RNA-Seq analysis of mung bean (Vigna radiata L.) roots shows differential gene expression and predicts regulatory pathways responding to taxonomically different rhizobia.

Symbiotic interaction among legume and rhizobia is a complex phenomenon which results in the formation of nitrogen-fixing nodules. Mung bean is promiscuous host however expression profile of this important legume plant in response to rhizobial infection was particularly lacking and urgently needed. We have demonstrated the pattern of gene expression of mung bean roots inoculated with two symbionts Bradyrhizobium yuanmingense Vr50 and Sinorhizobium (Ensifer) aridi Vr33 and non-inoculated control (CK). The RNA-Seq data analyzed at two growth stages i.e., 1-3 h and 10-16 days post inoculation revealed significantly higher number of differentially expressed genes (DEGs) at nodulation stage. The DEGs encoding receptor kinases identified at early stage might be involved in perception of Nod factors produced by different rhizobia. At nodulation stage important genes involved in plant hormone signal transduction, nitrogen and sulfur metabolism were identified. KEGG pathway enrichment analysis showed that metabolic pathways were most prominent in both groups (Group 1: Vr33 vs CK; Group 2: Vr50 vs CK), followed by biosynthesis of secondary metabolites, plant hormone signal transduction and biosynthesis of amino acids. Furthermore, DEGs involved in cell communication and plant hormone signal transduction were found to be different among two symbiotic systems while DEGs involved in carbon, nitrogen and sulfur metabolism were similar but their expression varied in response to two rhizobial strains. This study provides the first insight into the mechanisms underlying interactions of mung bean host with two taxonomically different symbionts (Bradyrhizobium and Sinorhizobium) and the candidate genes for better understanding the mechanisms of symbiotic host-specificity.

Exploring biocontrol and growth-promoting potential of multifaceted PGPR isolated from natural suppressive soil against the causal agent of chickpea wilt.

Chickpea is an important nutritive food crop both for humans and animals. Chickpea wilt caused by Fusarium oxysporum f.sp. ciceris (Foc) results in huge yield losses every year. Chickpea being a food crop requires the development of an eco-friendly bio-pesticide to effectively control the chickpea wilt disease. In this study, more than 50 bacterial stains isolated from the rhizosphere of healthy plants growing in wilt sick soil were examined for their Foc antagonist activities. Out of these, 17 strains showing > 90% growth inhibition of Foc were then characterized for their plant growth-promoting (PGP) and biocontrol traits. The biocontrol and PGP traits identified include amylase, hydrogen cyanide, protease, cellulase, chitinase activities, p-solubilization, nitrogen-fixing, and indole-3-acetic acid production. Two bacterial strains, IR-27 and IR-57, exhibiting the highest Foc proliferation inhibition and the PGP potential along with a consortium of four different strains (Serratia sp. IN-1, Serratia sp. IS-1, Enterobacter sp. IN-2, Enterobacter sp. IN-6) were used for controlling the chickpea wilt disease and growth promotion of the chickpea plants. Confocal laser scanning microscopy revealed their root colonization ability with partial or complete elimination of broken Foc mycelia and hyphae from roots. The bacterial inoculations particularly the consortium significantly suppressed the disease and improved the overall root morphology traits (root length, root surface area, root volume, forks, tips, and crossings), resulting in enhanced growth of the chickpea plants. Significant changes in growth (107% increase in root length, 23% increase in shoot length, and 54% increase in branches) in Foc-challenged plants were observed when inoculated with the consortium. Further investigations revealed that the chickpea plants inoculated with bacterial strains induced the expression of a number of key defence enzymes, including the phenylalanine ammonia lyase, peroxidase, polyphenol peroxidase, ß-1,3 glucanase, which might have helped the plants to thwart the pathogen attack. These findings indicate the potential of our identified bacterial strains to be used as a natural biopesticide for controlling the chickpea wilt disease.

Diazotrophs for Lowering Nitrogen Pollution Crises: Looking Deep Into the Roots.

During and after the green revolution in the last century, agrochemicals especially nitrogen (N) were extensively used. However, it resulted in a remarkable increase in crop yield but drastically reduced soil fertility; increased the production cost, food prices, and carbon footprints; and depleted the fossil reserves with huge penalties to the environment and ecological sustainability. The groundwater, rivers, and oceans are loaded with N excess which is an environmental catastrophe. Nitrogen emissions (e.g., ammonia, nitrogen oxide, nitrous oxide) play an important role in global climate change and contribute to particulate matter and acid rain causing respiratory problems, cancers, and damage to forests and buildings. Therefore, the nitrogen-polluted planet Earth needs concerted global efforts to avoid the disaster. Improved agricultural N management focuses on the synchronization of crop N demand and N supply along with improving the N-use efficiency of the crops. However, there is very little focus on the natural sources of N available for plants in the form of diazotrophic bacteria present inside or on the root surface and the rhizosphere. These diazotrophs are the mini-nitrogen factories that convert available (78%) atmospheric N2 to ammonia through a process known as "biological nitrogen fixation" which is then taken up by the plants for its metabolic functioning. Diazotrophs also stimulate root architecture by producing plant hormones and hence improve the plant's overall ability to uptake nutrients and water. In recent years, nanotechnology has revolutionized the whole agri-industry by introducing nano-fertilizers and coated/slow-releasing fertilizers. With this in mind, we tried to explore the following questions: To what extent can the crop N requirements be met by diazotroph inoculation? Can N input to agriculture be managed in a way leading to environmental benefits and farmers saving money? Can nanotechnology help in technological advancement of diazotroph application? The review suggests that an integrated technology based on slow-releasing nano-fertilizer combined with diazotrophs should be adopted to decrease nitrogen inputs to the agricultural system. This integrated technology would minimize N pollution and N losses to much extent.

Seed inoculation of desert-plant growth-promoting rhizobacteria induce biochemical alterations and develop resistance against water stress in wheat.

Water shortage limits agricultural productivity, so strategies to get higher yields in dry agricultural systems is vital to circumvent the effect of climate change and land-shortage. The plant rhizosphere harbors beneficial bacteria able to confer biotic/abiotic tolerance along with a positive impact on plant growth. Herein, three bacterial strains, Proteus mirabilis R2, Pseudomonas balearica RF-2 and Cronobacter sakazakii RF-4 (accessions: LS975374, LS975373, LS975370, respectively) isolated from native desert-weeds were investigated for their response to improve wheat growth under drought stress. The bacteria showed drought tolerance up to 20% polyethylene glycol (PEG; -0.6 MPa), and salt (65-97 g l-1 ), 1-aminocyclopropane-1-carboxylate (ACC)-deaminase activity, P/Zn/K-solubilization, calcite degradation, IAA, and siderophore production. The plant growth-promoting rhizobacteria (PGPR) were evaluated on wheat under water stress. The P. balearica strain RF-2 primed seeds showed a maximum promptness index and germination index under PEG-stress, that is, 68% and 100%, respectively. Inoculation significantly improved plant growth, leaf area, and biomass under water stress. P. mirabilis R2 inoculated plant leaves showed the highest water contents as compared to the plants inoculated with other strains. C. sakazakii RF-4 inoculated plants showed minimum cell injury, electrolyte leakage, and maximum cell membrane stability at PEG stress. After 13 days exposure to drought, C. sakazakii RF-4 treated plants showed an overall higher expression of cytosolic ascorbate peroxidase (cAPX) and ribulose-bisphosphate carboxylase (rbcL) genes. The activity of stress-induced catalase and polyphenol oxidase was reduced, while that of peroxidase and superoxide dismutase increased after inoculation but the response was temporal. Taken together, this data explains that different PGPR (especially C. sakazakii RF-4) modulate differential responses in wheat that eventually leads towards drought tolerance, hence, it has the potential to enhance crop production in arid regions.

Tailored Bioactive Compost from Agri-Waste Improves the Growth and Yield of Chili Pepper and Tomato.

An extensive use of chemical fertilizers has posed a serious impact on food and environmental quality and sustainability. As the organic and biofertilizers can satisfactorily fulfill the crop's nutritional requirement, the plants require less chemical fertilizer application; hence, the food is low in chemical residues and environment is less polluted. The agriculture crop residues, being a rich source of nutrients, can be used to feed the soil and crops after composting and is a practicable approach to sustainable waste management and organic agriculture instead of open-field burning of crop residues. This study demonstrates a feasible strategy to convert the wheat and rice plant residues into composted organic fertilizer and subsequent enrichment with plant-beneficial bacteria. The bioactive compost was then tested in a series of in vitro and in vivo experiments for validating its role in growing organic vegetables. The compost was enriched with a blend of micronutrients, such as zinc, magnesium, and iron, and a multi-trait bacterial consortium AAP (Azospirillum, Arthrobacter, and Pseudomonas spp.). The bacterial consortium AAP showed survival up to 180 days post-inoculation while maintaining their PGP traits. Field emission scanning electron microscopic analysis and fluorescence in situ hybridization (FISH) of bioactive compost further elaborated the morphology and confirmed the PGPR survival and distribution. Plant inoculation of this bioactive compost showed significant improvement in the growth and yield of chilies and tomato without any additional chemical fertilizer yielding a high value to cost ratio. An increase of ≈35% in chlorophyll contents, ≈25% in biomass, and ≈75% in yield was observed in chilies and tomatoes. The increase in N was 18.7 and 25%, while in P contents were 18.5 and 19% in chilies and tomatoes, respectively. The application of bioactive compost significantly stimulated the bacterial population as well as the phosphatase and dehydrogenase activities of soil. These results suggest that bioactive compost can serve as a source of bioorganic fertilizer to get maximum benefits regarding vegetable yield, soil quality, and fertilizer saving with the anticipated application for other food crops. It is a possible win-win situation for environmental sustainability and food security.

Plant survival under drought stress: Implications, adaptive responses, and integrated rhizosphere management strategy for stress mitigation.

In many regions of the world, the incidence and extent of drought spells are predicted to increase which will create considerable pressure on global agricultural yields. Most likely among all the abiotic stresses, drought has the strongest effect on soil biota and plants along with complex environmental effects on other ecological systems. Plants being sessile appears the least resilient where drought creates osmotic stress, limits nutrient mobility due to soil heterogeneity, and reduces nutrient access to plant roots. Drought tolerance is a complex quantitative trait controlled by many genes and is one of the difficult traits to study and characterize. Nevertheless, existing studies on drought have indicated the mechanisms of drought resistance in plants on the morphological, physiological, and molecular basis and strategies have been devised to cope with the drought stress such as mass screening, breeding, marker-assisted selection, exogenous application of hormones or osmoprotectants and or engineering for drought resistance. These strategies have largely ignored the role of the rhizosphere in the plant's drought response. Studies have shown that soil microbes have a substantial role in modulation of plant response towards biotic and abiotic stress including drought. This response is complex and involves alteration in host root system architecture through hormones, osmoregulation, signaling through reactive oxygen species (ROS), induction of systemic tolerance (IST), production of large chain extracellular polysaccharides (EPS), and transcriptional regulation of host stress response genes. This review focuses on the integrated rhizosphere management strategy for drought stress mitigation in plants with a special focus on rhizosphere management. This combinatorial approach may include rhizosphere engineering by addition of drought-tolerant bacteria, nanoparticles, liquid nano clay (LNC), nutrients, organic matter, along with plant-modification with next-generation genome editing tool (e.g., CRISPR/Cas9) for quickly addressing emerging challenges in agriculture. Furthermore, large volumes of rainwater and wastewater generated daily can be smartly recycled and reused for agriculture. Farmers and other stakeholders will get a proper knowledge-exchange and an ideal road map to utilize available technologies effectively and to translate the measures into successful plant-water stress management. The proposed approach is cost-effective, eco-friendly, user-friendly, and will impart long-lasting benefits on agriculture and ecosystem and reduce vulnerability to climate change.

Phylogenetic diversity analysis reveals Bradyrhizobium yuanmingense and Ensifer aridi as major symbionts of mung bean (Vigna radiata L.) in Pakistan.

The present study was carried out to evaluate the diversity of rhizobia associated with nodules of mung bean in Pakistan, because this information is necessary for inoculum development. Based on sequence analysis of 16S rRNA gene of thirty-one bacteria, 11 were assigned to genus Bradyrhizobium, 17 to Ensifer, and 3 to Rhizobium. Phylogenetic analyses on the basis of 16S-23S ITS region, atpD, recA, nifH, and nodA of representative strains revealed that B. yuanmingense is the predominant species distributed throughout different mung bean-growing areas. Among the fast-growing rhizobia, Ensifer aridi was predominant in Faisalabad, Layyah, and Rawalpindi, while E. meliloti in Thal desert. Sequence variations and phylogeny of nifH and nodA genes suggested that these genes might have been co-evolved with the housekeeping genes and maintained by vertical gene transfer in rhizobia detected in the present study. Host infectivity assay revealed the successful nodulation of host by rhizobia related to genera Bradyrhizobium, Ensifer and Rhizobium. Among all, Bradyrhizobium and Ensifer spp. inoculation exhibited a significantly higher number of nodules (11-34 nodules plant-1) and nitrogenase activity (nodule ARA 60-110 µmol g-1 h-1). Contrary to the previous studies, our data reveal that B. yuanmingense and E. aridi are predominant species forming effective nodules in mung bean in Pakistan. Furthermore, to the best of our knowledge, this is the first report showing the effective symbiosis of E. aridi, E. meliloti, and Rhizobium pusense with mung bean. The diversity of rhizobia in different habitats revealed in the present study will contribute towards designing site-specific inocula for mung bean.

Illumina sequencing of 16S rRNA tag shows disparity in rhizobial and non-rhizobial diversity associated with root nodules of mung bean (Vigna radiata L.) growing in different habitats in Pakistan.

In Rhizobium-legume symbiosis, the nodule is the most frequently studied compartment, where the endophytic/symbiotic microbiota demands critical investigation for development of specific inocula. We identified the bacterial diversity within root nodules of mung bean from different growing areas of Pakistan using Illumina sequencing of 16S rRNA gene. We observed specific OTUs related to specific site where Bradyrhizobium was found to be the dominant genus comprising of 82-94% of total rhizobia in nodules with very minor fraction of sequences from other rhizobia at three sites. In contrast, Ensifer (Sinorhizobium) was single dominant genus comprising 99.9% of total rhizobial sequences at site four. Among non-rhizobial sequences, the genus Acinetobacter was abundant (7-18% of total sequences), particularly in Bradyrhizobium-dominated nodule samples. Rhizobia and non-rhizobial PGPR isolated from nodule samples include Ensifer, Bradyrhizobium, Acinetobacter, Microbacterium and Pseudomonas strains. Co-inoculation of multi-trait PGPR Acinetobacter sp. VrB1 with either of the two rhizobia in field exhibited more positive effect on nodulation and plant growth than single-strain inoculation which favors the use of Acinetobacter as an essential component for development of mung bean inoculum. Furthermore, site-specific dominance of rhizobia and non-rhizobia revealed in this study may contribute towards decision making for development and application of specific inocula in different habitats.

Retrieved 16S rRNA and nifH sequences reveal co-dominance of Bradyrhizobium and Ensifer (Sinorhizobium) strains in field-collected root nodules of the promiscuous host Vigna radiata (L.) R. Wilczek.

In the present study, the relative distribution of endophytic rhizobia in field-collected root nodules of the promiscuous host mung bean was investigated by sequencing of 16S ribosomal RNA (rRNA) and nifH genes, amplified directly from the nodule DNA. Co-dominance of the genera Bradyrhizobium and Ensifer was indicated by 32.05 and 35.84% of the total retrieved 16S rRNA sequences, respectively, and the sequences of genera Mesorhizobium and Rhizobium comprised only 0.06 and 2.06% of the recovered sequences, respectively. Sequences amplified from rhizosphere soil DNA indicated that only a minor fraction originated from Bradyrhizobium and Ensifer strains, comprising about 0.46 and 0.67% of the total retrieved sequences, respectively. 16S rRNA gene sequencing has also identified the presence of several non-rhizobial endophytes from phyla Proteobacteria, Actinobacteria, Bacteroides, and Firmicutes. The nifH sequences obtained from nodules also confirmed the co-dominance of Bradyrhizobium (39.21%) and Ensifer (59.23%) strains. The nifH sequences of the genus Rhizobium were absent, and those of genus Mesorhizobium comprised only a minor fraction of the sequences recovered from the nodules and rhizosphere soil samples. Two bacterial isolates, identified by 16S rRNA gene sequence analysis as Bradyrhizobium strain Vr51 and Ensifer strain Vr38, successfully nodulated the original host (mung bean) plants. Co-dominance of Bradyrhizobium and Ensifer strains in the nodules of mung bean indicates the potential role of the host plant in selecting specific endophytic rhizobial populations. Furthermore, successful nodulation of mung bean by the isolates showed that strains of both the genera Bradyrhizobium and Ensifer can be used for production of inoculum.