Psychrotrophic plant beneficial bacteria from the glacial ecosystem of Sikkim Himalaya: Genomic evidence for the cold adaptation and plant growth promotion.

Commercial biofertilizers tend to be ineffective in cold mountainous regions due to reduced metabolic activity of the microbial inoculants under low temperatures. Cold-adapted glacier bacteria with plant growth-promoting (PGP) properties may prove significant in developing cold-active biofertilizers for improving mountain agriculture. With this perspective, the cultivable bacterial diversity was documented from the East Rathong glacier ecosystem lying above 3900 masl of Sikkim Himalaya. A total of 120 bacterial isolates affiliated to Gammaproteobacteria (53.33%), Bacteroidetes (16.66%), Actinobacteria (15.83%), Betaproteobacteria (6.66%), Alphaproteobacteria (4.16%), and Firmicutes (3.33%) were recovered. Fifty-two isolates showed many in vitro PGP activities of phosphate solubilization (9-100 µg/mL), siderophore production (0.3-100 psu) and phytohormone indole acetic acid production (0.3-139 µg/mL) at 10 °C. Plant-based bioassays revealed an enhancement of shoot length by 21%, 22%, and 13% in ERGS5:01, ERMR1:04, and ERMR1:05, and root length by 14%, 17%, 11%, and 22% in ERGS4:06, ERGS5:01, ERMR1:04, and ERMR1:05 treated seeds respectively. An increased shoot dry weight of 4-29% in ERMR1:05 and ERMR1:04, and root dry weight of 42-98% was found in all the treatments. Genome analysis of four bacteria from diverse genera predicted many genes involved in the bacterial PGP activity. Comparative genome study highlighted the presence of PGP-associated unique genes for glucose dehydrogenase, siderophore receptor, tryptophan synthase, phosphate metabolism (phoH, P, Q, R, U), nitrate and nitrite reductase, TonB-dependent receptor, spermidine/putrescine ABC transporter etc. in the representative bacteria. The expression levels of seven cold stress-responsive genes in the cold-adapted bacterium ERGS4:06 using real-time quantitative PCR (RT-qPCR) showed an upregulation of all these genes by 6-17% at 10 °C, and by 3-33% during cold-shock, which indicates the cold adaptation strategy of the bacterium. Overall, this study signifies the psychrotrophic bacterial diversity from an extreme glacier environment as a potential tool for improving plant growth under cold environmental stress.