Deep Chandra Suyal

Differential Proteomics in Response to Low Temperature Diazotrophy of Himalayan Psychrophilic Nitrogen Fixing Pseudomonas migulae S10724 Strain

Himalayas are considered as a reservoir of diversified and dynamic gene pool. This study describes the response of a Himalayan psychrophilic diazotroph to low temperature diazotrophy. Seven cold adaptive N2 fixing bacteria were isolated and identified as Bacillus sp., Arthrobacter sp., Rhodococcus sp., Pseudomonas sp., etc. In order to examine the physiological response to low temperature diazotrophy, differential proteomic analysis of Pseudomonas migulae S10724 strain was carried out using two dimensional electrophoresis and MALDI–TOF–MS. Functional assessment of 66 differentially expressed proteins revealed several mechanisms thought to be involved in low temperature adaptation and nitrogen fixation, including general stress adaptation, protein and nucleic acid synthesis, energy metabolism, cell growth/maintenance, etc. Major fraction of the upregulated proteins was stress proteins, while majority of the downregulated proteins were related to cell division. Furthermore, MALDI–TOF–MS-based identification of randomly selected peptides encountered two exclusively expressed proteins: NifU family SUF system FeS assembly protein and membrane protein, suppressor for copper-sensitivity B precursor which might have a crucial role at low temperature nitrogen fixation. To the best of our knowledge, this is the first report of the isolation and differential proteomic analysis of psychrophilic diazotroph from Himalayan high altitude rhizospheric soil.

Bacterial diversity and community structure of Western Indian Himalayan red kidney bean (Phaseolus vulgaris) rhizosphere as revealed by 16S rRNA gene sequences

Abstract Agriculture is an important livelihood activity in the Himalayan regions. Our previous studies revealed the presence of diverse diazotrophic assemblage in indigenous red kidney bean (RKB) rhizospheric soil from two different locations of Western Indian Himalaya, namely S1 (Chhiplakot, 30.70◦ N/80.30° E) and S2 (Munsyari, 30.60◦ N/80.20° E), selected on the basis of real-time PCR analysis. In this study, two 16S rRNA gene clone libraries (SB1 and SB2, respectively) were constructed using the same rhizospheric soil samples for assessing the total bacterial diversity and their community structure. A total of 760 clones were obtained, with ∼54-59% of these sequences belonging to the phylum Proteobacteria. While sequences belonging to Bacteroidetes, Chloroflexi, Acidobactria, Planctomycetes, Firmicutes, Nitrospira, Gemmatimonadetes, Cyanobacteria, Verrucomicrobia, OD1, OP11 and Actinobacteria were encountered in both the soils, sequences belonging to bacteria from the classes Chlorobi and BRC1 were only detected in the S1 soil. Both the libraries showed similar bacterial community compositions, with Pseudomonas (∼33-34%) as predominant genus. Phylogenetic analysis revealed that all the clone sequences were clustered in different bacterial groups as per their resemblance with their respective phylogenetic neighbours. Major clusters were formed by Gammapreoteobacteria followed by Bacteroidetes and Alphaproteobacteria. A good fraction of the clone sequences has no resemblance with existing groups, thereby suggesting the need of culture-dependent studies from Himalayan regions. To the best of our knowledge, this study is the first major metagenomic effort on Himalayan RKBs rhizobacteria revealing fundamental information that needs to be explored for functional studies.

Diversified diazotrophs associated with the rhizosphere of Western Indian Himalayan native red kidney beans (Phaseolus vulgaris L.)

Red kidney beans (RKBs) are one of the major components in the human diet of Western Indian Himalaya (WIH). Their cultivation in these habitats is strongly influenced by various biotic and abiotic stresses and therefore, there must be a selection of RKB associated microorganisms that are adapted to these harsh conditions. Seven cold adaptive diazotrophs from the same rhizosphere were isolated in our previous study to reveal the low-temperature associated proteins and mechanisms. However, the diversity and phylogenetic affiliations of these rhizosphere diazotrophs are still unknown. In this study, RKB rhizospheric soil from two different agro-ecosystems of WIH namely S1 (Chhiplakot, 30.70°N/80.30°E) and S2 (Munsyari, 30.60°N/80.20°E) were explored for the assessment of nitrogenase reductase gene (nifH) diversity by plating respective clone libraries SN1 and SN2. The RKB rhizosphere diazotroph assemblage was very diverse and apparently consists mainly of the genera Rhizobium, followed by unknown diazotrophic microorganisms. Deduced amino acid sequence analysis revealed the presence of diverse nifH sequences, affiliated with a wide range of taxa, encompassing members of the Proteobacteria, Actinobacteria and Firmicutes. Members of cyanobacteria, methanotrophs and archaea were also detected. To the best of our knowledge, this is the first major metagenomic effort that revealed the presence of diverse nitrogen-fixing microbial assemblages in indigenous RKB rhizospheric soil which can further be explored for improved crop yield/productivity.

Cold Stress and Nitrogen Deficiency Affected Protein Expression of Psychrotrophic Dyadobacter psychrophilus B2 and Pseudomonas jessenii MP1

Nitrogen (N) deficiency and low temperature conditions are the prominent facet of Western Himalayan agro-ecosystems. A slight change in the environment alters the protein expression of the microorganisms. Therefore, proteomes of the two psychrotrophs Dyadobacter psychrophilus B2 and Pseudomonas jessenii MP1 were analyzed using two dimensional electrophoresis and MALDI–TOF–MS, to determine the physiological response of altitudinally different but indigenous microorganisms in response to cold stress under N depleting conditions. Functional assessment of 150 differentially expressed proteins from both the psychrotrophs revealed several mechanisms might be involved in cold stress adaptation, protein synthesis/modifications, energy metabolism, cell growth/maintenance, etc. In both the proteomes, abundance of the proteins related to energy production and stress were significantly increased while, proteins related to biosynthesis and energy consuming processes decreased. ATP synthase subunit alpha, beta, ATP-dependent Clp protease, Enolase, groL HtpG and N(2)-fixation sustaining protein CowN proteins were found to be expressed in both B2 and MP1, similarly to previously studied diazotrophs under low temperature N2 fixing conditions and therefore, can be considered as a biomarker for monitoring the nitrogen fixation in cold niches. Nevertheless, in both the diazotrophs, a good fraction of the proteins were related to hypothetical proteins which are still uncharacterized, thereby, suggesting the need for in-depth studies on cold adapted diazotrophs and their adaptive mechanisms.

Isolation and characterization of phosphate solubilizing bacteria from Western Indian Himalayan soils

Previous studies confirmed the existence of diversified microbial flora in the rhizosphere of Himalayan Red Kidney Bean (RKB) (Phaseolus vulgaris L.). Therefore, fifteen different temperate and subtropical regions of Western Indian Himalaya (WIH) were explored for the isolation of RKB rhizosphere-associated Phosphorus (P) solubilizing bacteria. On the basis of qPCR analysis, three soils, i.e., Munsyari, Kandakhal and Nainital soils were selected for the isolation of P solubilizers. Among 133 isolates, three bacteria viz. Lysinibaccilus macroides ST-30, Pseudomonas palleroniana N-26 and Pseudomonas jessenii MP-1 were selected based on their P solubilization potential. Moreover, in vitro seed germination assay was performed to investigate their effectiveness against four native crops viz. (Cicer arietrinum L.), (Vigna radiata L.), (Pisum sativum L.) and (Zea mays L.). Treated seeds showed significant increase in germination efficiency over their respective controls. The results suggest that Lysinibaccilus macroides ST-30, strain is a potential plant growth-promoting bacterium for chickpea (Cicer arietrinum L.) and, therefore, could be implemented as a low-cost bio-inoculant in hill agriculture system.

Microbial Inoculants as Biofertilizer

Increasing application of chemical fertilizers in agriculture make country self dependent in food production but it depreciate environment and cause harmful impacts on living beings. The excess uses of these fertilizers in agriculture are costly and have various adverse effects on soil fertility. Further, soil microorganisms play an important role in the plant growth and development by various means viz. nitrogen fixation, phosphate solubilisation, phytohormone production etc. Therefore, bio-inoculants for agriculture purpose i.e. bio-fertilizers could be a better alternative to chemical fertilizers for agricultural as well as environmental sustainability.

Plant growth promoting potential of psychrotolerant Dyadobacter sp. for pulses and finger millet and impact of inoculation on soil chemical properties and diazotrophic abundance

ABSTRACT Western Indian Himalaya is very rich in biodiversity. Being a cold climatic region, it possesses various psychrotolerant and psychrophilic microorganisms. Psychrotolerant bacterium Dyadobacter sp. was isolated from this region and studied for its plant growth promoting potential against four legumes and finger millet. This bacterium was able to grow at nitrogen (N) deficient medium at both 10°C and 28°C and gave positive nifH amplification that confirms the psychrotolerant and diazotrophic nature of this bacterium. Pot trial-based study showed that this bacterium was able to promote plant growth by fixing atmospheric nitrogen (N2) and making it available to plants. Agronomical parameters, leaf nitrate reductase activity, and total chlorophyll content were recorded at 30, 45, 60, and 90 days after sowing and found to be increased over their respective controls. The 16S rDNA and nifH genes were quantified by q-PCR to study the dynamics of total bacterial and diazotrophic abundance due to inoculation of Dyadobacter sp. in soil. Soil chemical properties related to soil fertility were also studied at different time intervals after sowing. We found positive correlation among soil pH, soil nifH gene abundance, soil nitrate concentration, and plant leaf nitrate reductase activity. PCR-DGGE was performed to study persistence of Dyadobacter sp. in soil after inoculation, which showed good persistence of plant growth promoting rhizobacteria (PGPR). Hence, it is concluded that Dyadobacter sp. has potential to promote plant growth by fixing atmospheric N2 and making it available to plant. Further, psychrotolerant nature of this bacterium can be exploited to enhance plant growth in cold climate agriculture due to its ability to fix atmospheric N2 at low temperature.

Metagenomics of Plant Rhizosphere Microbiome

The rhizosphere is a specific microbial habitat in the soil ecosystem. This is the area where soil swayed by plant roots through plant exudates deposition. Further, rhizosphere and root microbiota provide useful services to their host plant, such as protection from pathogen and enhanced mineral acquirement from nearby soil for plant growth. Microbial communities, usually, interact with each other and their host, so it is important to detain as much of the microbial diversity as possible. It requires the use of modern analytical tools such as metagenomics, which can reveal the functional potential of a rhizosphere microbiome.

Elucidating stress proteins in rice (Oryza sativa L.) genotype under elevated temperature: a proteomic approach to understand heat stress response

Rice is one of the widely consumed staple foods among the world’s human population. Its production is adversely affected by high temperature and is more pronounced at flowering stage. Elucidating elevated temperature stress-related proteins as well as associated mechanisms is inevitable for improving heat tolerance in rice. In the present study, a proteomic analysis of heat-sensitive rice genotype, IET 21405 was conducted. Two-dimensional electrophoresis (2-DE) and MALDI-TOF/MS-based proteomics approaches revealed a total of 73 protein spots in rice leaf. The protein profiles clearly indicated variations in protein expression between the control and heat treated rice genotypes. Functional assessment of 73 expressed proteins revealed several mechanisms thought to be involved in high temperature including their putative role in metabolism, energy, protein synthesis, protein transport/storage, etc. Besides these, some proteins are expected to involve in photosynthesis, tricarboxylic acid (TCA) cycle, glycolysis and other proteins for energy production. The proteins identified in the present study provide a strong basis to elucidate gene function of these proteins and to explain further the molecular mechanisms underlying the adaptation of rice to high temperature stress.

Toward the Unculturable Microbes for Sustainable Agricultural Production

The microbes are elemental to maintain the life on the Earth, yet we have very little understanding about the majority of microbial forms present in various environments like soils. As per the information available from published researches, a big portion of microbial wealth is unculturable which may contain several beneficial traits, including the plant growth-promoting activities for sustainable agricultural production. Exploitation of these unculturable microbes can enhance our understanding of present practices of organic agriculture. The only way to exploit this unculturable wealth is the “metagenomics,” the culture-independent approach where we are analyzing microbial DNA extracted directly from an environmental sample.