Abstract
To study culturable bacterial diversity under subzero temperature conditions and their possible functional annotation, soil and water samples from Leh Ladakh region were analysed. Ten different nutrient combinations were used to isolate the maximum possible culturable morphotypes. A total of 325 bacterial isolates were characterized employing 16S rDNA-Amplified Ribosomal DNA Restriction Analysis with three restriction endonucleases AluI, MspI and HaeIII, which led to formation of 23–40 groups for the different sites at 75 % similarity index, adding up to 175 groups. Phylogenetic analysis based on 16S rRNA gene sequencing led to the identification of 175 bacteria, grouped in four phyla, Firmicutes (54 %), Proteobacteria (28 %), Actinobacteria (16 %) and Bacteroidetes (3 %), and included 29 different genera with 57 distinct species. Overall 39 % of the total morphotypes belonged to the Bacillus and Bacillus derived genera (BBDG) followed by Pseudomonas (14 %), Arthrobacter (9 %), Exiguobacterium (8 %), Alishewanella (4 %), Brachybacterium, Providencia, Planococcus (3 %), Janthinobacterium, Sphingobacterium, Kocuria (2 %) and Aurantimonas, Citricoccus, Cellulosimicrobium, Brevundimonas, Desemzia, Flavobacterium, Klebsiella, Paracoccus, Psychrobacter, Sporosarcina, Staphylococcus, Sinobaca, Stenotrophomonas, Sanguibacter, Vibrio (1 %). The representative isolates from each cluster were screened for their plant growth promoting characteristics at low temperature (5–15 °C). Variations were observed among strains for production of ammonia, hydrogen cyanide, indole-3-acetic acid and siderophore, solubilisation of phosphate, 1-aminocyclopropane-1-carboxylate deaminase activity and biocontrol activity against Rhizoctonia solani and Macrophomina phaseolina. Cold adapted microbes may have application as inoculants and biocontrol agents in crops growing at high altitudes under cold climate condition.
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References
Amato P, Hennebelle R, Magand O, Sancelme M, Delort AM, Barbante C, Boutron C, Ferrari C (2007) Bacterial characterization of the snow cover at Spitzberg, Svalbard. FEMS Microbiol Ecol 59:255–264
Bakker AW, Schippers B (1987) Microbial cyanide production in the rhizosphere in relation to potato yield reduction and Pseudomonas SPP-mediated plant growth-stimulation. Soil Biol Biochem 19:451–457
Bhromsiri C, Bhromsiri A (2010) Isolation, screening of growth-promoting activities and diversity of rhizobacteria from vetiver grass and rice plants. Thai J Agric Sci 43:217–230
Bric JM, Bostock RM, Silverstone SE (1991) Rapid in situ assay for indoleacetic acid production by bacteria immobilized on a nitrocellulose membrane. Appl Environ Microbiol 57:535–538
Brown ME, Burlingham SK (1968) Production of plant growth substances by Azotobacter chroococcum. J Gen Microbiol 53:135–144
Cappucino JC, Sherman N (1992) Nitrogen cycle. Microbiology: a laboratory manual, 4th edn. Benjamin/Cumming Pub. Co., New York, pp 311–312
Chaturvedi P, Shivaji S (2006) Exiguobacterium indicum sp. nov., a psychrophilic bacterium from the Hamta glacier of the Himalayan mountain ranges of India. Int J Syst Evol Microbiol 56:2765–2770
Chaturvedi P, Prabahar V, Manorama R, Pindi PK, Bhadra B, Begum Z, Shivaji S (2008) Exiguobacterium soli sp. nov., a psychrophilic bacterium from the McMurdo Dry Valleys, Antarctica. Int J Syst Evol Microbiol 58:2447–2453
Deming JW (2002) Psychrophiles and polar regions. Curr Opin Microbiol 5:301–309
Edwards U, Rogall T, Blöcker H, Emde M, Böttger EC (1989) Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res 17:7843–7853
Fruhling A, Schumann P, Hippe H, Straubler B, Stackebrandt E (2002) Exiguobacterium undae sp. nov. and Exiguobacterium antarcticum sp. nov. Int J Syst Evol Microbiol 52:1171–1176
Glick B, Patten C, Holguin G, Penrose D (1999) Overview of plant growth-promoting bacteria. In: Biochemical and genetic mechanisms used by plant gowth promoting bacteria. Imperial College Press, London, pp 1–13
Gordon SA, Weber RP (1951) Colorimetric estimation of indoleacetic acid. Plant Physiol 26:192
Jacobson CB, Pasternak J, Glick BR (1994) Partial purification and characterization of 1-aminocyclopropane-1-carboxylate deaminase from the plant growth promoting rhizobacterium Pseudomonas putida GR12-2. Can J Microbiol 40:1019–1025
Jurado V, Gonzalez JM, Laiz L, Saiz-Jimenez C (2006) Aurantimonas altamirensis sp. nov., a member of the order Rhizobiales isolated from Altamira Cave. Int J Syst Evol Microbiol 56:2583–2585
Kim WI, Kim SN, Ryu KY, Park CS (2011) Genetic diversity of cultivable plant growth-promoting rhizobacteria in Korea. J Microbiol Biotechnol 21:777–790
Kishore KH, Begum Z, Pathan AAK, Shivaji S (2010) Paenibacillus glacialis sp. nov., isolated from the Kafni glacier of the Himalayas, India. Int J Syst Evol Microbiol 60:1909–1913
Kumar M, Yadav AN, Tiwari R, Prasanna R, Saxena AK (2014) Deciphering the diversity of culturable thermotolerant bacteria from Manikaran hot springs. Ann Microbiol 64:741–751
Liu Y, Yao T, Jiao N, Kang S, Huang S, Li Q, Wang K, Liu X (2009) Culturable bacteria in glacial meltwater at 6,350 m on the East Rongbuk glacier, Mount Everest. Extremophiles 13:89–99
Mehta S, Nautiyal CS (2001) An efficient method for qualitative screening of phosphate-solubilizing bacteria. Curr Microbiol 43:51–56
Miteva VI, Brenchley JE (2005) Detection and isolation of ultrasmall microorganisms from a 120,000-year-old Greenland glacier ice core. Appl Environ Microbiol 71:7806–7818
Pandey S, Singh S, Yadav AN, Nain L, Saxena AK (2013) Phylogenetic diversity and characterization of novel and efficient cellulase producing bacterial isolates from various extreme environments. Biosci Biotech Biochem 77:1474–1480
Pikovskaya R (1948) Mobilization of phosphorus in soil in connection with vital activity of some microbial species. Mikrobiologiya 17:362–370
Prasad S, Manasa P, Buddhi S, Tirunagari P, Begum Z, Rajan S, Shivaji S (2014) Diversity and bioprospective potential (cold-active enzymes) of cultivable marine bacteria from the subarctic glacial fjord, Kongsfjorden. Curr Microbiol 68:233–238
Reddy G, Prakash J, Vairamani M, Prabhakar S, Matsumoto G, Shivaji S (2002) Planococcus antarcticus and Planococcus psychrophilus spp. nov. isolated from cyanobacterial mat samples collected from ponds in Antarctica. Extremophiles 6:253–261
Reddy GS, Matsumoto GI, Schumann P, Stackebrandt E, Shivaji S (2004) Psychrophilic pseudomonads from Antarctica: Pseudomonas antarctica sp. nov., Pseudomonas meridiana sp. nov. and Pseudomonas proteolytica sp. nov. Int J Syst Evol Microbiol 54:713–719
Reddy G, Uttam A, Shivaji S (2008) Bacillus cecembensis sp. nov., isolated from the Pindari glacier of the Indian Himalayas. Int J Syst Evol Microbiol 58:2330–2335
Rico A, Ortiz-Barredo A, Ritter E, Murillo J (2004) Genetic characterization of Erwinia amylovora strains by amplified fragment length polymorphism. J Appl Microbiol 96:302–310
Russell N, Fukunaga N (1990) A comparison of thermal adaptation of membrane lipids in psychrophilic and thermophilic bacteria. FEMS Microbiol Lett 75:171–182
Sahay H, Babu BK, Singh S, Kaushik R, Saxena AK, Arora DK (2013) Cold-active hydrolases producing bacteria from two different sub-glacial Himalayan lakes. J Basic Microbiol 53:703–714
Sattler B, Puxbaum H, Psenner R (2001) Bacterial growth in supercooled cloud droplets. Geophys Res Lett 28:239–242
Schwyn B, Neilands J (1987) Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160:47–56
Shivaji S, Pratibha M, Sailaja B, Kishore KH, Singh AK, Begum Z, Anarasi U, Prabagaran S, Reddy G, Srinivas T (2011) Bacterial diversity of soil in the vicinity of Pindari glacier, Himalayan mountain ranges, India, using culturable bacteria and soil 16S rRNA gene clones. Extremophiles 15:1–22
Sijam K, Dikin A (2005) Biochemical and physiological characterization of Burkholderia cepacia as biological control agent. Int J Agric Biol 7:385–388
Srinivas TNR, Nageswara Rao SSS, Vishnu Vardhan Reddy P, Pratibha MS, Sailaja B, Kavya B, Hara Kishore K, Begum Z, Singh SM, Shivaji S (2009) Bacterial diversity and bioprospecting for cold-active lipases, amylases and proteases, from culturable bacteria of Kongsfjorden and Ny-Ålesund, Svalbard, Arctic. Curr Microbiol 59:537–547
Srinivas T, Singh S, Pradhan S, Pratibha M, Kishore KH, Singh AK, Begum Z, Prabagaran S, Reddy G, Shivaji S (2011) Comparison of bacterial diversity in proglacial soil from Kafni glacier, Himalayan Mountain ranges, India, with the bacterial diversity of other glaciers in the world. Extremophiles 15:673–690
Steven B, Briggs G, McKay CP, Pollard WH, Greer CW, Whyte LG (2007) Characterization of the microbial diversity in a permafrost sample from the Canadian high Arctic using culture-dependent and culture-independent methods. FEMS Microbiol Ecol 59:513–523
Stibal M, Sabacka M, Zarsky J (2012) Biological processes on glacier and ice sheet surfaces. Nat Geosci 5:771–774
Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599
Tilak K, Ranganayaki N, Pal K, De R, Saxena A, Nautiyal CS, Mittal S, Tripathi A, Johri B (2005) Diversity of plant growth and soil health supporting bacteria. Curr Sci 89:136–150
Van Trappen S, Vandecandelaere I, Mergaert J, Swings J (2004) Flavobacterium degerlachei sp. nov., Flavobacterium frigoris sp. nov. and Flavobacterium micromati sp. nov., novel psychrophilic bacteria isolated from microbial mats in Antarctic lakes. Int J Syst Evol Microbiol 54:85–92
Verma P, Yadav AN, Kazy SK, Saxena AK, Suman A (2014) Evaluating the diversity and phylogeny of plant growth promoting bacteria associated with wheat (Triticum aestivum) growing in central zone of India. Int J Curr Microbiol Appl Sci 3:432–447
Yadav S, Kaushik R, Saxena AK, Arora DK (2011) Diversity and phylogeny of plant growth-promoting bacilli from moderately acidic soil. J Basic Microbiol 51:98–106
Yadav AN, Verma P, Kumar M, Pal KK, Dey R, Gupta A, Padaria JC, Gujar GT, Kumar S, Suman A, Prasanna R, Saxena AK (2014) Diversity and phylogenetic profiling of niche-specific Bacilli from extreme environments of India. Ann Microbiol. doi:10.1007/s13213-014-0897-9
Yoon J-H, Kim I-G, Kang KH, Oh T-K, Park Y-H (2003) Bacillus marisflavi sp. nov. and Bacillus aquimaris sp. nov., isolated from sea water of a tidal flat of the Yellow Sea in Korea. Int J Syst Evol Microbiol 53:1297–1303
Yoon J-H, Kang S-J, Lee J-S, Oh T-K (2006) Brevundimonas terrae sp. nov., isolated from an alkaline soil in Korea. Int J Syst Evol Microbiol 56:2915–2919
Acknowledgments
The authors are grateful to the Division of Microbiology, Indian Agricultural Research Institute (IARI), New Delhi and National Agricultural Innovation Project on “Diversity analysis of Bacillus and other predominant genera in extreme environments and its utilization in Agriculture”, Indian Council of Agricultural Research for providing the facilities and financial support, to undertake the investigations. There are no conflicts of interest.
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The experiments undertaken comply with the current laws of India, the country where the investigation was undertaken.
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Yadav, A.N., Sachan, S.G., Verma, P. et al. Culturable diversity and functional annotation of psychrotrophic bacteria from cold desert of Leh Ladakh (India). World J Microbiol Biotechnol 31, 95–108 (2015). https://doi.org/10.1007/s11274-014-1768-z
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DOI: https://doi.org/10.1007/s11274-014-1768-z