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Bioprospecting of Plant Growth Promoting Psychrotrophic Bacilli from the Cold Desert of North Western Indian Himalayas

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Bioprospecting of Plant Growth Promoting Psychrotrophic Bacilli from the Cold Desert of North Western Indian Himalayas Indian Journal of Experimental Biology Vol. 54, February 2016, pp. 142-150 Bioprospecting of plant growth promoting psychrotrophic Bacilli from the cold desert of north western Indian Himalayas Ajar Nath Yadav1,2, Shashwati Ghosh Sachan2, Priyanka Verma1 & Anil Kumar Saxena1* 1Division of Microbiology, Indian Agricultural Research Institute, New Delhi-110 012, Delhi, India 2Department of Bio-Engineering, Birla Institute of Technology, Mesra, Ranchi-835 215, Jharkhand, India The plant growth promoting psychrotrophic Bacilli were investigated from different sites in north western Indian Himalayas. A total of 247 morphotypes were obtained from different soil and water samples and were grouped into 43 clusters based on 16S rDNA-RFLP analysis with three restriction endonucleases. Sequencing of representative isolates has revealed that these 43 Bacilli belonged to different species of 11 genera viz., Desemzia, Exiguobacterium, Jeotgalicoccus, Lysinibacillus, Paenibacillus, Planococcus, Pontibacillus, Sinobaca, Sporosarcina, Staphylococcus and Virgibacillus. With an aim to develop microbial inoculants that can perform efficiently at low temperatures, all representative isolates were screened for different plant growth promoting traits at low temperatures (5-15°C). Among the strains, variations were observed for production (%) of indole-3-acetic acid (20), ammonia (19), siderophores (11), gibberellic acid (4) and hydrogen cyanide (2); solubilisation (%) of zinc (14), phosphate (13) and potassium (7); 1-aminocyclopropane-1- carboxylate deaminase activity (6%) and biocontrol activity (4%) against Rhizoctonia solani and Macrophomina phaseolina. Among all the strains, Bacillus licheniformis, Bacillus muralis, Desemzia incerta, Paenibacillus tylopili and Sporosarcina globispora were found to be potent candidates to be developed as inoculants as they exhibited multiple PGP traits at low temperature. Keywords: Abiotic stress, Beneficial microbes, Cold adaptation, Crop production, Extremophiles, Hill agriculture, PGPB, Sub-glacial Lakes Extreme environments such as high salt, drought genes imparting tolerance to abiotic stresses to and low temperature affect productivity of plants following inoculation with certain several commercial crop plants. Worldwide, 20% microorganisms11,12. Among cold adapted plant of the Earth’s surface is covered with frozen growth promoting bacteria (PGPB), Pseudomonas soils (permafrost), glaciers and ice sheets1. The and Exiguobacterium were well characterised and low temperature affects agricultural production. reported from low temperature environment2,3,5. The Inoculation with efficient microbes exhibiting widely studied Bacillus genus represents one of the multiple plant growth promoting (PGP) traits at low most diverse genera in the class Bacilli13. Numerous temperature could be a viable solution to enhance Bacilli strains express PGP activities and a number of crop production. Microbes isolated from rhizosphere these strains have already been commercially of crop plants growing in north-western (NW) Indian developed as biological specific plant growth Himalayan region have been shown to improve the promoters and fungicides2,4,14,15. growth of wheat2,3. There are several studies The NW Indian Himalayas ecosystem harbors indicating the role of microbes in alleviating cold a variety of beneficial microbes that positively stress4,5. The microbes can exert their influence on influences plant growth and development through a plant growth by production of hormones like auxin, cascade of processes that include increased plant cytokinin and gibberellic acid; solubilization of biomass, which in turn affects the nutrient uptake and P, production of siderophores, 1-aminocyclopropane- ultimately the plant productivity. Explorations in this 1-carboxylate (ACC) deaminase activity and region have revealed the presence and utility of antagonism towards deleterious microorganisms6-10. novel cold adaptive plant growth promoting bacterial In addition, there are reports on upregulation of species viz., Exiguobacterium, Pseudomonas and Serratia2,5,16. Pseudomonads are most-dominant at —————— low temperature and play an explicit role in nutrient * Correspondence: Phone: +91 11 25847649; Fax: +91 11 25846420 mobilization and disease control. In the present study, E-mail: [email protected] we screened diverse population of Bacilli isolated YADAV et al: BIOPROSPECTING OF PLANT GROWTH PROMOTING PSYCHROTROPHIC BACILLI 143 from cold deserts of NW Indian Himalayas that endonucleases Alu I, Hae III and Msp I (Bangalore exhibit PGP traits at low temperature so as to develop GeNei) in 25 µL reaction volumes, using the effective inoculants for hill agriculture. manufacturer’s recommended buffer and temperature. The digested product together with marker Material and Methods (100 bp, Bangalore GeNei) were resolved by Sampling site and sample collection gel electrophoresis (60V cm-1) in 2.5% agarose gels Water, soil and sediment samples were collected in 1X TAE buffer containing 10µg ml-1 ethidium from the six different sites from cold desert of bromide (EB). The numerical taxonomy analysis NW Indian Himalayas (32° 22′ 17″N:78° 53′ 48″ E). program (NTYSIS) package (version 2.02e, Exeter A total of 45 samples were collected from the sites Software, Setauket, NY) was used to score similarity at altitudes from 3979-5359 m, with temperatures and cluster analysis using the binary data. Jaccard’s ° ranging from -10-+15 C, pH 6.5-8.9, salinity coefficient was used to calculate the similarity among 0.030-1.875 mS/cm. Soil samples from different the isolates and dendrogram was constructed using altitudes of Khardungla Pass (5359 m), Chumathang the UPGMA method21. (4050 m) and Rohtang Pass (3979 m) were collected. Prior to collection, 1 cm of the surface soil 16S rRNA gene sequencing was removed with a sterile spatula and using another PCR amplified 16S rRNA gene were purified sterile spatula the soil was collected and transferred and sequenced with fluorescent terminators into sterile polythene bags. Samples from subglacial (Big Dye, Applied Biosystems) and run in 3130xl lakes (Pangong Lake, Chandratal Lake and Dashair Applied Biosystems ABI prism automated DNA Lake), comprised of surface water (10-60 cm from the sequence at SCI Genome Chennai, India. The 16S surface), sub-surface (100-200 cm from surface) rRNA gene sequences were aligned using the multiple water and deep sediments (10-50 cm from the alignment program Clustal W22 and the consensus bottom), and were collected in sterilized bottles. sequence was generated and checked for chimeric The bottles were labelled, transported on ice and artefacts with the Check Chimera program available stored at 4°C until analysis. in the Ribosomal Database Project23. The sequences were compared with the NCBI GenBank database, Enumeration and characterization of Bacilli The population of Bacilli in the water and sediment using the BLASTn program available in the samples were enumerated through enrichment National Centre for Biotechnology Information using the standard serial dilution plating technique (NCBI), USA (http://www.ncbi.nlm.nih.gov/BLAST). as described earlier15. Colonies that appeared Bacilli were identified based on percentage of ≥ were purified by repeated re-streaking to obtain sequence similarity ( 97%) with that of a prototype isolated colonies using nutrient agar plates. The strain sequence in the GenBank. The partial pure cultures were maintained at 4°C as slant and 16S rRNA gene sequences were submitted to glycerol stock (20%) at −80°C for further use. NCBI GenBank and were assigned the following All the isolates were screened in triplicates for accession numbers, KJ433613-KJ433631 and tolerance to temperatures, salt and pH following the KJ713308-KJ713331. All the 43 strains were procedure described earlier17. deposited at National Bureau of Agriculturally Important Microorganisms (NBAIM) culture PCR amplification of 16S rDNA and amplified rDNA collection facility. restriction analysis (ARDRA) Genomic DNA was extracted by the procedure Screening for plant growth promoting attributes as described earlier18. Amplification of 16S rRNA The representative isolates were initially screened gene was done using the universal primers pA qualitatively for PGP attributes which included (5'-AGAGTTTGATCCTGGCTCAG-3') and pH production of 1-aminocyclopropane-1-carboxylate (5'-AAGGAGGTGATCCAGCCGCA-3')19. The (ACC) deaminase24, ammonia25, gibberellic amplification conditions were used as described acid26, HCN27, indole-3-acetic acid (IAA)28 and earlier20. The PCR amplified 16S rDNA were siderophores29. Solubilization of phosphorus, purified by QIA quick PCR product purification potassium and zinc were carried according to kit (Qiagen). Purified PCR products (100 ng) methods described by Pikovskaya30, Hu et al.31 were digested separately with three restriction and Fasim et al.32, respectively. All assays were 144 INDIAN J EXP BIOL, FEBRUARY 2016 done in triplicates at different temperatures 4, 15 and 30°C. Quantitative estimation of phosphate and IAA was done according to method described by Mehta and Nautiyal33 and Gordon34, respectively. In vitro antagonistic activity of bacterial isolates was evaluated against two fungal pathogens Rhizoctonia solani and Macrophomina phaseolina according to the method described by Sijam and Dikin35. Results and Discussion The extreme environments of low temperature are rich source of cold adapted microbes. Fig. 1— Distribution of Bacilli on the basis of tolerance to
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