Co-existence of halo-tolerant Pseudomonas fluorescens and Enterococcus hirae with multifunctional growth promoting traits to ameliorate salinity stress in Vigna radiata.

Soil salinization has become a prominent obstacle in diverse arid and semi-arid region damaging agricultural productivity globally. From this perspective, present investigation was aimed to compare the potential compatible consortium of bio-inoculants for improving Plant Growth Promoting (PGP) attributes, anti-oxidative enzymes, grain yield and profitability of Vigna radiata in saline soil conditions. A total of 101 rhizobacterium isolated from salt affected regions of Punjab, India were screened for their ability to induce salt tolerance, multifunctional PGP traits and antagonistic activities. The 16S rRNA sequencing identified the strains LSMR-29 and LSMRS-7 as Pseudomonas flourescens and Enterococcus hirae, respectively. In-vitro compatible halo-tolerant dual inoculant (LSMR-29 + LSMRS-7) as bio-inoculants mitigated salt stress in Vigna radiata (spring mungbean) seedling with improved seed germination, biomass and salt tolerance index together with the presence of nifH, acds, pqq and ipdc gene under salinity stress as compared to single inoculants. Further, the potential of single and dual bio-inoculants were also exploited for PGP attributes in pot and field experiments. Results indicated that a significant improvement in chlorophyll content (2.03 fold), nodulation (1.24 fold), nodule biomass (1.23 fold) and leghemoglobin content (1.13 fold) with dual inoculant of LSMR-29 + LSMRS-7 over the LSMR-29 alone. The concentrations of macro & micronutrients, proline, soil enzyme activities i.e. soil dehydrogenase, acid & alkaline phosphatases and antioxidant enzymes such as superoxide dismutase, catalase and peroxidase also found to be high for LSMR-29 + LSMRS-7 as compared to un-inoculated control. The high grain yield thereby leading to Benefit: Cost (B: C) ratio at field scale was indicative of the commercial use bio-inoculants under salt affected Vigna radiata (spring mungbean) to improvement of productivity and soil health. The current finding reveals a co-inoculation of halo-tolerating Pseudomonas fluorescens and Enterococcus hirae containing ACC deaminase could prove to be novel approach for inducing salt tolerance and improving productivity of Vigna radiata (spring mungbean).

Marker trait association for biological nitrogen fixation traits in an interspecific cross of chickpea (Cicer arietinum × Cicer reticulatum).

A set of 165 Recombinant inbred lines (RILs) derived from an interspecific cross of chickpea was used to identify QTLs for key biological nitrogen fixation (BNF) traits. The phenotyping of BNF and related traits was done at two different agroclimatic zones viz., Central plain zone (Ludhiana) and Sub-Mountainous undulating zone (Gurdaspur) for 2 consecutive rabi seasons (2018-2020). Wild parent C. reticulatum ILWC292 showed significantly high performance in terms of biological nitrogen fixation (BNF) traits over the cultivated C. arietinum GPF-2. The triple interaction of genotypes × locations × years was significant (p 0.05) for all BNF traits in parental lines. Highly significant positive correlation was obtained between grain yield and key growth and symbiotic parameters at both the sites. Phenotypic analysis revealed nodule dry weight and leghaemoglobin content as key traits for BNF efficiency and contrasting DNA bulks were constituted on the basis of these traits. Out of 535 SSR markers, 139 exhibited polymorphism between the parental lines on polyacrylamide gel electrophoresis. A total of 30 SSR markers showed polymorphism between the higher and lower bulks for nodule dry weight and leghaemoglobin content. Out of these, 20 SSRs did not show any segregation distortion in RIL population as determined by chi square analysis (p < 0.05) and were used for quantitative trait loci (QTL) analysis. Using QTL cartographer, markers- CAGM02697, CAGM09835, CAGM09777, CAGM09227, CAGM09021, CAGM08679 were found linked with QTLs for BNF. These markers can be validated further for identification of genes for BNF traits and marker assisted selection in chickpea. To the best of our knowledge this is the first report on identification of genomic regions associated with key BNF traits in chickpea across different agro-climatic zones. Supplementary information: The online version contains supplementary material available at 10.1007/s12298-023-01335-3.

Exploring the molecular basis of resistance to Botrytis cinerea in chickpea genotypes through biochemical and morphological markers.

Chickpea (Cicer arietinum L.) is an important pulse crop around the globe and a valuable source of protein in the human diet. However, it is highly susceptible to various plant pathogens such as fungi, bacteria, and viruses, which can cause significant damage from the seedling phase until harvest, leading to reduced yields and affecting its production. Botrytis cinerea can cause significant damage to chickpea crops, especially under high humidity and moisture conditions. This fungus can cause grey mould disease, which can lead to wilting, stem and pod rot, and reduced yields. Chickpea plants have developed specific barriers to counteract the harmful effects of this fungus. These barriers include biochemical and structural defences. In this study, the defence responses against B. cinerea were measured by the quantification of biochemical metabolites such as antioxidant enzymes, malondialdehyde (MDA), proline, glutathione (GSH), H2O2, ascorbic acid (AA) and total phenol in the leaf samples of chickpea genotypes (one accession of wild Cicer species, viz. Cicer pinnatifidum188 identified with high level of resistance to Botrytis grey mould (BGM) and a cultivar, Cicer arietinumPBG5 susceptible to BGM grown in the greenhouse). Seedlings of both the genotypes were inoculated with (1 × 104 spore mL-1) inoculum of isolate 24, race 510 of B. cinerea and samples were collected after 1, 3, 5, and 7 days post-inoculation (dpi). The enhanced enzymatic activity was observed in the pathogen-inoculated leaf samples as compared to uninoculated (healthy control). Among inoculated genotypes, the resistant one exhibited a significant change in enzymatic activity, total phenolic content, MDA, proline, GSH, H2O2, and AA, compared to the susceptible genotype. The study also examined the isozyme pattern of antioxidant enzymes at various stages of B. cinerea inoculation. Results from scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy revealed that BGM had a more significant impact on susceptible genotypes compared to resistant ones when compared to the control (un-inoculated). In addition, SEM and FTIR spectroscopy analyses confirmed the greater severity of BGM on susceptible genotypes compared to their resistant counterparts. Our results suggest the role of antioxidant enzymes and other metabolites as defence tools and biochemical markers to understand compatible and non-compatible plant-pathogen interactions better. The present investigation will assist future plant breeding programs aimed at developing resistant varieties.

Investigating the impact of fungicides and mungbean genotypes on the management of pod rot disease caused by Fusarium equiseti and Fusarium chlamydosporum.

Introduction: Mungbean is a vital pulse crop in India that can thrive in dry-land conditions and is grown in three seasons, with the added benefit of being used as green manure due to its ability to fix atmospheric nitrogen. Recently, pod rot disease has emerged as a serious threat to mungbean cultivation in India. Methods: In this study, morpho-molecular identification of associated pathogens and the bio-efficacy of systemic and non-systemic fungicides as well as genotype screening was performed during the years 2019 and 2020. The pathogens associated with this disease were confirmed on the basis of morphological and molecular characterization. For the molecular characterization, the translation elongation factor 1-alpha (tef-1) gene sequences were amplified by using primers (EF1 and EF2). Results: Under in vitro conditions, trifloxystrobin + tebuconazole 75% WG was found to be the most effective against Fusarium equiseti (ED50 2.39 µg ml-1) and Fusarium chlamydosporum (ED50 4.23 µg ml-1) causal agents of pod rot of mungbean. Under field conditions, three applications of trifloxystrobin + tebuconazole 75% WG at 0.07% as a foliar application at fortnightly intervals starting from the last week of July proved to be the most effective against pod rot disease on mungbean cultivars, i.e., ML 2056 and SML 668. To identify the potential resistance sources, 75 interspecific derivative and mutant lines of mungbean were screened for disease reaction to pod rot under natural epiphytotic conditions for the years 2019 and 2020. Genotypic differences were observed for resistance to pod rot disease. The study revealed that among the tested genotypes, ML 2524 exhibited resistance to pod rot disease, with a disease incidence of 15.62% and disease severity of 7.69%. In addition, 41 other genotypes were found to be moderately resistant (MR) to the disease. Conclusion: Altogether, the identified management options will offer an immediate solution to manage this disease under recent outbreak conditions and pave a path for futuristic disease management using identified resistant sources in breeding programs.

Ecological performance of multifunctional pesticide tolerant strains of Mesorhizobium sp. in chickpea with recommended pendimethalin, ready-mix of pendimethalin and imazethpyr, carbendazim and chlorpyrifos application.

The present study was designed to screen the Mesorhizobium strains (50) for tolerance with four recommended pesticides in chickpea. In-vitro, robust pesticide tolerant strains were developed in pesticides amended media over several generations. Further, verification of the multifunctional traits of pesticide tolerant mesorhizobia under pesticide stress was conducted in-vitro. Among different pesticides, significantly high tolerance in Mesorhizobium strains was observed with recommended doses of pendimethalin (37%) and ready-mix (36%) followed by chlorpyrifos (31%) and carbendazim (30%), on an overall basis. Based on multifunctional traits, Mesorhizobium strains viz. MR2, MR17 and recommended MR33 were the most promising. Ecological performance of the potential Mesorhizobium strains alone and in dual-inoculation with recommended PGP rhizobacterium strain RB-1 (Pseudomonas argenttinensis JX239745.1) was subsequently analyzed in field following standard pesticide application in PBG-7 and GPF-2 chickpea varieties for two consecutive rabi seasons (2015 and 2016). Dual-inoculant treatments; recommended RB-1 + MR33 (4.1%) and RB-1 + MR2 (3.8%) significantly increased the grain yield over Mesorhizobium alone treatments viz MR33 and MR2, respectively. Grain yield in PBG7 variety was significantly affected (7.3%) by the microbial inoculant treatments over GPF2 variety. Therefore, the potential pesticide tolerant strains MR2 and MR33 can be further explored as compatible dual-inoculants with recommended RB-1 for chickpea under environmentally stressed conditions (pesticide application) at multiple locations. Our approach using robust multifunctional pesticide tolerant Mesorhizobium for bio-augmentation of chickpea might be helpful in the formulation of effective bio-inoculants consortia in establishing successful chickpea-Mesorhizobium symbiosis.

Chickpea (Cicer arietinum L.) as model legume for decoding the co-existence of Pseudomonas fluorescens and Mesorhizobium sp. as bio-fertilizer under diverse agro-climatic zones.

Microbial co-inoculation strategy utilizes a combination of microbes to stimulate plant growth concomitant with an increased phytopathogen tolerance. In the present study, 15 endophytic bacterial isolates from rhizosphere and roots of wild chickpea accessions (Cicer pinnatifidum, C. judiacum, C. bijugum and C. reticulatum) were characterized for morphological, biochemical and physiological traits. Two promising isolates were identified as Pseudomonas fluorescens strain LRE-2 (KR303708.1) and Pseudomonas argentinensis LPGPR-1 (JX239745.1) based on 16S rRNA gene sequencing. Biocompatibility of selected endophytes with Mesorhizobium sp. CH1233, a standard isolate used as a national check in All India Coordinated Research Project (AICRP) was assessed to develop functional combinations capable of producing Indole acetic acid, gibberellins, siderophores and improving seed vigour (in vitro). In vivo synergistic effect of promising combinations was further evaluated under national AICRP, (Chickpea) at two different agro-climatic zones [North-West plain (Ludhiana and Hisar) and Central zones (Sehore)] for three consecutive Rabi seasons (2015-18) to elucidate their effect on symbiotic, soil quality and yield parameters. On the pooled mean basis across locations over the years, combination of Mrh+LRE-2 significantly enhanced symbiotic, soil quality traits and grain yield over Mrh alone and highly positive correlation was obtained between the nodulation traits and grain yield. Superior B: C ratio (1.12) and additional income of Rs 6,505.18 ha-1 was obtained by application of Mrh+LRE-2 over Mrh alone and un-inoculated control. The results demonstrate that dual combination of Mrh and Pseudomonas sp. from wild Cicer relatives can be exploited as a potential bio-fertilizer for increasing soil fertility and improving chickpea productivity under sustainable agriculture.

Dual Microbial Inoculation, a Game Changer? - Bacterial Biostimulants With Multifunctional Growth Promoting Traits to Mitigate Salinity Stress in Spring Mungbean.

Soil microbes play a vital role in improving plant growth, soil health, ameliorate biotic/abiotic stress and enhance crop productivity. The present study was aimed to investigate a coordinated effect of compatible consortium [salt tolerating Rhizobium and rhizobacterium with 1-aminocyclopropane-1-carboxylate (ACC) deaminase] in enhancing plant growth promoting (PGP) traits, symbiotic efficiency, nutrient acquisition, anti-oxidative enzymes, grain yield and associated profitability in spring mungbean. We identified a non-pathogenic compatible Rhizobium sp. LSMR-32 (MH644039.1) and Enterococcus mundtii LSMRS-3 (MH644178.1) from salt affected areas of Punjab, India and the same were assessed to develop consortium biofertilizer based on salt tolerance, multifarious PGP traits, antagonistic defense activities and presence of nifH, acds, pqq, and ipdc genes. Indole Acetic acid (IAA), P-solubilization, biofilm formation, exo-polysaccharides, siderophore, salt tolerance, ACC deaminase activities were all found highly significant in dual inoculant (LSMR-32 + LSMRS-3) treatment compared to LSMR-32 alone. Under saline soil conditions, dual inoculant showed a higher seed germination, plant height, biomass, chlorophyll content and macro and micro-nutrient uptake, than un-inoculated control. However, symbiotic (nodulation, nodule biomass and leghaemoglobin content) and soil quality parameters (phosphatase and soil dehydrogenase enzymes) increased numerically with LSMR-32 + LSMRS-3 over Rhizobium sp. LSMR-32 alone. Dual bacterial inoculation (LSMR-32 + LSMRS-3) increased the proline content (2.05 fold), anti-oxidative enzymes viz., superoxide dismutase (1.50 fold), catalase (1.43 fold) and peroxidase (3.88 folds) in contrast to control treatment. Decreased Na+ accumulation and increased K+ uptake resulted in favorable K+/Na+ ratio through ion homeostasis. Co-inoculation of Rhizobium sp. LSMR-32 and Enterococcus mundtii LSMRS-3 significantly improved the grain yield by 8.92% and led to superior B: C ratio over Rhizobium sp. alone under salt stress. To best of our knowledge this is perhaps the first field report from Indian soils that largely describes dual inoculation of Rhizobium sp. LSMR-32 and Enterococcus mundtii LSMRS-3 and the same can be considered as a game-changer approach to simultaneously induce salt tolerance and improve productivity in spring mungbean under saline stress conditions.

Co-existence of Leclercia adecarboxylata (LSE-1) and Bradyrhizobium sp. (LSBR-3) in nodule niche for multifaceted effects and profitability in soybean production.

The present study was designed with the objective of improving growth and nodulation of soybean [Glycine max (L.) Merill] with co-inoculation of native Bradyrhizobium sp. (LSBR-3) (KF906140) and non-rhizobial nodule endophytic diazotroph Leclercia adecarboxylata (LSE-1) (KX925974) with multifunctional plant growth promoting (PGP) traits in cereal based cropping system (Rice-Wheat). A total of 40 endophytic bacteria from cultivated and wild sp. of soybean were screened for multifarious PGP traits and pathogenicity test. Based on PGP traits, antagonistic activities and bio-safety test; L. adecarboxylata (LSE-1) was identified with 16 S rRNA gene sequencing along with the presence of nifH (nitrogen fixation) and ipdc (IAA production) genes. Dual inoculant LSE-1 and LSBR-3 increased indole acetic acid (IAA), P & Zn-solubilization, 1-aminocyclopropane-1-carboxylate deaminase (ACCD) activity, siderophore, biofilm formation and exo-polysaccharides in contrast to single inoculation treatment. Further, assessment of dual inoculant LSBR-3 + LSE-1 improved growth parameters, nodulation, soil enzymes activities, nutrient accumulation and yield as compared to single as well as un-inoculated control treatment under field conditions. Single inoculant LSBR-3 improved yield by 8.84% over control. Further, enhancement of 4.15% grain yield was noticed with LSBR-3 + LSE-1 over LSBR-3 alone treatment. Application of LSBR-3 + LSE-1 gave superior B:C ratio (1.29) and additional income approximately 116 USD ha-1 in contrast to control treatment. The present results thus, is the first report of novel endophytic diazotroph L. adecarboxylata (LSE-1) as PGPR from Indian conditions particularly in Punjab region for exploiting as potential PGPR along with Bradyrhizobium sp. (LSBR-3) in soybean.

Synergism of Pseudomonas aeruginosa (LSE-2) nodule endophyte with Bradyrhizobium sp. (LSBR-3) for improving plant growth, nutrient acquisition and soil health in soybean.

The present study was aimed to assess the scope of native potential endophyte Pseudomonas aeruginosa (LSE-2) strain (KX925973) with recommended Bradyrhizobium sp. (LSBR-3) (KF906140) for synergistic effect to develop as consortium biofertilizer of soybean. A total of 28 non-rhizobial endophytic bacteria were isolated from cultivated and wild sp. of soybean. All isolates were screened for multifarious PGP traits viz. Indole-3-acetic acid (IAA), phosphate (P) and zinc (Zn) solubilization, siderophore, cell wall degrading enzymes and pathogenicity. Compatible of LSBR-3 and LSE-2 enhanced IAA, P-solubilization, 1-aminocyclopropane-carboxylate deaminase and biofilm formation over the single inoculant treatment. Further, consortium was evaluated in vivo for growth, symbiotic traits, nutrient acquisition, soil quality parameters and yield attributes of soybean. Improvement in growth parameters were recorded with dual inoculant LSBR-3 + LSE-2 as compared to LSBR-3 alone and un-inoculated control treatments. Significantly (p ≥ 0.05) high symbiotic and soil quality parameters (phosphatase and soil dehydrogenase activity) was recorded with LSBR-3 + LSE-2 at vegetative and flowering stage as compared to LSBR-3 alone and un-inoculated control treatments. Single inoculation of LSBR-3 improved grain yield by 4.25% over the un-inoculated control treatment, further, enhancement in yield was recorded with consortium inoculant (LSBR-3 and LSE-2) by 3.47% over the LSBR-3 alone. Application of consortium inoculant (LSBR-3 + LSE-2) gave an additional income of Rs. 5089/ha over the un-inoculated control treatment. The results, thus strongly suggest that endophytic diazotroph LSE-2 can be used as potent bio-inoculant along with LSBR-3 as bio-enhancer for improving soybean productivity in a sustainable system.

Detection of quantitative trait loci for mungbean yellow mosaic India virus (MYMIV) resistance in mungbean (Vigna radiata (L.) Wilczek) in India and Pakistan.

Yellow mosaic disease (YMD) is one of the major diseases affecting mungbean (Vigna radiata (L.) Wilczek). In this study, we report the mapping of the quantitative trait locus (QTL) for mungbean yellow mosaic India virus (MYMIV) resistance in mungbean. An F8 recombinant inbred line (RIL) mapping population was generated in Thailand from a cross between NM10-12-1 (MYMIV resistance) and KPS2 (MYMIV susceptible). One hundred and twenty-two RILs and their parents were evaluated for MYMIV resistance in infested fields in India and Pakistan. A genetic linkage map was developed for the RIL population using simple sequence repeat (SSR) markers. Composite interval mapping identified five QTLs for MYMIV resistance: three QTLs for India (qYMIV1, qYMIV2 and qYMIV3) and two QTLs for Pakistan (qYMIV4 and qYMIV5). qYMIV1, qYMIV2, qYMIV3, qYMIV4 and qYMIV5 explained 9.33%, 10.61%, 12.55%, 21.93% and 6.24% of variation in disease responses, respectively. qYMIV1 and qYMIV4 appeared to be the same locus and were common to a major QTL for MYMIV resistance in India identified previously using a different resistant mungbean.