affected by the presence of Rhizobium and the bacteria could be observed after 30 days of incubation in soil. Various plant growth parameters, antioxidant metabolites and oxidative stress indicator were significantly influenced by bacterial treatment, which, overall, reduced the adverse effect of Cd. CONCLUSION: Heavy metal tolerant bacteria may be a good choice for the development of biofertilizers and may work well with the native soil microbes such as Rhizobium under the metal polluted soil. © 2019 Society of Chemical Industry.

Impact of abiotic stressors on native rhizospheric bacterial community of Cajanus cajan.

Salinity and drought are the major abiotic stresses that limit agricultural productivity. Application of plant growth promoting rhizobacteria (PGPR) is an attractive technology but with the bottlenecks of reduced efficacy and survivability in the environment. For increased efficiency of PGPR strains, the impact of stresses on the native bacterial community needs to be studied. Experimentally induced stresses would be ideal to assess the immediate perturbances in the structure of soil bacterial community. Hence, the study focused on the effect of experimentally-induced salinity, and drought stress on rhizospheric bacterial community of Cajanus cajan. A plant growth experiment was set up to induce salinity and drought stresses. Shifts in the bacterial community were assessed by a culture-independent technique of denaturing gradient gel electrophoresis using 16S ribosomal RNA gene and transcript as markers, leading to a comparison of the resident with the active bacterial community. The impact on plant was evaluated by measurement of plant biometrics. Further, salinity and drought-stressed conditions led to distinct shifts in native and active rhizospheric bacterial community, corresponding to the higher decline at induction of stresses, and stabilization at later time points. The study encompasses the perturbations in the active and resident rhizospheric bacterial community caused by the induction of two different abiotic stresses along the plant's growth.

Identification, characterization and expression profiles of Fusarium udum stress-responsive WRKY transcription factors in Cajanus cajan under the influence of NaCl stress and Pseudomonas fluorescens OKC.

The WRKY gene family has never been identified in pigeonpea (Cajanus cajan). Therefore, objective of the present study was to identify the WRKY gene family in pigeonpea and characterize the Fusarium udum stress-responsive WRKY genes under normal, NaCl-stressed and Pseudomonas fluorescens OKC (a plant growth-promoting bacterial strain) treated conditions. The aim was to characterize the Fusarium udum stress-responsive WRKY genes under some commonly occurring field conditions. We identified 97 genes in the WRKY family of pigeonpea, using computational prediction method. The gene family was then classified into three groups through phylogenetic analysis of the homologous genes from the representative plant species. Among the 97 identified WRKY genes 35 were further classified as pathogen stress responsive genes. Functional validation of the 35 WRKY genes was done through generating transcriptional profiles of the genes from root tissues of pigeonpea plants under the influence of P. fluorescens OKC after 24 h of stress application (biotic: Fusarium udum, abiotic: NaCl). The entire experiment was conducted in two pigeonpea cultivars Asha (resistant to F. udum) and Bahar (susceptible to F. udum) and the results were concluded on the basis of transcriptional regulation of the WRKY genes in both the pigeonpea cultivars. The results revealed that among the 35 tentatively identified biotic stress responsive CcWRKY genes, 26 were highly F. udum responsive, 17 were better NaCl responsive compared to F. udum and 11 were dual responsive to both F. udum and NaCl. Application of OKC was able to enhance transcript accumulation of the individual CcWRKY genes to both the stresses when applied individually but not in combined challenge of the two stresses. The results thus indicated that CcWRKY genes play a vital role in the defense signaling against F. udum and some of the F. udum responsive CcWRKYs (at least 11 in pigeonpea) are also responsive to abiotic stresses such as NaCl. Further, plant beneficial microbes such as P. fluorescens OKC also help pegionpea to defend itself against the two stresses (F. udum and NaCl) through enhanced expression of the stress responsive CcWRKY genes when the stresses are applied individually.

Classical Soybean (Glycine max (L.) Merr) Symbionts, Sinorhizobium fredii USDA191 and Bradyrhizobium diazoefficiens USDA110, Reveal Contrasting Symbiotic Phenotype on Pigeon Pea (Cajanus cajan (L.) Millsp).

Pigeon pea (Cajanus cajan (L.) Millspaugh) is cultivated widely in semiarid agricultural regions in over 90 countries around the world. This important legume can enter into symbiotic associations with a wide range of rhizobia including Bradyrhizobium and fast-growing rhizobia. In comparison with other major legumes such as soybean and common bean, only limited information is available on the symbiotic interaction of pigeon pea with rhizobia. In this study, we investigated the ability of two classical soybean symbionts-S. fredii USDA191 and B. diazoefficiens USDA110-and their type 3 secretion system (T3SS) mutants, to nodulate pigeon pea. Both S. fredii USDA191 and a T3SS mutant S. fredii RCB26 formed nitrogen-fixing nodules on pigeon pea. Inoculation of pigeon pea roots with B. diazoefficiens USDA110 and B. diazoefficiens Δ136 (a T3SS mutant) resulted in the formation of Fix- and Fix+ nodules, respectively. Light and transmission electron microscopy of Fix- nodules initiated by B. diazoefficiens USDA110 revealed the complete absence of rhizobia within these nodules. In contrast, Fix+ nodules formed by B. diazoefficiens Δ136 revealed a central region that was completely filled with rhizobia. Ultrastructural investigation revealed the presence of numerous bacteroids surrounded by peribacteroid membranes in the infected cells. Analysis of nodule proteins by one- and two-dimensional gel electrophoresis revealed that leghemoglobin was absent in B. diazoefficiens USDA110 nodules, while it was abundantly present in B. diazoefficiens Δ136 nodules. Results of competitive nodulation assays indicated that B. diazoefficiens Δ136 had greater competitiveness for nodulation on pigeon pea than did the wild type strain. Our results suggest that this T3SS mutant of B. diazoefficiens, due to its greater competitiveness and ability to form Fix+ nodules, could be exploited as a potential inoculant to boost pigeon pea productivity.