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 and low temperature affect productivity of several commercial crop plants. Worldwide, 20%
of the Earth’s surface is covered with frozen soils (permafrost), glaciers and ice sheets1. The low temperature affects agricultural production.
Inoculation with efficient microbes exhibiting multiple plant growth promoting (PGP) traits at low temperature could be a viable solution to enhance crop production. Microbes isolated from rhizosphere of crop plants growing in north-western (NW) Indian Himalayan region have been shown to improve the growth of wheat2,3. There are several studies indicating the role of microbes in alleviating cold stress4,5. The microbes can exert their influence on plant growth by production of hormones like auxin, cytokinin and gibberellic acid; solubilization of P, production of siderophores, 1-aminocyclopropane- 1-carboxylate (ACC) deaminase activity and antagonism towards deleterious microorganisms6-10. In addition, there are reports on upregulation of
genes imparting tolerance to abiotic stresses to plants following inoculation with certain microorganisms11,12. Among cold adapted plant growth promoting bacteria (PGPB), Pseudomonas and Exiguobacterium were well characterised and reported from low temperature environment2,3,5. The widely studied Bacillus genus represents one of the most diverse genera in the class Bacilli13. Numerous Bacilli strains express PGP activities and a number of these strains have already been commercially developed as biological specific plant growth promoters and fungicides2,4,14,15.
The NW Indian Himalayas ecosystem harbors a variety of beneficial microbes that positively influences plant growth and development through a cascade of processes that include increased plant biomass, which in turn affects the nutrient uptake and ultimately the plant productivity. Explorations in this region have revealed the presence and utility of novel cold adaptive plant growth promoting bacterial species viz., Exiguobacterium, Pseudomonas and Serratia2,5,16. Pseudomonads are most-dominant at low temperature and play an explicit role in nutrient mobilization and disease control. In the present study, we screened diverse population of Bacilli isolated
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* Correspondence:
Phone: +91 11 25847649; Fax: +91 11 25846420 E-mail: saxena461@yahoo.com
from cold deserts of NW Indian Himalayas that exhibit PGP traits at low temperature so as to develop effective inoculants for hill agriculture.
Material and Methods
Sampling site and sample collection
Water, soil and sediment samples were collected from the six different sites from cold desert of NW Indian Himalayas (32° 22′ 17″N:78° 53′ 48″ E).
A total of 45 samples were collected from the sites at altitudes from 3979-5359 m, with temperatures ranging from -10-+15°C, pH 6.5-8.9, salinity 0.030-1.875 mS/cm. Soil samples from different altitudes of Khardungla Pass (5359 m), Chumathang (4050 m) and Rohtang Pass (3979 m) were collected.
Prior to collection, 1 cm of the surface soil was removed with a sterile spatula and using another sterile spatula the soil was collected and transferred into sterile polythene bags. Samples from subglacial lakes (Pangong Lake, Chandratal Lake and Dashair Lake), comprised of surface water (10-60 cm from the surface), sub-surface (100-200 cm from surface) water and deep sediments (10-50 cm from the bottom), and were collected in sterilized bottles.
The bottles were labelled, transported on ice and stored at 4°C until analysis.
Enumeration and characterization of Bacilli
The population of Bacilli in the water and sediment samples were enumerated through enrichment using the standard serial dilution plating technique as described earlier15. Colonies that appeared were purified by repeated re-streaking to obtain isolated colonies using nutrient agar plates. The pure cultures were maintained at 4°C as slant and glycerol stock (20%) at −80°C for further use.
All the isolates were screened in triplicates for tolerance to temperatures, salt and pH following the procedure described earlier17.
PCR amplification of 16S rDNA and amplified rDNA restriction analysis (ARDRA)
Genomic DNA was extracted by the procedure as described earlier18. Amplification of 16S rRNA gene was done using the universal primers pA (5'-AGAGTTTGATCCTGGCTCAG-3') and pH (5'-AAGGAGGTGATCCAGCCGCA-3')19. The amplification conditions were used as described earlier20. The PCR amplified 16S rDNA were purified by QIA quick PCR product purification kit (Qiagen). Purified PCR products (100 ng) were digested separately with three restriction
endonucleases Alu I, Hae III and Msp I (Bangalore GeNei) in 25 µL reaction volumes, using the manufacturer’s recommended buffer and temperature.
The digested product together with marker (100 bp, Bangalore GeNei) were resolved by gel electrophoresis (60V cm-1) in 2.5% agarose gels in 1X TAE buffer containing 10µg ml-1 ethidium bromide (EB). The numerical taxonomy analysis program (NTYSIS) package (version 2.02e, Exeter Software, Setauket, NY) was used to score similarity and cluster analysis using the binary data. Jaccard’s coefficient was used to calculate the similarity among the isolates and dendrogram was constructed using the UPGMA method21.
16S rRNA gene sequencing
PCR amplified 16S rRNA gene were purified and sequenced with fluorescent terminators (Big Dye, Applied Biosystems) and run in 3130xl Applied Biosystems ABI prism automated DNA sequence at SCI Genome Chennai, India. The 16S rRNA gene sequences were aligned using the multiple alignment program Clustal W22 and the consensus sequence was generated and checked for chimeric artefacts with the Check Chimera program available in the Ribosomal Database Project23. The sequences were compared with the NCBI GenBank database, using the BLASTn program available in the National Centre for Biotechnology Information (NCBI), USA (http://www.ncbi.nlm.nih.gov/BLAST).
Bacilli were identified based on percentage of sequence similarity (≥97%) with that of a prototype strain sequence in the GenBank. The partial 16S rRNA gene sequences were submitted to NCBI GenBank and were assigned the following accession numbers, KJ433613-KJ433631 and KJ713308-KJ713331. All the 43 strains were deposited at National Bureau of Agriculturally Important Microorganisms (NBAIM) culture collection facility.
Screening for plant growth promoting attributes
The representative isolates were initially screened qualitatively for PGP attributes which included production of 1-aminocyclopropane-1-carboxylate (ACC) deaminase24, ammonia25, gibberellic acid26, HCN27, indole-3-acetic acid (IAA)28 and siderophores29. Solubilization of phosphorus, potassium and zinc were carried according to methods described by Pikovskaya30, Hu et al.31 and Fasim et al.32, respectively. All assays were
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.
Diverse groups of microbes have been isolated from Indian Himalayas4,5,16,17. Many new and novel psychrotrophic Bacilli have been isolated from cold environments including Bacillus amyloliquefaciens16, B. cecembensis36, B. licheniformis16, Exiguobacterium antarcticum37, E. indicum38, E. soli39, E. undae37, Lysinibacillus fusiformis16, Paenibacillus glacialis40, P. terrae16 and Planococcus antarcticus41. Among cold adaptive microbes, only few Bacilli have been reported and characterised as PGPB at low temperature2,5,16.
In the present study, a total of 247 Bacilli were recovered from soil and water samples collected from different sites in NW Himalayas, India. Isolates, representing different morphotypes were characterized according to their phenotypic properties such as colony morphology, pigmentation and tolerance to temperatures, salt and pH. Out of 43 representative strains, 11, 29 and 12 were grouped as psychrophiles (5-20°C with an optimum temperature of 10°C), psychrotrophic (5-30°C with an optimum temperature of 15°C) and psychrotolerant (5-37°C with an optimum temperature of 20°C) bacteria, respectively. Bacterial isolates also exhibited tolerance to different NaCl concentrations varying from 3 to 10% (w/v) and low and high pH (Fig. 1). Bacilli were identified on the basis of 16S rRNA gene sequencing and BLASTn analysis led to identification of 43 distinct species of 11 genera, namely Desemzia, Exiguobacterium, Jeotgalicoccus, Lysinibacillus, Paenibacillus, Planococcus, Pontibacillus, Sinobaca, Sporosarcina, Staphylococcus and Virgibacillus. Among Bacilli, Bacillus and Bacillus derived genera (BBDG) were most dominant followed by Exiguobacterium and Sporosarcina (Table 1).
Plant growth promoting bacteria (PGPB) have a high potential for agriculture because they can improve plant growth, under limiting or stressful
Fig. 1— Distribution of Bacilli on the basis of tolerance to temperature, salt and pH
conditions of temperatures, salt, pH and drought2,9,16,42. Psychrotrophic PGPB were recently being used to improve cold stress in plant2,3,5. PGPB can directly facilitate proliferation of their plant host through production of the stimulatory phytohormones. The auxin, indole-3-acetic acid (IAA) particularly, is an important phytohormone produced by PGPB, and treatment with auxin- producing rhizobacteria has been shown to increase the plant growth10,43. Along with phytohormone production, plant growth promotion is known to be mediated by a variety of mechanisms including solubilization phosphorus, potassium and zinc;
production of ammonia, siderophores and HCN4,44,45. The representative strains were screened for plant growth promoting traits and differential results were obtained at different incubation temperatures of 4, 15 and 30°C. In general, psychrophilic and psychrotolerant strains showed higher activities for all the traits at 15 and 30°C, respectively as compared to other temperatures tested. Among plant growth promoting attributes, variations were observed among strains for production (%) of IAA (20), ammonia (19), siderophores (11), gibberellic acid (4) and hydrogen cyanide (2); solubilisation (%) of phosphate (13), zinc (14) and potassium (7); ACC deaminase activity (6%) and biocontrol activity (4%) against Rhizoctonia solani and Macrophomina phaseolina (Fig. 2, Table 1). Among 43 strains, nine strains identified as Bacillus cereus, Bacillus firmus, Bacillus licheniformis, B. muralis, B. thuringiensis, Desemzia incerta, Exiguobacterium antarcticum, Exiguobacterium sp., Lysinibacillus sphaericus, Paenibacillus tylopili, Planococcus donghaensis, Sporosarcina globispora and Staphylococcus xylosus exhibited more than four different plant growth
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
(µg mg-1day-1)#
IAA production (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.
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
Fig. 2— Plant growth promoting traits of Bacilli, isolated from different sites of NW Indian Himalayas
Fig. 3— Characterization of thirteen Bacilli showing different plant growth promoting attributes at low temperature
promoting activities at low temperature (Fig. 3).
Two strains, B. firmus and E. antarcticum exhibited four significant PGP traits of phosphate solubilization, siderophore production, IAA production and ACC deaminase activity (Fig. 4).
Phosphorus is an essential mineral nutrient that often limits plant growth because of its low solubility and fixation in the soil. The release of fixed and poorly soluble forms of phosphorus is an important aspect for increasing soil fertility. Phosphate- solubilizing bacteria increase plant growth under conditions of poor phosphorus availability by mobilization of insoluble phosphates in the soil45-50. The ability of bacteria to solubilize mineral phosphates has been of interest to microbiologists, as
Fig. 4— The Venn diagram illustrates the number of PGPB showing the PGP traits phosphate solubilization, ACC deaminase, siderophore production and indole production
it can enhance the availability of phosphorus for plant growth9,16,44. The phosphate solubilizing Bacilli has been employed for improving crop yield in agriculture in hill and mountain regions of earth. Among strains screened, 19 strains showed solubilisation of phosphorus in the range of 1.2±2.1 to 76.4±1.4 µ g mg-1 protein day-1. Paenibacillus tylopili (IARI-AR36) solublized highest amount of phosphorus 76.4±1.4 µ g mg-1 protein day-1 followed by Paenibacillus terrae (IARI-AR39) 56.4±2.2 µ g mg-1 protein day-1 at 30°C (Table 1). Among the nutrients, phosphorus, potassium and zinc are the major nutrient constraints in realizing sustainable productivity under cropping system50,51. It is well known that zinc is an essential
plant nutrient, a deficiency of which affects crop production and nutritional quality. Inoculation of zinc solubilising bacteria, decrease rhizosphere pH, increased dehydrogenase and β-glucosidase activity;
auxin production, microbial respiration and microbial biomass-C in the rhizosphere soil of different crops grown under microcosm conditions53. Three strains, Bacillus mojavensis (IARI-AL40), B.
thuringiensis (IARI-AR26) and B. pumilus (IARI- AL54) showed efficient zinc solubilization at low temperatures. Potassium (K) is an essential macronutrient and most abundantly absorbed cation that play an important role in the growth and development of plants50. Potassium solubilising bacteria (KSB) have been reported to play a key role in the natural potassium cycle and therefore, the presence of KSB in the soil make potassium available for uptake by plants31. Bacillus thuringiensis (IARI-AR26) showed highest solubilization of potassium at low temperature.
Among the PGP traits, IAA production by the Bacilli has a cascading effect on the plant development due to its ability to influence root growth, which in turn affects nutrient uptake and ultimately plant productivity2,3,10,51. IAA production is proposed as a major means of achieving plant growth promotion by the bacterium described in the present investigation. Among Bacilli screened, IAA production ranges from 10.2±1.2 to 99.3±1.5 µg mg-1 protein day-1. In particular, Sporosarcina psychrophila (IARI-AR110) was the most efficient IAA producer with 99.3±1.5 µg ml-1 protein day-1 on incubation at 15 and 30°C (Table 1). ACC deaminase activity by microbe can lower plant ethylene levels and in turn facilitate plant growth55. The production of siderophore by microbes influence plant growth by binding to the available iron form (Fe3+) in the rhizosphere and in this process, iron is made unavailable to the phytopathogens, and thus siderophore protects the plant health48. In the present investigation, 35 strains produced siderophore at low temperature. The four strains, Bacillus cereus (IARI- AL73), B. flexus (IARI-AL37), B. licheniformis (IARI-AL38) and B. psychrosaccharolyticus (IARI- AR2) showed antagonistic activity against Rhizoctonia solani and Macrophomina phaseolina (Table 1). HCN production by bacteria has been reported as a major means of control of diseases of crop plants. This may be attributed to the presence of a cyanide-resistant respiratory pathway in plants.
Further, we identified few strains that were tolerant to low temperature (4°C) and exhibited PGP activities. In addition, they also showed tolerance to low or high pH and high salt concentration. The two strains, B. amyloliquefaciens (IARI-AR25) and B. licheniformis (IARI-AL38) exhibited PGP activities of P-solubilization, siderophore and IAA production at low pH (3-4) and low temperatures (4°C). The two strains, Lysinibacillus fusiformis (IARI-AR8) and Virgibacillus halodenitrificans (IARI-AR18) produced IAA at high pH (8-11).
The strains Bacillus marisflavi (IARI-AL39), B. mojavensis (IARI-AL40) and Staphylococcus xylosus (IARI-AR1) produced IAA at 10% NaCl and at low temperature (4°C). These strains could prove more effective as microbial inoculants in hill and mountain region with acidic soils and saline soils. The members of Bacilli are ubiquitous bacteria that include both free-living PGPB and pathogenic species. PGPB belonging to Bacillus have been reported to enhance the growth of several plants such as wheat, tomato, sugar beet, sorghum, peanut, and onion under normal temperature condition (30°C)16. In the present study, we have observed BBDG such as Bacillus psychrosaccharolyticus, B. amyloliquefaciens, B. altitudinis, B. muralis, Paenibacillus lautus, P. pabuli, P. terrae and P. tylopili exhibiting PGP activity at low temperatures. This is possibly the first such report on BBDG. Cold adapted microbes have attracted the attention of the scientific community due to their ability to promote plant growth and produce cold active enzymes, with potential applications in a broad range of industrial, agricultural and medical processes2,16,42. Cold-tolerant microorganisms are widely distributed in the agro-ecosystem and play a variety of roles extending from nitrogen fixation, plant growth promotion and alleviation of cold stress in plants16.
In conclusion, utility of such cold adapted Bacilli strains in the context of hill and mountain agro ecosystems is immense considering the unique crop growing situations and the climatic conditions of the high-altitude agricultural systems. The selection of native functional plant growth promoting microorganisms is a mandatory step for reducing the use of energy intensive chemical fertilisers.
Some of promising strains identified in this study could be used as potential inoculants in cold environments as they have multiple plant growth promotion traits.
Acknowledgment
The authors are grateful to the Division of Microbiology, ICAR-Indian Agricultural Research Institute, 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.
Declaration
The experiments undertaken comply with the current laws of India, the country where the investigation was undertaken. Further, authors declare that there are no conflicts of interest.
References
1 Deming JW, Psychrophiles and polar regions. Curr Opin Microbiol, 5 (2002) 301.
2 Selvakumar G, Joshi P, Nazim S, Mishra PK, Kundu S
& Gupta HS, Exiguobacterium acetylicum strain 1P (MTCC 8707) a novel bacterial antagonist from the North Western Indian Himalayas. World J Microb Biotech, 25 (2009) 131.
3 Mishra PK, Bisht SC, Ruwari P, Selvakumar G, Joshi GK, Bisht JK, Bhatt JC & Gupta HS, Alleviation of cold stress in inoculated wheat (Triticum aestivum L.) seedlings with psychrotolerant Pseudomonads from NW Himalayas. Arch Microbiol, 193 (2011) 497.
4 Bisht SC, Mishra PK & Joshi GK, Genetic and functional diversity among root-associated psychrotrophic Pseudomonad’s isolated from the Himalayan plants. Arch Microbiol, 195 (2013) 605.
5 Verma P, Yadav AN, Khannam KS, Panjiar N, Kumar S, Saxena AK & Suman A, Assessment of genetic diversity and plant growth promoting attributes of psychrotolerant bacteria allied with wheat (Triticum aestivum) from the northern hills zone of India. Ann Microbiol, 65 (2015) 1885.
6 Glick B, Patten C, Holguin G & Penrose D, Overview of plant growth-promoting bacteria. In: Biochemical and Genetic Mechanisms Used by Plant Growth Promoting Bacteria, edited by Glick B R (Imperial College Press, London), 1999, 267.
7 Gupta A, Gopal M & Tilak KVBR, Mechanism of plant growth promotion by rhizobacteria. Indian J Exp Biol, 38 (2000) 856.
8 Verma P, Yadav AN, Kazy SK, Saxena AK & Suman A, Elucidating the diversity and plant growth promoting attributes of wheat (Triticum aestivum) associated acidotolerant bacteria from southern hills zone of India.
Natl J Life Sci, 10 (2013) 219.
9 Verma P, Yadav AN, Kazy SK, Saxena AK & Suman A, 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 App Sci, 3 (2014) 432.
10 Sethia B, Mustafa M, Manohar S, Patil SV, Jayamohan NS &
Kumudini BS, Indole acetic acid production by fluorescent Pseudomonas spp. from the rhizosphere of Plectranthus amboinicus (Lour.) Spreng. and their variation in extragenic repetitive DNA sequences. Indian J Exp Biol, 53 (2015) 342.
11 Zhang H, Kim MS, Sun Y, Dowd SE, Shi H & Pare PW, Soil bacteria confer plant salt tolerance by tissue-specific regulation of the sodium transporter HKT1. Mol Plant Microbe IN, 21 (2008) 737.
12 Sharma R, Sahoo A, Devendran R & Jain M, Over- expression of a rice tau class glutathione S-transferase gene improves tolerance to salinity and oxidative stresses in Arabidopsis. PloS one, 9 (2014) 2900.
13 Garbeva P, Van Veen J & Van Elsas J, Predominant Bacillus spp.
in agricultural soil under different management regimes detected via PCR-DGGE. Microb Ecol, 45 (2003) 302.
14 Gorashi N, Tripathi M, Kalia V & Gujar G, Identification and characterization of the Sudanese Bacillus thuringiensis and related bacterial strains for their efficacy against Helicoverpa armigera and Tribolium castaneum. Indian J Exp Biol, 52 (2014) 637.
15 Kalita M, Bharadwaz M, Dey T, Gogoi K, Dowarah P, Unni BG, Ozah D & Saikia I, Developing novel bacterial based bioformulation having PGPR properties for enhanced production of agricultural crops. Indian J Exp Biol, 53 (2015) 56.
16 Verma P, Yadav AN, Khannam KS, Kumar S, Saxena AK &
Suman A, Molecular diversity and multifarious plant growth promoting attributes of Bacilli associated with wheat rhizosphere from six agro-ecological zones of India. J Basic Microbiol, (2015) doi:10.1002/jobm.201500459
17 Yadav AN, Verma P, Kumar M, Pal KK, Dey R, Gupta A, Padaria JC, Gujar GT, Kumar S, Suman A, Prasanna R &
Saxena AK, Diversity and phylogenetic profiling of niche- specific Bacilli from extreme environments of India. Ann Microbiol, 65 (2015) 611.
18 Kumar M, Yadav AN, Tiwari R, Prasanna R & Saxena AK, Deciphering the diversity of culturable thermotolerant bacteria from Manikaran hot springs. Ann Microbiol, 64 (2014) 741.
19 Edwards U, Rogall T, Blöcker H, Emde M & Böttger EC, Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res, 17 (1989) 7843.
20 Pandey S, Singh S, Yadav AN, Nain L & Saxena AK, Phylogenetic diversity and characterization of novel and efficient cellulase producing bacterial isolates from various extreme environments. Biosci Biotechnol Biochem, 77 (2013) 1474.
21 Nei M & Li WH, Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc Natl Acad Sci, 76 (1979) 5269.
22 Thompson JD, Higgins DG & Gibson TJ, CLUSTAL W:
improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res, 22 (1994) 4673.
23 Maidak BL, Olsen GJ, Larsen N, Overbeek R, McCaughey MJ
& Woese CR, The RDP (ribosomal database project).
Nucleic Acids Res, 25 (1997) 109.
24 Jacobson CB, Pasternak J & Glick BR, Partial purification and characterization of 1-aminocyclopropane-1-carboxylate
deaminase from the plant growth promoting rhizobacterium Pseudomonas putida GR12-2. Can J Microbiol, 40 (1994) 1019.
25 Cappuccino JG & Sherman N, In: Microbiology: A Laboratory Manual. 3rd ed New York: Benjamin/Cumming Pub Co (NH3 test), 1992.
26 Brown ME & Burlingham SK, Production of plant growth substances by Azotobacter chroococcum. J Gen Microbiol, 53 (1968) 135.
27 Bakker AW & Schippers B, Microbial cyanide production in the rhizosphere in relation to potato yield reduction and Pseudomonas spp-mediated plant growth-stimulation. Soil Biol Biochem, 19 (1987) 451.
28 Bric JM, Bostock RM & Silverstone SE, Rapid in situ assay for indoleacetic acid production by bacteria immobilized on a nitrocellulose membrane. Appl Environ Microbiol, 57 (1991) 535.
29 Schwyn B & Neilands J, Universal chemical assay for the detection and determination of siderophores. Anal Biochem, 160 (1987) 47.
30 Pikovskaya R, Mobilization of phosphorus in soil in connection with vital activity of some microbial species.
Mikrobiologiya, 17 (1948) 362.
31 Hu X, Chen J & Guo J, Two phosphate- and potassium- solubilizing bacteria isolated from Tianmu Mountain, Zhejiang, China. World J Microb Biotech, 22 (2006) 983.
32 Fasim F, Ahmed N, Parsons R & Gadd GM, Solubilization of zinc salts by a bacterium isolated from the air environment of a tannery. FEMS Microbiol Lett, 213 (2002) 1.
33 Mehta S & Nautiyal CS, An efficient method for qualitative screening of phosphate-solubilizing bacteria. Curr Microbiol, 43 (2001) 51.
34 Gordon SA & Weber RP, Colorimetric estimation of indoleacetic acid. Plant Physiol, 26 (1951) 192.
35 Sijam K & Dikin A, Biochemical and physiological characterization of Burkholderia cepacia as biological control agent. Int J Agr Biol, 7 (2005) 385.
36 Reddy G, Prakash J, Vairamani M, Prabhakar S, Matsumoto G & Shivaji S, Planococcus antarcticus and Planococcus psychrophilus spp. nov. isolated from cyanobacterial mat samples collected from ponds in Antarctica. Extremophiles, 6 (2002) 253.
37 Fruhling A, Schumann P, Hippe H, Straubler B &
Stackebrandt E, Exiguobacterium undae sp. nov. and Exiguobacterium antarcticum sp. nov. Int J Syst Evol Microbiol, 52 (2002) 1171.
38 Chaturvedi P, Prabahar V, Manorama R, Pindi PK, Bhadra B, Begum Z & Shivaji S, Exiguobacterium soli sp. nov., a psychrophilic bacterium from the McMurdo Dry Valleys, Antarctica. Int J Syst Evol Microbiol, 58 (2008) 2447.
39 Reddy G, Uttam A & Shivaji S, Bacillus cecembensis sp.
nov., isolated from the Pindari glacier of the Indian Himalayas. Int J Syst Evol Microbiol, 58 (2008) 2330.
40 Chaturvedi P & Shivaji S, Exiguobacterium indicum sp. nov., a psychrophilic bacterium from the Hamta glacier of the Himalayan mountain ranges of India. Int J Syst Evol Microbiol, 56 (2006) 2765.
41 Kishore KH, Begum Z, Pathan AAK & Shivaji S, Paenibacillus glacialis sp. nov., isolated from the Kafni glacier of the Himalayas, India. Int J Syst Evol Microbiol, 60 (2010) 1909.
42 Mishra PK, Mishra S, Selvakumar G, Bisht SC, Bisht JK, Kundu S & Gupta HS, Characterisation of a psychrotolerant plant growth promoting Pseudomonas sp. strain PGERs17 (MTCC 9000) isolated from North Western Indian Himalayas. Ann Microbiol, 58 (2008) 561.
43 Poopathi S, Thirugnanasambantham K, Mani C, Ragul K
& Sundarapandian S, Isolation of mosquitocidal bacteria (Bacillus thuringiensis, B. sphaericus and B. cereus) from excreta of arid birds. Indian J Exp Biol, 52 (2014) 739.
44 Tilak KBR, Ranganayaki N, Pal KK, De R, Saxena AK, Nautiyal CS, Mittal S, Tripathi AK & Johri BN, Diversity of plant growth and soil health supporting bacteria. Curr Sci, 89 (2005) 136.
45 Yadav AN, Sharma D, Gulati S, Singh S, Dey R, Pal KK
& Saxena AK, Haloarchaea endowed with phosphorus solubilization attribute implicated in phosphorus cycle.
Sci Rep, 5 (2015) 12293.
46 Rodrı́guez H & Fraga R, Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnol Adv, 17 (1999) 319.
47 Das J, Ramesh KV, Maithri U, Mutangana D & Suresh CK, Response of aerobic rice to Piriformospora indica. Indian J Exp Biol, 52 (2014) 237.
48 Sayantan D & Shardendu, Phosphorus amendment competitively prevents chromium uptake and mitigates its toxicity in Spinacea oleracea L. Indian J Exp Biol, 53 (2015) 395.
49 Ramesh A, Sharma S, Sharma M, Yadav N & Joshi O, Plant growth-promoting traits in Enterobacter cloacae subsp.
dissolvens MDSR9 isolated from soybean rhizosphere and its impact on growth and nutrition of soybean and wheat upon inoculation. Agri Res, 3 (2014) 53.
50 Suman A, Yadav AN & Verma P, Endophytic Microbes in Crops: Diversity and Beneficial impact for Sustainable Agriculture. In: Microbial Inoculants in Sustainable Agricultural Productivity, Research Perspectives, (Eds.
Singh DP, Abhilash PC & Prabha R, Springer-Verlag, Germany), 2015. doi:10.1007/978-81-322-2647-5_7.
51 Glick BR, Penrose DM & Li J, A model for the lowering of plant ethylene concentrations by plant growth-promoting bacteria. J Theo Biol, 190 (1998) 63.
