Mocrobial Diversity and Metagenomic Analysis of Rhizosphere of Plants Growing in Extremely Halophytic Environment (Khewra Salt Mines)

Abstract

Natural hypersaline environments such as Khewra Salt Mines seem to be a promising source of bacteria able to alleviate salt stress in important crops such as wheat, rice and maize. Bacteria belonging to various genera have been able to impart salt tolerance to plants growing in such places. Saline soils get more attention these days as a result of shortage of arable lands and demands for environmental restoration. Therefore it is important to get better understanding of the microbial diversity and their ecological role in saline environments. The goal of the present study was to evaluate the diversity of bacterial communities in the rhizosphere of halophytes (Salsola stocksii and Atriplex amnicola), non-rhizospheric soil and hypersaline lake-bank soil samples by culture dependent and metagenomic approaches. This study included assessment of microbial diversity by isolation of bacteria from the different fractions of the rhizosphere of halophytes, to unlock the massive uncultured microbial diversity present in the rhizosphere of halophytes from moderately and highly saline environments by pyrosequencing analysis of 16S rRNA gene, comparison of culturable and non-culturable microbial diversity from the rhizosphere of halophytes. Additionally, megaplasmids were isolated from different halophilic bacterial strains and plasmids conferring salt tolerant genes were characterized. To study culturable microbial diversity from the rhizosphere of halophytes (S. stocksii and A. amnicola), non-rhizospheric soil and hypersaline lake-bank soil samples, two media were used; (1) HaP (halophilic medium) with 2M NaCl concentration and (2) hypersaline MGM (minimal growth medium) specific for haloarchaea with 4M NaCl concentrations. On the basis of salt tolerance ability, 47 halophilic isolates from the rhizosphere of Salsola, 42 isolates from Atriplex, 27 isolates from non-rhizospheric and 27 isolates from hypersaline lake-bank soils were identified by using 16S rRNA gene analysis. Phylogenetic analysis showed that bacterial strains belonging to Bacillus, Pseudomonas, Halobacillus and Kocuria were dominant in the rhizosphere of halophytes (Salsola and Atriplex) and non-rhizospheric and hypersaline lake-bank soils. Halobacterium and Halococcus were dominant archaeal genera identified from all soils. To characterize plasmid conferring salt tolerant genes, selected halophilic strains with salt tolerance greater than 2.5M NaCl were plasmid cured by using heat shock method, 3% SDS and sodium benzoate. These plasmids were isolated and transformed into E. coli strains (Top10, DH10α and BL21). The growth response of wild type, plasmid cured and transformed E. coli strains was compared at 1.5-4M NaCl concentration. Plasmids from halophilic Bacillus strain HL2HP6 (Bacillus endophyticus) were sequenced by using whole genome sequencing technique at Hirsch’s lab, UCLA in collaboration with Joint Genome Institute lab (JGI), USA. Results of plasmid curing showed that some halophilic bacterial strains such as Bacillus strain HL2HP6, Oceanobacillus strain AT3HP15 and Oceanobacillus strain LK3HaP7 lost their ability to grow in halophilic medium but they grew well on LB medium. Functional analysis of plasmid sequences showed different proteins and enzymes which are known to be involved in genetic information processing, carbohydrate metabolism, amino acid metabolism, xenobiotics biodegradation and metabolism, glycan biosynthesis and metabolism and human diseases. To study the plant growth promoting effect of phosphate solubilizing bacteria, five bacterial isolates; three Bacillus (HL1HP11, HL3RS14 and LK1HaP9), one Enterobacter aerogenes (AT1HP4) and one Aeromonas veronii (AT1RP10) strains were used as inoculants; in the form of seed coat and enriched soil based phosphate biofertilizers. All bacterial strains positively affected the plant growth as compared to non-inoculated control plants. Plants inoculated with Bacillus strain HL3RS14 based soil biofertilizers showed maximum increase in dry weights of root (51-104%) and shoot (35-114%) as compared to control (soil + rock phosphate, no inoculum). Rhizosphere microbiomes of halophytes Urochloa, Kochia, Salsola, and Atriplex living in moderately and highly saline environments and non-halophyte Triticum were analysed by using pyrosequencing technique. Metagenomic analysis of soil microbiomes indicated that Actinobacteria were dominant in the saline soils whereas Proteobacteria predominated in non-saline soils. Firmicutes, Acidobacteria, Bacteriodetes, Planctomycetes and Thaumarchaeota were predominant phyla in saline and non-saline soils, whereas Cyanobacteria, Verrucomicrobia, Choroflexi, Gemmatimonadetes and the unclassified WPS-2 were less abundant. Plant microbiome of halophyte (Salsola) and non-halophyte (wheat) was also studied through metagenomics approach. Proteobacteria and Actinobacteria were the most abundant phyla in the rhizosphere, root endosphere and phyllosphere of Salsola and wheat. However, Firmicutes, Acidobacteria, Bacteriodetes, Planctomycetes, Cyanobacteria, Verrucomicrobia, Choroflexi and Euryarchaeota were predominant groups from halophyte whereas Actinobacteria, Proteobacteria, Firmicutes, Cyanobacteria, Acidobacteria, Bacteriodetes, Planctomycetes and Verrucomicrobia were predominant phyla of wheat samples. Diversity and differences of microbial flora of Salsola and wheat suggested that functional interactions between plants and microorganisms contribute to salt stress tolerance.