Emerging Molecular Tools for Engineering Phytomicrobiome.

Microbial plant interaction plays a major role in the sustainability of plants. The understanding of phytomicrobiome interactions enables the gene-editing tools for the construction of the microbial consortia. In this interaction, microbes share several common secondary metabolites and terpenoid metabolic pathways with their host plants that ensure a direct connection between the microbiome and associated plant metabolome. In this way, the CRISPR-mediated gene-editing tool provides an attractive approach to accomplish the creation of microbial consortia. On the other hand, the genetic manipulation of the host plant with the help of CRISPR-Cas9 can facilitate the characterization and identification of the genetic determinants. It leads to the enhancement of microbial capacity for more trait improvement. Many plant characteristics like phytovolatilization, phytoextraction, phytodesalination and phytodegradation are targeted by these approaches. Alternatively, chemical communications by PGPB are accomplished by the exchange of different signal molecules. For example, quorum-sensing is the way of the cell to cell communication in bacteria that lead to the detection of metabolites produced by pathogens during adverse conditions and also helpful in devising some tactics towards understanding plant immunity. Along with quorum-sensing, different volatile organic compounds and N-acyl homoserine lactones play a significant role in cell to cell communication by microbe to plant and among the plants respectively. Therefore, it is necessary to get details of all the significant approaches that are useful in exploring cell to cell communications. In this review, we have described gene-editing tools and the cell to cell communication process by quorum-sensing based signaling. These signaling processes via CRISPR- Cas9 mediated gene editing can improve the microbe-plant community in adverse climatic conditions.

Low-cost media engineering for phosphate and IAA production by Kosakonia pseudosacchari TCPS-4 using Multi-objective Genetic Algorithm (MOGA) statistical tool.

The plant growth-promoting rhizobacteria (PGPR) can improve the biotic or abiotic stress condition by exploiting the productivity and plant growth of the plants under stressful conditions. This study examines the role of a rhizospheric bacterial isolate Kosakonia pseudosacchari TCPS-4 isolated from cluster bean plant (Cyamopsis tetragonoloba) under dryland condition. The low-cost media engineering was evaluated, and the phosphate-solubilizing and IAA-producing abilities of Kosakonia pseudosacchari TCPS-4 were improved using a hybrid statistical tool viz. Multi-objective Genetic Algorithm (MOGA). Further, the effect of carbon and nitrogen media constituents and their interactions on IAA production and phosphate solubilization were also confirmed by a single-factor experiment assay. This revealed that MOGA-based model depicted 47.5 mg/L inorganic phosphate as the highest phosphate concentration in media containing 45 g/L carbon source, 12 g/L nitrogen source and 0.20 g/L MgSO4. The highest IAA production was 18.74 mg/L in media containing 45 g/L carbon source, 12 g/L nitrogen source and 0.2 g/L MgSO4. These values were also confirmed and measured by the experiments with phosphate solubilization of 45.71 mg/L and IAA production of 18.71 mg/L with 1012 cfu/mL. This concludes that effective media engineering using these statistical tools can enhance the phosphate and IAA production by each model. A good correlation between measured and predicted values of each model confirms the validity of both responses. The present study gives an insight on media engineering for phosphate and IAA production by Kosakonia pseudosacchari TCPS-4.

Mechanism and application of Sesbania root-nodulating bacteria: an alternative for chemical fertilizers and sustainable development.

Chemical fertilizers are used in large-scale throughout the globe to satisfy the food and feed requirement of the world. Demanding cropping with the enhanced application of chemical fertilizers, linked with a decline in the recycling of natural or other waste materials, has led to a decrease in the organic carbon levels in soils, impaired soil physical properties and shrinking soil microbial biodiversity. Sustenance and improvement of soil fertility are fundamental for comprehensive food security and ecological sustainability. To feed the large-scale growing population, the role of biofertilizers and their study tends to be an essential aspect globally. In this review, we have emphasized the nitrogen-fixing plants of Sesbania species. It is a plant that is able to accumulate nitrogen-rich biomass and used as a green manure, which help in soil amelioration. Problems of soil infertility due to salinity, alkalinity and waterlogging could be alleviated through the use of biologically fixed nitrogen by Sesbania plants leading to the conversion of futile land into a fertile one. A group of plant growth-promoting rhizobacteria termed as "rhizobia" are able to nodulate a variety of legumes including Sesbania. The host-specific rhizobial strains can be used as potential alternative for nitrogenous fertilizers as they help the host plant in growth and development and enhance their endurance under stressed conditions. The review gives the depth understanding of how the agriculturally important microorganisms can be used for the reduction of broad-scale application of chemical fertilizers with special attention to Sesbania-nodulating rhizobia.

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.

Techniques for improving formulations of bioinoculants.

Bioinoculants are eco-friendly microorganisms having a variety of products commonly utilized for improving the potential of soil and providing the nutrient requirements to the host plant. The usage of chemical fertilizers is not beneficial because it affects the soil microbial communities on large scale. The toxicity of chemical fertilizer decreases the fertility of soil and causes microbial disruption. Bioinoculants that are used as PGPR play an important role in the enhancement of crop production and beneficial for both producers and consumers economically by protecting the soil during unfavourable conditions. The utilization of PGPR in the bioinoculant form imparts successfully sustain agricultural yield production and such formulated products contain living microbial cells of bioinoculants that also helps in seed treatment and enhances the mobilization process of nutrients by the low-cost process. This review mainly focuses on different bioinoculant formulations related to its recent approaches such as metabolite formulations, liquid formulations, solid carrier-based formulations and synthetic polymer-based formulations. This review also gives an overview of some aspects of the bioinoculant efficiency and their appropriate formulation, production and storage condition of microbial cells.

Deciphering the Potential of Rhizobium pusense MB-17a, a Plant Growth-Promoting Root Endophyte, and Functional Annotation of the Genes Involved in the Metabolic Pathway.

Plant growth-promoting rhizobacteria (PGPR) are root endophytic bacteria used for growth promotion, and they have broader applications in enhancing specific crop yield as a whole. In the present study, we have explored the potential of Rhizobium pusense MB-17a as an endophytic bacterium isolated from the roots of the mung bean (Vigna radiata) plant. Furthermore, this bacterium was sequenced and assembled to reveal its genomic potential associated with plant growth-promoting traits. Interestingly, the root endophyte R. pusense MB-17a showed all essential PGPR traits which were determined by biochemical and PGPR tests. It was noted that this root endophytic bacterium significantly produced siderophores, indole acetic acid (IAA), ammonia, and ACC deaminase and efficiently solubilized phosphate. The maximum IAA and ammonia produced were observed to be 110.5 and 81 µg/ml, respectively. Moreover, the PGPR potential of this endophytic bacterium was also confirmed by a pot experiment for mung bean (V. radiata), whose results show a substantial increase in the plant's fresh weight by 76.1% and dry weight by 76.5% on the 60th day after inoculation of R. pusense MB-17a. Also, there is a significant enhancement in the nodule number by 66.1% and nodule fresh weight by 162% at 45th day after inoculation with 100% field capacity after the inoculation of R. pusense MB-17a. Besides this, the functional genomic annotation of R. pusense MB-17a determined the presence of different proteins and transporters that are responsible for its stress tolerance and its plant growth-promoting properties. It was concluded that the unique presence of genes like rpoH, otsAB, and clpB enhances the symbiosis process during adverse conditions in this endophyte. Through Rapid Annotation using Subsystem Technology (RAST) analysis, the key genes involved in the production of siderophores, volatile compounds, indoles, nitrogenases, and amino acids were also predicted. In conclusion, the strain described in this study gives a novel idea of using such type of endophytes for improving plant growth-promoting traits under different stress conditions for sustainable agriculture.

Gordonia sp.: a salt tolerant bacterial inoculant for growth promotion of pearl millet under saline soil conditions.

In the resent study, a diazotrophic bacterial isolate JPA2 having the ability to tolerate salinity (6 % NaCl) and plant growth-promoting features was isolated from rhizospheric soil of weed Chenopodium murale growing in saline soil of Pindara (EC 11.47 dS m-1), district Jind (Haryana). The nitrogen fixing ability of the isolate was confirmed by nifH gene amplification and acetylene reduction assay (38.9 nmol ethylene h-1 mg-1 protein). The potential of strain JPA2 to promote growth of pearl millet was investigated by inoculation experiment which showed significant increase in plant height (51.1, 39.9 and 28.8 %) and dry weight (55.9, 36.4 and 35.5 %) over uninoculated control plants at EC 0, 6, 8 dS m-1, respectively. The strain JPA2 was Gram +ve and identified as Gordonia sp. on the basis of partial 16S rRNA gene sequencing and biochemical characterization. It is concluded that salt tolerant diazotrophic Gordonia sp. can be considered as a beneficial microbe for agriculture in saline soils.

Isolation of a multi-trait plant growth promoting Brevundimonas sp. and its effect on the growth of Bt-cotton.

Arid regions pose a serious problem for crop production by suppressing plant growth. The use of plant growth promoting rhizobacteria (PGPR) as bioinoculant may be promising to enhance the crop yield in arid conditions. In the present investigation, four strains of cultivable bacteria associated with the rhizosphere of Saccharum L. grown in arid region were isolated using N-free media. Assessment of their nitrogen-fixing ability through amplification of nifH gene showed the presence of nifH gene (390 bp) in only one (MDB4) of the four isolates. The nitrogen-fixing potential of this isolate was confirmed by the presence of nitrogenase activity determined using acetylene reduction assay. The diazotrophic MDB4 isolate also exhibited other PGPR traits, such as the production of indole-3-acetic acid (IAA) and ammonia. In pot experiments, inoculation of Bt-cotton seeds with MDB4 enhanced the growth of plants as shown by significant increase in plant height (68.41 %), shoot dry weight (58.44 %) and root dry weight (64.81 %) over untreated control. The MDB4 strain was Gram negative and identified as Brevundimonas sp. on the basis of phenotypic, biochemical, phylogenetic and 16S rRNA gene sequencing data. It is concluded that the MDB4 bacterial strain having different plant growth promoting activities can be considered as a beneficial microbe for sustainable agriculture in arid regions.

Studies on salinization in Haryana soils on free-living nitrogen-fixing bacterial populations and their activity.

A total of 26 soil samples from saline soils of Haryana were collected. Based on their electrical conductivity (EC) values, which varied from 1.04 to 21.00 dS m(-1), the soils were categorized into non-saline soils (EC 0-2 dS m(-1)), weakly saline soils (EC 2-4 dS m(-1)), saline soils (EC 4-8 dS m(-1)), strongly saline soils (EC 8-16 dS m(-1)), and very strongly saline soils (EC >16 dS m(-1)). The pH values of these soil samples ranged from 6.03 to 8.62, while organic C, total N, and available P were in the range of 0.06-0.94%, 0.07-0.15%, and 0.11-0.29 µg g(-1) soil, respectively. As a measure of the impact of salinity on free-living N(2) fixers and their activity, the total bacterial populations on four media (Jensen's nitrogen-free medium, malate medium, Burk's medium, and soil extract agar medium) decreased from 6.12 to 3.70 log CFU g(-1) soil with increasing salinity level. PCR amplification of the nifH region of the DNA from 234 selected morphotypes from all the media showed the presence of nifH in 71 isolates. Out of these, 37% of the isolates were obtained using Jensen's medium; 35, 28, and 21% of the isolates were obtained using soil extract medium, Burk's medium, and malate medium, respectively. The majority of the free-living N(2) fixers (67%) were Gram negative. Apart from the acetylene reduction assay (ARA) activity in these isolates, other beneficial traits like ammonia excretion and indole acetic acid (IAA) production were also present. A decreasing trend in the activities was observed with increasing salinity levels. Isolates JN6, BP8, and MJ4 showed the highest ARA activity, ammonia excretion, and IAA production. The performance of isolates like BNC2 with good ARA activity, ammonia excretion, and IAA production and isolated from a very strongly saline soil should be further evaluated under high-saline conditions.