Indian Journal of Experimental Biology Vol. 54, February 2016, pp. 142-150

Bioprospecting of plant growth promoting psychrotrophic 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 of 11 genera viz., , 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, , 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 (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 L 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 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 Diverse groups of microbes have been isolated from temperature, salt and pH 4,5,16,17 Indian Himalayas . Many new and novel psychrotrophic Bacilli have been isolated from cold conditions of temperatures, salt, pH and 16 environments including Bacillus amyloliquefaciens , drought2,9,16,42. Psychrotrophic PGPB were recently 36 16 B. cecembensis , B. licheniformis , Exiguobacterium being used to improve cold stress in plant2,3,5. PGPB 37 38 39 37 antarcticum , E. indicum , E. soli , E. undae , can directly facilitate proliferation of their plant 16 40 Lysinibacillus fusiformis , Paenibacillus glacialis , host through production of the stimulatory 16 41 P. terrae and Planococcus antarcticus . Among cold phytohormones. The auxin, indole-3-acetic acid adaptive microbes, only few Bacilli have been reported (IAA) particularly, is an important phytohormone 2,5,16 and characterised as PGPB at low temperature . produced by PGPB, and treatment with auxin- In the present study, a total of 247 Bacilli producing rhizobacteria has been shown to increase were recovered from soil and water samples the plant growth10,43. Along with phytohormone collected from different sites in NW Himalayas, production, plant growth promotion is known to India. Isolates, representing different morphotypes be mediated by a variety of mechanisms including were characterized according to their phenotypic solubilization phosphorus, potassium and zinc; properties such as colony morphology, pigmentation production of ammonia, siderophores and HCN4,44,45. and tolerance to temperatures, salt and pH. Out of The representative strains were screened for plant 43 representative strains, 11, 29 and 12 were grouped growth promoting traits and differential results were as psychrophiles (5-20°C with an optimum obtained at different incubation temperatures ° temperature of 10 C), psychrotrophic (5-30°C with of 4, 15 and 30°C. In general, psychrophilic and ° an optimum temperature of 15 C) and psychrotolerant psychrotolerant strains showed higher activities for (5-37°C with an optimum temperature of 20°C) all the traits at 15 and 30°C, respectively as compared bacteria, respectively. Bacterial isolates also to other temperatures tested. Among plant growth exhibited tolerance to different NaCl concentrations promoting attributes, variations were observed among varying from 3 to 10% (w/v) and low and high strains for production (%) of IAA (20), ammonia (19), pH (Fig. 1). Bacilli were identified on the basis siderophores (11), gibberellic acid (4) and hydrogen of 16S rRNA gene sequencing and BLASTn analysis cyanide (2); solubilisation (%) of phosphate (13), zinc led to identification of 43 distinct species of (14) and potassium (7); ACC deaminase activity (6%) 11 genera, namely Desemzia, Exiguobacterium, and biocontrol activity (4%) against Rhizoctonia Jeotgalicoccus, Lysinibacillus, Paenibacillus, solani and Macrophomina phaseolina (Fig. 2, Planococcus, Pontibacillus, Sinobaca, Sporosarcina, Table 1). Among 43 strains, nine strains identified Staphylococcus and Virgibacillus. Among Bacilli, as Bacillus cereus, Bacillus firmus, Bacillus Bacillus and Bacillus derived genera (BBDG) were licheniformis, B. muralis, B. thuringiensis, most dominant followed by Exiguobacterium and Desemzia incerta, Exiguobacterium antarcticum, Sporosarcina (Table 1). Exiguobacterium sp., Lysinibacillus sphaericus, Plant growth promoting bacteria (PGPB) have a Paenibacillus tylopili, Planococcus donghaensis, high potential for agriculture because they can Sporosarcina globispora and Staphylococcus xylosus improve plant growth, under limiting or stressful exhibited more than four different plant growth YADAV et al: BIOPROSPECTING OF PLANT GROWTH PROMOTING PSYCHROTROPHIC BACILLI 145

Table 1—Plant growth promoting attributes of psychrotrophic Bacilli, isolated from the cold desert of NW Indian Himalayas Strain number Nearest phylogenetic relative Phosphate solubilization IAA production (µg mg-1day-1)# (µg mg-1 protein day-1)# 4 °C 15 °C 30 °C 4 °C 15 °C 30 °C IARI-AR41 Bacillus altitudinis - - - 21.4±1.5 42.5±1.6 52.2±1.8 IARI-AR25 Bacillus amyloliquefaciens 36.1±0.9 39.4±2.4 54.2±0.6 17.0±0.4 24.2±1.0 30.8±1.1 IARI-AL36 Bacillus baekryungensis ------IARI-AL73 Bacillus cereus - - - 16.6±1.0 38.5±0.9 52.7±2.6 IARI-AL21 Bacillus firmus 29.3±2.0 35.2±3.3 40.5±0.4 22.8±0.8 35.2±1.0 39.7±0.7 IARI-AL37 Bacillus flexus 25.8±0.6 33.1±1.9 36.2±0.6 - - - IARI-AL38 Bacillus licheniformis 19.2±1.0 29.3±2.0 39.3±1.0 13.2±1.0 16.6±1.0 19.7±1.0 IARI-AL39 Bacillus marisflavi - - - 17.2±1.1 28.6±1.0 35.1±1.0 IARI-AR44 Bacillus megaterium 14.4±2.4 22.3±1.4 44.2±2.1 22.4±1.2 33.3±2.1 42.5±1.7 IARI-AL40 Bacillus mojavensis 11.2±2.1 15.4±2.0 34.3±2.5 24.4±2.4 28.4±2.2 34.4±2.5 IARI-AR28 Bacillus muralis - - - 22.5±0.5 29.4±3.2 35.4±1.2 IARI-AR2 Bacillus psychrosaccharolyticus - - - 11.7±0.8 15.5±0.3 22.5±1.3 IARI-AL54 Bacillus pumilus 24.4±2.1 36.1±0.8 41.4±1.1 40.8±1.5 48.3±1.2 55.2±1.6 IARI-AR3 Bacillus simplex 27.4±0.7 34.5±1.5 42.5±1.1 - - - IARI-AR49 Bacillus subtilis 26.0±1.0 38.0±1.3 45.0±1.2 20.0±1.0 25.4±1.6 29.6±1.2 IARI-AR26 Bacillus thuringiensis 19.4±2.4 29.9±2.1 37.5±2.1 19.4±1.4 32.4±1.1 39.6±1.6 IARI-AL46 Desemzia incerta 27.5±1.5 47.5±1.2 57.5±1.2 14.2±1.0 28.6±1.0 35.6±1.2 IARI-AL70 Exiguobacterium antarcticum 21.1±1.8 31.1±1.8 42.1±1.8 17.2±1.3 27.3±1.3 36.3±1.1 IARI-AR137 Exiguobacterium indicum 20.8±0.4 35.8±0.4 55.8±0.4 36.4±0.5 58.4±0.5 68.4±1.5 IARI-AR40 Exiguobacterium marinum 37.4±0.7 42.4±0.7 52.4±0.7 - - - IARI-AR140 Exiguobacterium sp. 15.2±0.5 22.2±0.5 41.2±0.5 28.4±1.5 48.4±0.5 78.4±0.5 IARI-AL116 Exiguobacterium undae - - - 15.5±1.8 25.5±1.1 35.5±0.2 IARI-AR5 Jeotgalicoccus halotolerans - - - - - IARI-AR8 Lysinibacillus fusiformis - - - 41.7±0.8 46.7±1.8 53.7±1.2 IARI-AR11 Lysinibacillus sphaericus 14.4±1.2 24.4±1.2 29.6±1.1 IARI-AR27 Paenibacillus lautus - - - 31.7±0.8 38.7±1.1 49.7±1.6 IARI-AR43 Paenibacillus pabuli 29.0±2.0 38.0±2.2 45.0±2.5 13.4±1.1 22.4±1.2 29.6±1.1 IARI-AR39 Paenibacillus terrae 39.4±2.0 43.4±2.1 56.4±2.2 16.5±1.2 30.4±1.6 34.6±1.3 IARI-AR36 Paenibacillus tylopili 48.4±2.4 68.4±1.4 76.4±1.4 39.4±2.4 42.4±2.4 49.6±1.0 IARI-AL76 Paenibacillus xylanexedens 31.1±1.2 35.1±1.5 42.2±1.1 16.2±1.5 25.1±1.4 32.2±1.5 IARI-AL9 Planococcus antarcticus ------IARI-AN39 Planococcus donghaensis 20.2±0.8 28.2±0.5 30.2±0.9 28.2±1.2 35.2±0.3 40.2±1.8 IARI-AL3 Planococcus kocurii - - - 13.2±1.0 23.2±1.0 43.2±1.5 IARI-AL11 Pontibacillus sp. - - - 15.4±1.4 22.4±1.5 29.6±1.2 IARI-AL18 Sinobaca beijingensis - - - 10.2±1.2 18.2±1.2 28.2±1.3 IARI-AL77 Sporosarcina aquimarina - - - 15.2±1.5 22.2±1.0 35.2±1.2 IARI- AR111 Sporosarcina globispora - - - 37.6±0.3 57.6±0.3 87.6±0.3 IARI- AR37 Sporosarcina pasteurii 23.1±1.5 29.1±1.2 33.1±1.5 12.2±1.0 22.2±1.2 34.2±1.5 IARI- AR110 Sporosarcina psychrophila - - - 75.3±0.5 88.3±0.5 99.3±1.5 IARI- AL33 Staphylococcus arlettae 39.4±2.0 43.4±2.1 56.4±2.2 - - - IARI- AR29 Staphylococcus cohnii 30.2±0.5 36.2±0.5 40.2±0.9 - - - IARI- AR1 Staphylococcus xylosus - - - 56.5±1.2 75.5±1.1 89.5±1.3 IARI-AR18 Virgibacillus halodenitrificans - - - 35.7±1.0 39.3±1.5 47.7±1.4 Contd. 146 INDIAN J EXP BIOL, FEBRUARY 2016

Table 1 (Continued) Solubilization Production Other activities

Strains number Potassium* Zinc* Siderophores* GA NH3 HCN ACC Bio-control IARI-AR41 3.4±1.5 5.6±.05 - - + - - - IARI-AR25 - - 4.8±1.2 - + - - - IARI-AL36 - 4.5±0.5 - - - - + - IARI-AL73 2.3±0.5 6.3±1.5 - - + - - + IARI-AL21 4.7±0.9 1.3±0.6 6.0±0.8 - + - + - IARI-AL37 - - - - + - + + IARI-AL38 - 5.3±0.6 4.5±0.5 - + - - + IARI-AL39 - 1.3±2.1 - - + - + - IARI-AR44 - 6.8±0.9 - - + - - - IARI-AL40 - 8.3±0.6 ------IARI-AR28 3.8±0.6 5.3±0.6 5.7±1.2 + + - - - IARI-AR2 - 5.3±1.2 - - + - - + IARI-AL54 - 7.3±1.2 - - + - - - IARI-AR3 - 6.6±0.8 4.9±0.7 - - - - - IARI-AR49 - 6.6±0.5 ------IARI-AR26 5.4±0.5 7.7±1.2 ------IARI-AL46 3.2±1.2 - 4.7±0.5 + + - - - IARI-AL70 - - 8.7±0.5 - + - + - IARI-AR137 - - 2.6±0.5 - + - - - IARI-AR40 - 6.6±1.2 5.9±0.7 - - - - - IARI-AR140 - 2.2±0.5 4.5±0.7 - + - - - IARI-AL116 - 4.2±0.5 - - + - - - IARI-AR5 - - 4.5±0.7 + + - - - IARI-AR8 - - - - + - - - IARI-AR11 1.3±0.5 3.2±0.5 2.8±1.2 - + - + - IARI-AR27 - - - - + - - + IARI-AR43 - - - - + - - + IARI-AR39 ------+ IARI-AR36 1.5±0.5 4.3±1.4 3.8±1.2 - + - - + IARI-AL76 3.7±0.8 2.3±1.1 - - + - + - IARI-AL9 - - 5.5±0.7 - + + - IARI-AN39 - - 4.7±0.5 - + - - - IARI-AL3 - - 5.4±0.5 - - + + - IARI-AL11 - - - - + - - - IARI-AL18 - 2.3±1.4 - - + - - - IARI-AL77 - - 6.4±0.5 + + - + - IARI- AR111 1.2±0.8 3.3±1.2 - + + - + - IARI- AR37 1.5±0.5 1.3±1.2 2.7±0.5 - - - - - IARI- AR110 - - - - + - + - IARI- AL33 - - - - + - - - IARI- AR29 - - - + + - - - IARI- AR1 3.8±1.2 4.5±1.0 - + + - - - IARI-AR18 - - - - + + - - #Numerical values are mean ± SD of three independent observations; *Radius of halo zone in mm; -, negative for the attributes; +, positive for the attributes; IAA, Indole 3-acetic acid; GA, Gibberellic acid; HCN, Hydrogen cyanide; ACC,1-aminocyclopropane- 1-carboxylate deaminase; E., Exiguobacterium; V. virgibacillus

YADAV et al: BIOPROSPECTING OF PLANT GROWTH PROMOTING PSYCHROTROPHIC BACILLI 147

Fig. 2— Plant growth promoting traits of Bacilli, isolated from different sites of NW Indian Himalayas

Fig. 4— The Venn diagram illustrates the number of PGPB Fig. 3— Characterization of thirteen Bacilli showing different showing the PGP traits phosphate solubilization, ACC deaminase, plant growth promoting attributes at low temperature siderophore production and indole production promoting activities at low temperature (Fig. 3). it can enhance the availability of phosphorus for Two strains, B. firmus and E. antarcticum exhibited plant growth9,16,44. The phosphate solubilizing four significant PGP traits of phosphate solubilization, Bacilli has been employed for improving crop siderophore production, IAA production and ACC yield in agriculture in hill and mountain regions deaminase activity (Fig. 4). of earth. Among strains screened, 19 strains Phosphorus is an essential mineral nutrient that showed solubilisation of phosphorus in the range often limits plant growth because of its low solubility of 1.2±2.1 to 76.4±1.4 µg mg-1 protein day-1. and fixation in the soil. The release of fixed and Paenibacillus tylopili (IARI-AR36) solublized poorly soluble forms of phosphorus is an important highest amount of phosphorus 76.4±1.4 µg mg-1 aspect for increasing soil fertility. Phosphate- protein day-1 followed by Paenibacillus terrae solubilizing bacteria increase plant growth under (IARI-AR39) 56.4±2.2 µg mg-1 protein day-1 at 30°C conditions of poor phosphorus availability by (Table 1). Among the nutrients, phosphorus, mobilization of insoluble phosphates in the soil45-50. potassium and zinc are the major nutrient constraints The ability of bacteria to solubilize mineral in realizing sustainable productivity under cropping phosphates has been of interest to microbiologists, as system50,51. It is well known that zinc is an essential 148 INDIAN J EXP BIOL, FEBRUARY 2016

plant nutrient, a deficiency of which affects crop Further, we identified few strains that were tolerant production and nutritional quality. Inoculation of to low temperature (4°C) and exhibited PGP zinc solubilising bacteria, decrease rhizosphere pH, activities. In addition, they also showed tolerance to increased dehydrogenase and β-glucosidase activity; low or high pH and high salt concentration. The two auxin production, microbial respiration and strains, B. amyloliquefaciens (IARI-AR25) and microbial biomass-C in the rhizosphere soil of B. licheniformis (IARI-AL38) exhibited PGP different crops grown under microcosm conditions53. activities of P-solubilization, siderophore and IAA Three strains, Bacillus mojavensis (IARI-AL40), B. production at low pH (3-4) and low temperatures thuringiensis (IARI-AR26) and B. pumilus (IARI- (4°C). The two strains, Lysinibacillus fusiformis AL54) showed efficient zinc solubilization at low (IARI-AR8) and Virgibacillus halodenitrificans temperatures. Potassium (K) is an essential (IARI-AR18) produced IAA at high pH (8-11). macronutrient and most abundantly absorbed cation The strains Bacillus marisflavi (IARI-AL39), that play an important role in the growth and B. mojavensis (IARI-AL40) and Staphylococcus development of plants50. Potassium solubilising xylosus (IARI-AR1) produced IAA at 10% NaCl and bacteria (KSB) have been reported to play a key role at low temperature (4°C). These strains could prove in the natural potassium cycle and therefore, the more effective as microbial inoculants in hill and presence of KSB in the soil make potassium mountain region with acidic soils and saline soils. The available for uptake by plants31. Bacillus members of Bacilli are ubiquitous bacteria that thuringiensis (IARI-AR26) showed highest include both free-living PGPB and pathogenic solubilization of potassium at low temperature. species. PGPB belonging to Bacillus have been Among the PGP traits, IAA production by the reported to enhance the growth of several plants such Bacilli has a cascading effect on the plant as wheat, tomato, sugar beet, sorghum, peanut, and development due to its ability to influence root onion under normal temperature condition (30°C)16. growth, which in turn affects nutrient uptake and In the present study, we have observed BBDG such as ultimately plant productivity2,3,10,51. IAA production is Bacillus psychrosaccharolyticus, B. amyloliquefaciens, proposed as a major means of achieving plant B. altitudinis, B. muralis, Paenibacillus lautus, growth promotion by the bacterium described in the P. pabuli, P. terrae and P. tylopili exhibiting PGP present investigation. Among Bacilli screened, IAA activity at low temperatures. This is possibly the first production ranges from 10.2±1.2 to 99.3±1.5 µg mg-1 such report on BBDG. Cold adapted microbes have protein day-1. In particular, Sporosarcina attracted the attention of the scientific community due psychrophila (IARI-AR110) was the most efficient to their ability to promote plant growth and produce IAA producer with 99.3±1.5 µg ml-1 protein day-1 on cold active enzymes, with potential applications in a incubation at 15 and 30°C (Table 1). ACC deaminase broad range of industrial, agricultural and medical activity by microbe can lower plant ethylene levels processes2,16,42. Cold-tolerant microorganisms are and in turn facilitate plant growth55. The production of widely distributed in the agro-ecosystem and play a siderophore by microbes influence plant growth by variety of roles extending from nitrogen fixation, binding to the available iron form (Fe3+) in the plant growth promotion and alleviation of cold stress rhizosphere and in this process, iron is made in plants16. unavailable to the phytopathogens, and thus In conclusion, utility of such cold adapted Bacilli siderophore protects the plant health48. In the present strains in the context of hill and mountain agro investigation, 35 strains produced siderophore at low ecosystems is immense considering the unique crop temperature. The four strains, Bacillus cereus (IARI- growing situations and the climatic conditions of AL73), B. flexus (IARI-AL37), B. licheniformis the high-altitude agricultural systems. The selection (IARI-AL38) and B. psychrosaccharolyticus (IARI- of native functional plant growth promoting AR2) showed antagonistic activity against microorganisms is a mandatory step for reducing Rhizoctonia solani and Macrophomina phaseolina the use of energy intensive chemical fertilisers. (Table 1). HCN production by bacteria has been Some of promising strains identified in this study reported as a major means of control of diseases of could be used as potential inoculants in cold crop plants. This may be attributed to the presence of environments as they have multiple plant growth a cyanide-resistant respiratory pathway in plants. promotion traits. YADAV et al: BIOPROSPECTING OF PLANT GROWTH PROMOTING PSYCHROTROPHIC BACILLI 149

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