Research Paper Culturable diversity and functional ...

Research Paper Culturable diversity and functional annotation of psychrotrophic bacteria from cold desert of Leh Ladakh (India)

Ajar Nath Yadav1, 2, Shashwati Ghosh Sachan2, Priyanka Verma1, Satya Prakash Tyagi1, Rajeev Kaushik1 and Anil K. Saxena1* 1

Division of Microbiology, Indian Agricultural Research Institute, New Delhi- 110012, India

2

Department of Bio-Engineering, Birla Institute of Technology, Mesra, Ranchi-835215, India

World Journal of Microbiology and Biotechnology Doi: 10.1007/s11274-014-1768-z

Supplementary Table 1 The different media used in this study for isolation of bacteria from cold desert S. N

Media and composition per liter

1.

Nutrient agar: 5 g peptone; 5 g NaCl; 3 g beef extract; 20 g agar

2.

T3 agar: 3 g Tryptone; 2 g Tryptose; 1.5 g Yeast Extract; 0.005 g MnCl2 ; 0.05 Sodium Phosphate; 20 g agar; pH-6.8

3.

Soil extract agar: 2 g glucose; 1 g Yeast Extract; 0.5 g K2HPO4; 100ml Soil extract*; 20 g agar; pH-7.2

4.

Tryptic soy agar: 17 g tryptone; 3 g soya meal; 2.5 g dextrose; 5 g NaCl; 2.5 g K 2HPO4; 20 g agar; pH-7.2

5.

King’s B agar: 20 g Protease Peptone; 1.5 g K2HPO4; 1.5 MgSO4.7H2O; 10 ml Glycerol; 20 g agar; pH 7.2

6.

Jensen’s agar: 20 g Sucrose;1 g K2HPO4; 0.5 g Mg2SO4 ; 0.5 g NaCl; 0.001 g Na2MoO4; 0.01 g FeSO4; 2 g CaCO3; 20 g agar; pH 7.2

7.

R2 agar; 0.5 g Proteose peptone; 0.5 g Casmino acids; 0.5 g Yeast extract; 0.5 g Dextrose; 0.5 g Soluble starch; 0.3 g Dipotassium phosphate; 0.05 g Magnesium sulphate 7H2O; 0.3 g Sodium pyruvate; agar 20 g agar; pH 7.2 ± 0.2

8.

Chemically defined medium: 5 g casamino acids; 5 g yeast extract; 1 g sodium glutamate; 3 g tri-sodium citrate; 20 g MgSO4; 2 g KCl; 100 g NaCl; 36 mg FeCl2; 0.36 mg MgCl2; 20 g agar pH-7.0-7.2

9.

Standard growth media;7.5 g Casamino Acids; 4 g MgSO4; 2 g KCl; 150 g NaCl; 3 g Tri-Sodium citrate; 2.3 mg FeCl2;7 mg CaCl2; 0.044 mg MnSO4; 0.05 mg CuSO4; 20 g agar; pH 7.2

10.

Halophilic medium: 100 g NaCl; 2 g KCl; 1 g MgSO4·7H2O; 0.36 g CaCl2·2H2O; 0.23 g NaBr; 0.06 g NaHCO3; 5 g protease-peptone; 10 g yeast extract; 1g glucose; trace FeCl3; 20 g agar; pH 7.2–7.4

*Soil extract: 250 g soil from sampling site + 1L H20, Autoclave and filter

Supplementary Table 2 Total viable count of bacteria in water and sediment samples from Leh Ladakh on different growth media. Sites Khardungla Pass Indus river

I-Z confluence

Zanskar river Pangong Lake Chumathang

Type of samples Soil Water Sediment Average Water Sediment Average Water Sediment Average Water Sediment Average Soil

NA 5.12 4.85 5.55 5.20 3.96 5.39 4.68 2.98 4.8 3.89 4.82 6.33 5.58 2.90

Total viable count (cfu g-1 sediment or ml-1 water × 106) on different media T 3A SEA TSA KB JA R2A CDM SGM 0.011 1.34 5.68 0.90 0.09 0.78 0.005 0.003 0.009 0.82 3.59 0.78 0.68 0.83 0.004 0.001 0.015 1.12 5.56 0.98 0.96 0.88 0.008 0.003 0.012 0.97 4.58 0.88 0.08 0.86 0.006 0.002 0.01 0.98 3.2 0.84 0.02 0.62 0.0073 0.001 0.016 1.23 5.6 1.12 0.05 0.63 0.0092 0.0015 0.013 1.10 4.40 0.98 0.04 0.60 0.008 0.001 0.007 1.19 3.95 0.89 0.045 0.52 0.001 0.001 0.015 1.45 5.26 1.35 0.081 0.56 0.005 0.003 0.011 1.32 4.6 1.1 0.06 0.54 0.003 0.002 0.014 1.01 5.98 0.98 0.098 1.68 0.011 0.012 0.021 1.98 7.25 1.69 0.15 0.87 0.013 0.016 0.018 1.50 6.62 1.34 0.12 1.28 0.012 0.014 0.012 0.96 3.46 1.14 0.03 0.62 0.002 0.002

Nutrient agar (NA); T3 agar (T3A); Soil extract agar (SEA); Tryptic soy agar (TSA); King’s B agar (KB); Jensen’s agar (JA); R2 agar (R2A); chemically defined medium (CDM); Standard growth media (SGM); Halophilic medium (HM). *No of clusters after digestion with three restriction enzymes -AluI, HaeIII and MspI

HM 0.008 0.002 0.004 0.003 0.0045 0.0098 0.007 0.0006 0.0021 0.001 0.0082 0.018 0.013 0.003

No of isolates

Total isolates 62

No of clusters* 32

47

26

43

25

36

23

85 52

40 29

20 27 15 28 16 20 38 47

Supplementary table 3 Distribution of bacteria from different site showing niche specificity and their phenotypic characterizations

Strain name IARI-L-120 IARI-L-16 IARI-L-60 IARI-ABL-35 IARI-ABL-30 IARI-ABL-2 IARI-ABL-31 IARI-ABL-32 IARI-L-33 IARI-ABL-45 IARI-ABL-47 IARI-L-24 IARI-ABL-36 IARI-L-73 IARI-L-21 IARI- ABL-37 IARI- ABL-38 IARI- ABL-39 IARI- ABL-40 IARI-L-74 IARI-L-54 IARI-L-118 IARI-L-14 IARI-L-69 IARI-L-46 IARI-L-70 IARI-L-62 IARI-L-116 IARI-L-2 IARI-L-65 IARI-L-3 IARI-L-128 IARI-L-127 IARI-L-76

Nearest phylogenetic relative Arthrobacter sp.(DQ310481) Arthrobacter sulfonivorans (FM955888) Arthrobacter sulfureus (AY392127) Brachybacterium sp.(HQ407433) Cellulosimicrobium cellulans (EU931556) Citricoccus sp.(AB594473) Kocuria kristinae (AB616677) Kocuria palustris (HM218471) Sanguibacter antarcticus (JX869974) Flavobacterium antarcticum (FM163401) Sphingobacterium sp. (KC252807) Bacillus anthracis (JN999848) Bacillus baekryungensis (JN210568) Bacillus cereus (JX155390) Bacillus firmus (GQ903380) Bacillus flexus (GU397395) Bacillus licheniformis (GQ280023) Bacillus marisflavi (AB617551) Bacillus mojavensis (JF901760) Bacillus muralis (JX035939) Bacillus pumilus (JF803782) Bacillus simplex (DQ514314) Bacillus sp.(JQ956513) Bacillus subtilis (GU125629) Desemzia incerta (HQ336336) Exiguobacterium antarcticum (NR043476) Exiguobacterium sp.(DQ019169) Exiguobacterium undae (FN870073) Lysinibacillus fusiformis (EU187498) Lysinibacillus sp.(GQ199724) Lysinibacillus sphaericus (JX067902) Paenibacillus sp.(JF694817) Paenibacillus terrae (EF690404) Paenibacillus xylanexedens (NR044524)

Sampling sites and number of isolated bacteria KP

IR

IZ

ZR

PL

LC

3 2 3 2 3* 2 2 1 2 2 2 2 3 2 1 2 1 2 3 2 2 -

2 1 2 1 3 2 2 1 2 1 1 2 2 2 2 1 -

2 1 2 2 1 2 2 1 1 2 2 2 2 3 1 1 2 -

1 2 1 2 1 1 2 1 2 2 1 1 1 2 2 1 2 -

2 3 2 3 3 5 3 2 3 3 1 3 3 2 2 2 2 1 1 4 2 1 3 2 1 -

1 1 2 1 1 1 1 1 1 1 1 3 3 1 3 2 2 3

Phenotypic characteristics Pigmentation /color Bright yellow Light yellow Yellow Red Creamy white Yellow Light yellow Yellow Yellow Yellow Yellow Cream Off white Light yellow Yellow Off white Creamy Bright white Yellow Cream White White Cream White White Bright orange Orange Orange Peach Peach Peach Creamy white Cream Cream

Temp Range (optimum) 0-20 (12) 0-20 (12) 0-20 (12) 4-37 (18) 4-37 (16) 4-37 (18) 4-37 (18) 4-37 (16) 0-20 (10) 4-30 (14) 4-30 (14) 4-30 (16) 4-30 (16) 4-30 (16) 0-30 (14) 4-37 (16) 4-37 (18) 4-37 (16) 4-37 (16) 0-20 (10) 4-30 (16) 0-30 (14) 4-30 (14) 0-30 (14) 4-30 (14) 0-20 (12) 0-20 (10) 0-20 (10) 4-30 (16) 4-30 (16) 4-30 (16) 4-30 (16) 0-30 (14) 0-20 (10)

NaCl Tolerance (%) 5 5 5 10 7 10 10 10 3 5 5 5 10 5 10 7 7 10 10 5 5 3 5 3 5 3 3 3 3 3 3 5 3 5

pH range 6-9 6-9 6-9 6-9 6-9 6-8 6-9 6-8 5-9 6-8 6-8 5-9 5-9 5-9 5-9 5-9 6-9 5-9 5-11 5-9 5-8 6-9 5-9 5-9 6-8 6-9 6-9 6-8 6-9 5-8 6-8 5-9 6-9 5-9

IARI-ABL-9 IARI-L-39 IARI-ABL-3 IARI- ABL-41 IARI-ABL-18 IARI-L-77 IARI-ABL-33 IARI-ABL-26 IARI-ABL-27 IARI-ABL-28 IARI-L-110 IARI-L-23 IARI-ABL-29 IARI-L-6 IARI-L-28 IARI-L-117 IARI-L-109 IARI-L-108 IARI-L-71 IARI-L-119 IARI-L-112 IARI-ABL-34 IARI-L-9

Planococcus antarcticus (FR691465) Planococcus donghaensis (NR_044073) Planococcus kocurii (AB680986) Pontibacillus sp. (AB305321) Sinobaca beijingensis (DQ344635) Sporosarcina aquimarina (FJ944660) Staphylococcus arlettae (JN315890) Aurantimonas altamirensis (AB682666) Brevundimonas terrae (NR043726) Paracoccus sp.(AY864654) Janthinobacterium sp.(GU244366) Alishewanella sp. (EU574916.) Klebsiella sp. (GU290319) Providencia sp. (HM468083) Pseudomonas frederiksbergensis (JF343187) Pseudomonas peli (HM371423) Pseudomonas putida (AF094742) Pseudomonas reactans (JF343208) Pseudomonas sp. (AM403657) Pseudomonas stutzeri (EF429003) Psychrobacter glacincola (AY771724) Stenotrophomonas maltophilia (GU737688) Vibrio metschnikovii (NR029258) Number of clusters (175) Number of isolates (325)

1 1 1 2 3 2 2 1 2 2 1 32 62

2 2 2 1 2 2 2 2 2 3 26 47

2 2 2 2 2 1 2 1 25 43

3 2 2 1 1 2 23 36

1 1 2 1 2 2 1 3 2 2 2 1 2 1 3 40 85

2 3 3 2 2 2 2 2 2 2 1 29 52

Orange Orange Light orange Yellow Red Bright orange Off white Bright yellow Light yellow Orange Light black Off white Cream Peach Pink Yellow Yellow Yellow Cream Red White Off white Orange

0-20 (12) 0-20 (12) 4-30 (16) 4-30 (16) 4-30 (16) 0-20 (12) 4-30 (16) 4-30 (16) 4-37 (18) 4-37 (18) 0-30 (14) 0-30 (14) 4-37 (18) 0-30 (14) 0-30 (14) 0-30 (14) 0-30 (14) 0-30 (14) 4-30 (16) 0-20 (8) 0-20 (10) 4-37 (18) 4-30 (16)

Khardungla Pass (KP); Indus River (IR); I-Z confluence (IZ); Zanskar River (ZR); Pangong Lake (PL); Chumathang (LC); *Niche-specific bacteria

10 5 7 10 10 10 10 10 10 5 3 3 7 5 3 3 3 5 3 3 3 10 3

5-11 6-9 6-9 5-9 5-11 5-9 5-9 5-8 6-11 5-8 5-8 5-8 5-8 6-9 6-9 6-9 6-8 5-9 6-8 5-8 6-8 6-11 6-9

Culturable diversity and functional annotation of psychrotrophic bacteria from cold desert of Leh Ladakh (India) Ajar Nath Yadav, Shashwati Ghosh Sachan, Priyanka Verma, Satya Prakash Tyagi, Rajeev Kaushik & Anil K. Saxena World Journal of Microbiology and Biotechnology ISSN 0959-3993 Volume 31 Number 1 World J Microbiol Biotechnol (2015) 31:95-108 DOI 10.1007/s11274-014-1768-z

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ORIGINAL PAPER

Culturable diversity and functional annotation of psychrotrophic bacteria from cold desert of Leh Ladakh (India) Ajar Nath Yadav • Shashwati Ghosh Sachan Priyanka Verma • Satya Prakash Tyagi • Rajeev Kaushik • Anil K. Saxena

•

Received: 15 August 2014 / Accepted: 28 October 2014 / Published online: 5 November 2014 Ó Springer Science+Business Media Dordrecht 2014

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, Electronic supplementary material The online version of this article (doi:10.1007/s11274-014-1768-z) contains supplementary material, which is available to authorized users. A. N. Yadav P. Verma S. P. Tyagi R. Kaushik A. K. Saxena (&) Division of Microbiology, Indian Agricultural Research Institute, New Delhi 110012, India e-mail: [email protected] A. N. Yadav S. G. Sachan Department of Bio-Engineering, Birla Institute of Technology, Mesra, Ranchi 835215, India

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. Keywords Cold desert Diversity Leh Ladakh PGPB Psychrotrophic bacteria 16S rRNA gene

Introduction The microorganisms from extreme environments are of particular importance in global ecology since the majority of terrestrial and aquatic ecosystems of our planet is permanently or seasonally submitted to cold temperatures: the world’s oceans occupy 71 % of the earth surface and 90 % of their volume is below 4 °C; the polar regions represent 14 % of the earth surface and if one includes alpine soils and lakes, snow and ice fields, fresh waters and caves, more than 80 % of the earth biosphere is below 4 °C (Stibal et al. 2012). Microorganisms capable of coping with low temperatures are widespread in these natural environments where they often represent the dominant flora and they should therefore be regarded as the most successful colonizers of our planet (Russell and Fukunaga 1990). Psychrophilic microorganisms are adapted to thrive well at low temperatures close to the freezing point of water (Shivaji et al. 2011). Microbial

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activity of psychrophiles has even been reported at subzero temperatures. In general, psychrophilic microorganisms exhibit higher growth yield and microbial activity at low temperatures compared to temperatures close to the maximum temperature of growth and has more often been put forth as an explanation to successful microbial adaptation to the natural cold environment (Prasad et al. 2014; Sahay et al. 2013; Shivaji et al. 2011). In the past few years, the diversity of microorganisms inhabiting cold environments has been extensively investigated with a focus on culture dependent techniques. Coldadapted microorganisms have been reported from Antarctic sub-glacial and permanently ice-covered lakes, cloud droplets, ice cap cores from considerable depth, snow and glaciers (Deming 2002). However, little information is available on the use of these organisms in agriculture at low temperature. Understanding of permafrost microbial communities led to the exploitation of potential biotechnological applications (cold-adapted enzymes and compounds) of indigenous microorganisms. Many psychrophilic species have been isolated from cold environments including Planococcus antarcticus (Reddy et al. 2002), Exiguobacterium undae, Exiguobacterium antarcticum (Fruhling et al. 2002), Pseudomonas aeruginosa, P. fluorescens, P. putida, P. syringae and P. antarctica (Reddy et al. 2004), Exiguobacterium soli (Chaturvedi et al. 2008), Pseudoaltermonas arctica (Prasad et al. 2014) from Antarctica, Bacillus sp. (Reddy et al. 2008), Exiguobacterium indicum (Chaturvedi and Shivaji 2006), Paenibacillus glacialis (Kishore et al. 2010), Janthinobacterium lividum, Sphingobacterium antarcticus, Psychrobacter valli (Shivaji et al. 2011) and Alishewanella sp., Brevundimonas sp. (Sahay et al. 2013) from Indian Himalayas. Leh Ladakh (Jammu and Kashmir), which represent cold deserts and a niche for cold adapted microorganisms, besides psychrophilic microorganisms which have immense significance in the field of biotechnology because of their distinct metabolism from other organisms. Psychrophilic microorganisms are also potential sources of novel pigments (as food additives), cold active enzymes, antifreeze compounds, which can be valuable in agriculture as inoculants and biocontrol agents in extreme habitats. The bacterial diversity from the cold environment could serve as a database for selection of inoculants with plant growth promoting ability and could be used for improving the growth and yield of crops grown at high altitudes with prevailing low temperatures. Plant growth promoting bacteria (PGPB) promote plant growth directly by either facilitating resource acquisition or modulating plant hormone levels, or indirectly by decreasing the inhibitory effects of various pathogenic agents on plant growth and development, that is, by acting as biocontrol bacteria (Kim et al. 2011; Tilak et al. 2005; Verma et al. 2014). Though

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the beneficial effects of inoculation of such PGPB have been well documented at several locations, there is an urgent need to evolve location specific inoculant strains. The use of psychrophiles as biofertilizers, biocontrol agent and bioremediators would be of great use in Indian agriculture under cold climate condition. The present investigation deals with the isolation, characterization, phylogenetic analysis and the plant growth promoting potential of cold-adapted bacteria isolated from Leh Ladakh region, Jammu and Kashmir, India.

Materials and methods Study area and sampling sites Water, soil and sediment samples were collected from the cold desert of Leh Ladakh regions of Jammu and Kashmir including, Khardungla Pass, Indus and Zanskar River confluence, Pangong Lake and Chumathang (Fig. 1), whose details are given in Table 1. Khardungla Pass is a high mountain pass located in Ladakh region, situated at a height of about 5,602 m (18,379 ft). The Zanskar River is a north-flowing tributary of the Indus River, which is a major river in Asia and flows through Pakistan. The Indus is one of the few rivers in the world to exhibit a tidal bore. Pangong Lake in the Himalayas is situated at a height of about 4,250 m (13,900 ft) and is one of the largest brackish water snow capped lake (salinity 25 ppt). It is 134 km long and extends from India to China. In winter, the lake surface freezes completely despite being salt water. A total of forty-nine samples were collected from six sites of Leh Ladakh region. Samples were collected in sterile polythene bags/bottles labelled, transported on ice and stored at 4 °C until analysis. The pH and conductivity of the samples was recorded on site. Enumeration and isolation of bacteria The population of culturable psychrophilic and psychrotolerant bacteria in the water, sediments and soil samples were enumerated through enrichment using the standard serial dilution plating technique. Ten different nutrient combinations were used to isolate the maximum possible culturable morphotypes (Supplementary Table 1). All the media were used at full strength and also after diluting 10, 50 and 100 times. Psychrophilic/psychrotolerant Bacillus sp. was isolated by following heat enrichment technique and isolated using nutrient agar media (Yadav et al. 2014). The plates were incubated at 4 °C and the population was counted after 15–20 days. Colonies that appeared were purified by repeated streaking to obtain isolated colonies using respective medium plates. The pure cultures were

Author's personal copy World J Microbiol Biotechnol (2015) 31:95–108

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Fig. 1 Map of Jammu and Kashmir depicting sampling locations

Table 1 Geographic details and physico-chemical characteristics of collection sites S. no.

Sampling location

No. of samples

Altitude (m)

Latitude: longitude

Temperature (°C)

pH

Conductivity (mS/cm)

1

Khardungla Pass

KP (8)

5,359

34°160 4200 N:77°360 1500 E

-20 to ?05

6.8–7.4

0.376–0.495

2

Indus River

IR (8)

5,175

34°120 4000 N:77°350 0300 E

-10 to ?05

6.8–7.6

0.346–0.572

3

I-Z confluence

IZ (8)

5,095

34°080 3100 N:77°340 1100 E

-10 to ?05

6.8–7.8

0.486–0.485

4

Zanskar River

ZR (8)

4,985

33°460 1900 N:76°500 4300 E

-10 to ?05

6.8–7.4

0.486–0.552

5

Pangong Lake

PL (9)

4,350

33°430 0400 N:78°530 4800 E

-10 to ?05

6.8–89

0.825–1.875

-10 to ?15

6.5–8.4

0.486–0.552

6

Chumathang

LC(8)

4,050

0

00

0

00

33°18 00 N:78°24 00 E

maintained at 4 °C as slant and glycerol stock (20 %) at -80 °C for further use. Physiological characteristics of bacterial isolates All the isolates were screened for tolerance to temperature, salt and pH as method described earlier (Yadav et al. 2014). The pigment extraction was done as method described earlier by Srinivas et al. (2009). All the isolates were screened in triplicates and depending on the temperature range for optimal growth. PCR amplification of 16S rDNA and amplified rDNA restriction analysis (ARDRA) Genomic DNA was extracted by the method described earlier by Kumar et al. (2014). DNA samples were subjected to PCR amplification of 16S rRNA gene using the universal primers

pA (50 -AGAGTTTGATCCTGGCTCAG-30 ) and pH (50 AAGGAGGTGATCCAGCCGCA-30 ) (Edwards et al. 1989). The amplification was carried out in a 100 lL volume and amplification conditions were used as described earlier (Pandey et al. 2013). The PCR amplified 16S rDNA were purified with a Quiaquick purification kit (Qiagen). Aliquots of purified 16S rDNA PCR products were digested separately with three restriction endonucleases AluI, HaeIII and MspI in 25 lL reaction volumes, using the manufacturer’s recommended buffer and temperature. Restricted DNA was analyzed by horizontal electrophoresis in 2.5 % agarose gels. The gels were visualized and gel images were digitalized. Strong and clear bands were scored for similarity and clustering analysis undertaken using the software, NTSYS-2.02e package (Numerical taxonomy analysis program package, Exeter software, USA). Similarity among the isolates was calculated by Jaccard’s coefficient and dendrogram was constructed using the UPGMA method.

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16S rDNA sequencing and phylogenetic analysis One representative strain from each ARDRA group was selected for phylogenetic analysis. PCR products of partial 16S rRNA gene were sequenced with fluorescent terminators (Big Dye, Applied Biosystems) and run in 3130xl Applied Biosystems ABI prism automated DNA sequencer at SCI Genome Chennai, India. 16S rRNA gene sequences were analysed using codon code aligner v.4.0.4. The 16S rRNA gene sequences were aligned to those of closely related bacterial species available at GenBank database using BLASTn program. Bacterial isolates were identified based on percentage of sequence similarity (C97 %) with that of a prototype strain sequence in the GenBank. The phylogenetic tree was constructed on the aligned datasets using the neighbour-joining method (NJ) implemented in the program MEGA 4.0.2 (Tamura et al. 2007). Bootstrap analysis was performed on 1,000 random samples taken from the multiple alignments. Accession numbers The partial 16S rRNA gene sequences of 175 strains were submitted to NCBI GenBank and accession numbers assigned were HQ653597, JF343177-79, JF343183-85, JF343187, JF343191, JF343193, JF343196-99, JF343203-06, JF343208, JF343210-11, JN411434, JN411436, JN411453, JN411455, JN411457-59, JN411464-65, JX428960-61, JX428995, JX429000, JX429003-04, JX460849-50, JX512196, KC581669-75, KC581677-88 and KM878217-KM878334. All the 175 strains were deposited at National Bureau of Agriculturally Important Microorganisms (NBAIM) culture collection facility, Mau, Uttar Pradesh, India.

Statistical analysis In order to compare the bacterial diversity within the six sampling sites, the 16S rRNA gene sequences of the isolates showing C97 % sequence similarity were grouped into the same OTU (phylotype). The software Shannon–Wiener Diversity Index/Shannon Entropy Calculator and Rarefaction Calculator were used to calculate Shannon index (H), Evenness (J) and the Simpson’s index (D). Using 16S rRNA gene sequences, the Rarefaction curves were generated to compare the relative diversity and coverage of each sample. Principal coordinate analysis (PCA) was used to determine the statistical correlation between population diversity of six sites survey (Rico et al. 2004). PCA was performed for different parameters (temperature, pH and conductivity) and sampling sites. PCA analysis was carried out using the XLSTAT (version 2013) program.

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World J Microbiol Biotechnol (2015) 31:95–108 Fig. 2 Phylogenetic tree showing the relationship among 57 psy- c chrotrophic bacteria, 16S rRNA gene sequences with reference sequences obtained through BLAST analysis. The sequence alignment was performed using the CLUSTAL W program and trees were constructed using Neighbor Joining (NJ) with algorithm using MEGA4 software (Tamura et al. 2007). One thousand bootstrap replicates were performed. Bootstrap values are indicated on the branches

Screening for plant growth promoting attributes Representative isolates from each cluster were initially screened qualitatively for PGP attributes. The cultures were screened for production of gibberllic acid (Brown and Burlingham 1968), siderophore (Schwyn and Neilands 1987), HCN (Bakker and Schippers 1987), indole-3acetic acid (Bric et al. 1991), ammonia (Cappucino and Sherman 1992) and 1-Aminocyclopropane-1-carboxylate deaminase (Jacobson et al. 1994). Phosphate solubilizing activity of the isolate was carried out on Pikovskaya agar (Pikovskaya 1948). All assays were done in triplicate at 4, 15 and 30 °C. Quantitative estimation of phosphate solubilization was done at three different incubation temperatures: 4, 15 and 30 °C, by inoculating 1 mL of bacterial suspension in 50 mL of National Botanical Research Institute phosphate medium broth in Erlenmeyer flasks (150 mL), and incubating for 14 days, at the desired temperature. At the end of the incubation period the culture suspension was centrifuged at 10,0009g for 10 min and the P content in the supernatant was spectrophotometrically estimated by the ascorbic acid method (Mehta and Nautiyal 2001). Indole acetic acid concentration was estimated by inoculating 1 mL of bacterial suspension (3 9 107 cfu/mL) in 50 mL Luria–Bertani (LB) broth containing L tryptophan at 100 lg/mL (from a filter-sterilized L tryptophan stock prepared in ethyl alcohol; HiMedia, Mumbai), and incubated at 4, 15, and 30 °C for 72 h. The IAA concentration in the culture supernatant was estimated by the procedure of Gordon and Weber (1951).

Screening for bioprotectant properties (indirect PGP) 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 Dikin (2005). The fungal pathogens (R. solani and M. phaseolina), involved in root-rot complex in crops, were obtained from the well-characterized culture stock being maintained in the Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi, India. Representative bacterial isolates were spotted


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Fig. 2 continued

on respective medium plates and incubated at three different temperatures 4, 15 and 30 °C. Control plates with only the mycelial plug were set up and, when the pathogen had grown across these control plates, the diameter of growth in the challenge plates was measured. Dual culture assays were repeated three times per isolate and for each fungus.

123

Results Enumeration and isolation of bacteria The population of heterotrophic bacteria were enumerated in different samples collected from Leh Ladakh region of Jammu and Kashmir, India (Table 1). Significant variations


were observed among the culturable bacterial population of each sample on ten different media. The abundance of bacteria in the samples varied from 9.12 9 105 to 1.65 9 106 cfu g-1 sediment or mL-1 water, with the lowest values recorded for Chumathang and the highest for Pangong Lake. Among the media used, trypticase soy agar (4.89 9 106 cfu g-1 sediment or mL-1 water) supported more population of bacteria followed by nutrient agar (4.56 9 106 cfu g-1 sediment or mL-1 water), whereas standard growth medium (4.6 9 103 cfu mL-1 or g-1 soil followed by halophilic agar medium 5.8 9 103 cfu mL-1 or g-1 soil) supported the least for all sediment and water samples (Supplementary Table 2). The pure colonies obtained from each sample on different media were isolated based on colony morphology and cultural characteristics. A total of 325 distinct bacterial colonies were obtained from six different sites of Leh Ladakh region (Table 3). Growth temperature range, sodium chloride tolerance and pH range All 325 isolates were screened for tolerance to range of temperatures, salinities and pH and the results are presented for 57 representative strains. Fourteen strains were psychrophilic and could grow in the temperature range of 0–20 °C but not above 20 °C. Further, 30 strains could grow between 0 or 4 and 30 °C and 13 strains between 4 and 37 °C indicating that they were psychrotolerant. Bacterial isolates also exhibited tolerance to different NaCl concentrations varying from 3 to 15 % (w/v). Out of 57 representative isolates, 37 were tolerant to 5 % NaCl, while 21 and 16 isolates could tolerate 7 and 10 % NaCl respectively. Isolates from Pangong Lake could grow at 10 % NaCl concentration. Strains could grow in the pH range of 5–11, however majority showed growth in the range of 5–9. Out of 57 representatives, 26 strains could grow at pH 5 while only 5 strains could grow at pH 11. Most of the isolates recovered were pigmented and formed different coloured colonies (red, pink, orange, black, yellow, creamy-yellow and creamywhite) on different media (Supplementary Table 3). PCR amplification of 16S rDNA and amplified rDNA restriction analysis (ARDRA) PCR amplification of 16S rRNA gene followed by ARDRA with three restriction endonucleases revealed species variation among the morphotypes selected. The 16S rDNA amplicons when digested with restriction enzymes, generated profiles having 3–7 fragments ranging in size from 100 to 820 base pairs. ARDRA results revealed that among the restriction endonucleases, AluI was more discriminatory as compared to other two restriction enzymes. A combined dendrogram was constructed for each sampling

101

site to determine the percent similarity among the isolates. At a level of 75 % similarity (Supplementary Table 2), the isolates were grouped into clusters; and the number of clusters ranged from 23 (for Zanskar River) to 40 (for Pangong Lake). The total number of clusters was 175, summed up for all the six sites. 16S rRNA gene sequencing and phylogenetic analysis 16S rRNA gene sequencing and phylogenetic analysis of a representative isolate from each cluster revealed that all the isolates showed [99–100 % similarity with the sequences within the GenBank. One sequence from each group was selected as a representative operational taxonomic unit (OTU) and all the isolates were classified into 175 groups using a C97 % sequence similarity cut-off value, which led to identification of 57 distinct bacteria. The phylogenetic tree of 57 identified bacteria was constructed to determine their affiliations (Fig. 2a, b). Analysis of the 16S rRNA gene sequences revealed that 30 strains belonged to Firmicutes (53 %), 15 strains to Proteobacteria (25 %), 9 strains to Actinobacteria (19 %) and 2 strains to Bacteroidetes (3 %) (Fig. 2a, b). The Firmicutes were further distributed into seven families namely, Bacillaceae, Bacillales incertae sedis, Carnobacteriaceae, Panenibacillaceae, Planococcaceae, Sporolactobacillaceae and Staphylococcaceae. Out of 57 OTUs, 17 strains belonged to family Bacillaceae and included members of Bacillus cereus group (B. cereus and B. anthracis), Bacillus simplex group (B. simplex and B. muralis) and Bacillus subtilis group (B. pumilus, B. subtilis, B. mojavensis, B. licheniformis and B. flexus). Bacillus baekryungensis, B. marisflavi, Lysinibacillus and Pontibacillus formed a separate group along with Sinobaca beijingensis (a member of family Sporolactobacillaceae) (Fig. 2a). Three separate clusters each comprising of four strains of Sporosarcina aquimarina, Planococcus kocurii, P. antarcticus and Planococcus donghaensis (family Planococcaceae), 3 strains Paenibacillus terrae, Paenibacillus xylanexedens and Paenibacillus sp. (family Panenibacillaceae) and 3 strains of E. undae, E. antarcticum and Exiguobacterium sp. [no rank family Bacillales family XII (Bacillales incertae sedis)] were identified. Nine strains belonged to the phylum Actinobacteria, represented by Arthrobacter sp., Arthrobacter sulfonivorans, Arthrobacter sulfureus, Brachybacterium sp., Cellulosimicrobium cellulans, Citricoccus sp., Kocuria kristinae, Kocuria palustris and Sanguibacter antarcticus and two belonged to the phylum Bacteroidetes and was represented by Flavobacterium antarcticum and Sphingobacterium sp. (Figure 2b). The sixteen strains belonged to the phylum Proteobacteria (a, b and c-Proteobacteria). The twelve strains (Alishewanella sp., Klebsiella sp., Providencia sp.,

123


Pseudomonas frederiksbergensis, Pseudomonas peli, Pseudomonas putida, Pseudomonas reactans, Pseudomonas sp., Pseudomonas stutzeri, Psychrobacter glacincola, Stenotrophomonas maltophilia and Vibrio metschnikovii) belonged to the class gamma-proteobacteria, three strains (Aurantimonas altamirensis, Brevundimonas terrae, and Paracoccus sp.) to the class alpha-proteobacteria and one strain (Janthinobacterium sp.) to the class beta-proteobacteria (Fig. 2b). Overall Arthrobacter from Actinobacteria, Bacillus and Paenibacillus from Firmicutes, Pseudomonas from Proteobacteria were the most frequently recovered genera (Table 2). Statistical analysis The 325 isolates from the six different sampling sites could be categorised into 23–40 clusters, based on similarity index of[97 % of the 16S rRNA gene sequence (Table 2). Of the 57 strains identified, nine strains Arthrobacter sp., B. cereus, Bacillus firmus, Bacillus flexus, Bacillus pumilus, B. subtilis, Paenibacillus sp., Providencia sp., and P. reactans were common to all six sites (Supplementary Table 3). Shannon’s diversity index (H0 = 3.6) and species richness was highest for Pangong Lake, whereas Zanskar River recorded the lowest value (H0 = 3.06). These observations are supported by bacterial diversity parameters, such as Simpson’s index, Chao-1, Evenness and Shannon Entropy and individual rarefaction curves (Table 3; Fig. 3a). Correlation analysis proved existence of significant relationship between the different parameters and sampling sites. The first two factorial axes of biplot represent 63.83–98.49 % variance in the data (Fig. 3b). Principal coordinate analysis was used to investigate relationships between bacterial diversity (Shannon’s diversity index). The first two dimensions of PCA (PCA1 and PCA2) explained 70.16 % of the total variation, with component 1 accounting for 54.71 % and component 2 for 15.45 % of the variance (Fig. 3c). Plant growth promoting (PGP) traits The representative strains were screened for PGP traits at three different temperatures 4, 15 and 30 °C. Differential results were obtained for various traits at three incubation temperatures. 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. Out of 57 representatives, 27 were positive for solubilisation of phosphorus (Table 2). Production of ammonia, gibberellic acid, and indole-3-acetic acid was exhibited by 50, 11 and 35 strains respectively (Table 2). Twenty-one strains were positive for ACC deaminase activity. Isolate IARI-L-108 solubilized highest amount of phosphorus

123


(55.9 ± 0.6 lg mg-1 day-1) followed by IARI-ABL-2 (55.4 ± 1.0 lg mg-1 day-1) at 30 °C. However at 4 °C, highest P-solubilization was recorded for strain IARI-L-46. Strains IARI-L-54 and IARI-L-6 showed highest IAA production at 4 and 30 °C respectively (Table 2). Twenty and nine strains were exhibited production of siderophores and HCN respectively, while ten strains (IARI-L-2, IARIL-73, IARI-L-110, IARI-L-118, IARI-L-120, IARI-L-127, IARI-L-128, IARI-ABL-34, IARI-ABL-38 and IARIABL-41) showed antagonistic activity against R. solani and M. phaseolina. Among fifty-seven strains, twelve strains identified as A. sulfonivorans, B. firmus, Bacillus muralis, B. pumilus, B. subtilis, Desemzia incerta, Exiguobacterium sp., Lysinibacillus sp., P. frederiksbergensis, P. reactans, Pseudomonas sp. and S. antarcticus exhibited more than five different plant growth promoting activities at low temperature. Four strains, Sanguibacter antarcticus, Bacillus firmus, Bacillus subtilis and Exiguobacterium sp. possess four PGP traits of phosphate solubilization, siderophore production, IAA production and ACC deaminase activity (Fig. 4).

Discussion The cold deserts represent hot spots of biodiversity and several novel cold-tolerant bacterial species have been isolated from these regions (Chaturvedi et al. 2008; Fruhling et al. 2002; Kishore et al. 2010; Reddy et al. 2008; Srinivas et al. 2011). Significant prokaryotic diversity has been detected, including heterotrophic bacteria, cyanobacteria and eukaryotes, with many related to known psychrophilic and psychrotolerant species (Amato et al. 2007). Diverse culturable psychrophilic and psychrotolerant bacteria were obtained from cold desert of Leh Ladakh. They had some common features with other low temperature tolerant bacteria like production of exopolysaccharide and pigment as reported earlier (Sahay et al. 2013). The presence of pigments have been suggested to play a role in providing protection from harmful effects of solar irradiation in the glacier environment (Prasad et al. 2014; Srinivas et al. 2011). The strains identified in the present study could be grouped phylogenetically with psychrophilic or psychrotolerant isolates reported from cold environments. It has been reported that cold environments exert selection pressure and choose similar bacteria (Miteva and Brenchley 2005). The presence of viable bacteria and fungi in ancient glacier ice has been widely documented in polar and non-polar locations. Remarkably, many isolates obtained from geographically diverse glacier samples of polar and non-polar origin belong to the same bacterial genera including representatives of the Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes (Prasad et al.


103

Table 2 Identification and characterization of the bacterial isolates from Leh Ladakh, J & K Strain number

Nearest phylogenetic relative

Plant growth promoting traits Phosphateb

IAAb

Siderob

GAÒ

NHa3

ACCÒ

HCN

Actinobacteria IARI-L-120

Arthrobacter sp.

23.5 ± 0.5

28.2 ± 1.2

-

-

?

-

-

IARI-L-16

Arthrobacter sulfonivorans

25.6 ± 1.2

27.6 ± 0.7

4.7 ± 0.9

?

??

-

-

IARI-L-60

Arthrobacter sulfureus

-

48.2 ± 1.2

-

?

?

?

-

IARI-ABL-35

Brachybacterium sp.

-

17.6 ± 0.7

3.7 ± 0.5

-

?

?

-

IARI-ABL-30

Cellulosimicrobium cellulans

15.5 ± 1.1

18.4 ± 0.8

-

-

???

?

-

IARI-ABL-2 IARI-ABL-31

Citricoccus sp. Kocuria kristinae

54.8 ± 0.8 -

28.4 ± 0.8

-

-

??? ??

?

-

IARI-ABL-32

Kocuria palustris

-

-

-

-

-

-

-

IARI-L-33

Sanguibacter antarcticus

20.1 ± 0.1

9.3 ± 0.9

10 ± 0.8

?

??

?

-

Bacteroidetes IARI-ABL-45

Flavobacterium antarcticum

-

8.4 ± 0.8

-

-

-

?

?

IARI-ABL-47

Sphingobacterium sp.

12.2 ± 0.1

18.7 ± 0.5

-

-

??

?

-

IARI-L-24

Bacillus anthracis

24.1 ± 0.7

28.2 ± 1.2

-

-

??

-

-

IARI-ABL-36

Bacillus baekryungensis

-

-

-

-

-

?

-

IARI-L-73

Bacillus cereus

-

38.5 ± 0.9

-

-

???

-

-

IARI-L-21

Bacillus firmus

35.2 ± 3.3

35.2 ± 1.0

6.0 ± 0.8

-

??

?

-

IARI-ABL-37

Bacillus flexus

33.1 ± 1.9

-

-

-

??

?

-

Firmicutes

-

IARI-ABL-38

Bacillus licheniformis

-

-

4.5 ± 0.5

IARI-ABL-39

Bacillus marisflavi

-

28.6 ± 1.0

-

-

?

?? ?

-

-

IARI-ABL-40 IARI-L-74

Bacillus mojavensis Bacillus muralis

-

28.6 ± 1.0

9.7 ± 1.2

?

?? ???

-

?

IARI-L-54

Bacillus pumilus

36.1 ± 0.8

32.3 ± 1.2

4.3 ± 0.9

?

?

-

?

IARI-L-118

Bacillus simplex

-

32.4 ± 1.3

-

-

?

-

-

IARI-L-14

Bacillus sp.

-

48.5 ± 1.5

-

-

?

?

-

IARI-L-69

Bacillus subtilis

19.8 ± 0.5

27.7 ± 0.9

5.3 ± 0.5

?

?

?

-

IARI-L-46

Desemzia incerta

47.5 ± 1.2

28.6 ± 1.0

4.7 ± 0.5

?

?

-

-

IARI-L-70

Exiguobacterium antarcticum

31.1 ± 1.8

27.3 ± 1.3

-

-

?

?

-

IARI-L-62

Exiguobacterium sp.

46.4 ± 0.6

25.3 ± 0.9

8.7 ± 0.5

-

?

?

?

IARI-L-116

Exiguobacterium undae

-

25.5 ± 0.8

-

-

-

-

-

IARI-L-2

Lysinibacillus fusiformis

20.6 ± 1.2

25.3 ± 1.3

7.0 ± 0.8

-

??

-

-

IARI-L-65

Lysinibacillus sp.

26.3 ± 1.1

28.2 ± 1.2

9.7 ± 0.9

-

?

-

?

IARI-L-3

Lysinibacillus sphaericus

-

22.6 ± 1.1

-

-

??

-

-

IARI-L-128

Paenibacillus sp.

-

-

-

-

??

-

-

IARI-L-127

Paenibacillus terrae

29.4 ± 1.2

-

-

-

??

?

-

IARI-L-76

Paenibacillus xylanexedens

-

-

-

-

?

-

-

IARI-ABL-9 IARI-L-39

Planococcus antarcticus Planococcus donghaensis

20.2 ± 0.8

28.2 ± 1.2

4.7 ± 0.5

-

?? ?

-

? -

IARI-ABL-3

Planococcus kocurii

-

-

-

-

??

?

?

IARI-ABL-41

Pontibacillus sp.

-

-

-

-

?

-

-

IARI-ABL-18

Sinobaca beijingensis

-

18.2 ± 1.2

-

-

?

-

-

IARI-L-77

Sporosarcina aquimarina

-

68.2 ± 1.2

-

?

???

-

-

IARI-ABL-33

Staphylococcus arlettae

-

-

-

-

?

-

-

IARI-ABL-26

Aurantimonas altamirensis

-

17.0 ± 1.7

-

-

?

-

-

IARI-ABL-27

Brevundimonas terrae

-

-

5.5 ± 1.2

-

?

-

-

Proteobacteria

123



Table 2 continued Strain number

Nearest phylogenetic relative

Plant growth promoting traits Phosphateb

IAAb

IARI-ABL-28

Paracoccus sp.

-

-

IARI-L-110

Janthinobacterium sp.

26.0 ± 0.3

27.0 ± 1.7

Siderob

GAÒ -

??

-

-

8.2 ± 0.8

-

?

-

-

NHa3

ACCÒ

HCN

IARI-L-23

Alishewanella sp.

-

72.1 ± 1.2

-

-

?

-

-

IARI-ABL-29

Klebsiella sp.

44.7 ± 0.9

-

5.3 ± 0.5

-

??

-

-

IARI-L-6

Providencia sp.

-

110.7 ± 1

-

-

-

-

-

IARI-L-28

P. frederiksbergensis

31.6 ± 0.4

15.7 ± 0.6

5.0 ± 0.8

-

??

-

?

IARI-L-117

Pseudomonas peli

20.3 ± 0.6

-

-

-

-

?

-

IARI-L-109

Pseudomonas putida

49.6 ± 0.6

-

4.3 ± 0.5

-

??

?

-

IARI-L-108

Pseudomonas reactans

54.3 ± 0.8

-

4.7 ± 1.2

-

?

?

-

IARI-L-71

Pseudomonas sp.

-

23.6 ± 0.8

-

?

?

?

?

IARI-L-119

Pseudomonas stutzeri

31.5 ± 1.1

-

-

-

-

-

-

IARI-L-112

Psychrobacter glacincola

23.4 ± 0.8

-

7.0 ± 0.8

?

??

-

-

IARI-ABL-34

Stenotrophomonas maltophilia

-

-

-

-

?

-

-

IARI-L-9

Vibrio metschnikovii

32.7 ± 1.1

48.2 ± 1.2

-

?

?

-

-

IAA indole 3-acetic acid, Sidero siderophore, GA gibberllic acid, ACC 1-aminocyclopropane-1-carboxylate, HCN hydrogen cyanide a

None -; weak ?; moderate ??; strong ???; P-solubilization (lg mg-1 day-1) and IAA (lg mg-1 protein day-1)

b

Numerical values are mean ± SD of three independent observations

Table 3 Diversity indices for the isolates from six locations in Leh Ladakh, J & K (India) KP

IR

IZ

ZR

PL

LC

No. of isolates

62

47

43

36

85

52

Species richness

32

26

25

23

40

29

0

Evenness (J )

0.94

0.95

0.95

0.94

0.91

0.91

Shannon (H)

3.40

3.20

3.16

3.06

3.60

3.27

Simpson’s (D) Chao-1 No. of niche-specific bacteria

0.96

0.95

0.95

0.95

0.97

0.96

33.4

27.1

26.6

27.5

43.2

34.5

2

3

2

4

7

6

KP Khardungla Pass, IR Indus River, IZ I-Z confluence, ZR Zanskar River, PL Pangong Lake, LC Chumathang

2014). In the present study, isolates of the Actinobacteria and Firmicutes are usually found to be predominant, followed by members of the Proteobacteria and Bacteroidetes. Two genera, F. antarcticum and Sphingobacterium sp., belonging to Bacteroidetes were isolated only from Pangong Lake. This could be attributed to high conductivity values for water samples collected from this site that allowed the growth of low temperature adapted and moderately halotolerant bacteria of the group Bacteroidetes. The members of Bacteroidetes isolated from cold environment have been reported to be halotolerant and may be due to adaptation of members of this group to moderate salt concentrations (up to 5 % NaCl) (Van Trappen et al. 2004). Many bacteria like B. baekryungensis, Bacillus marisflavi,

123

D. incerta, P. xylanexedens, Pontibacillus sp. and S. beijingensis from phylum Firmicutes and A. altamirensis, Alishewanella sp., Providencia sp., P. frederiksbergensis and V. metschnikovii from phylum Proteobacteria were first time isolated and reported from high altitude and low temperature environments. These species have been earlier isolated from different habitats. For example, B. marisflavi was earlier reported from sea water of a tidal flat of the Yellow Sea in Korea (Yoon et al. 2003). It is a slow growing bacterium, forms cream/white coloured colonies on nutrient agar medium, grows in a pH range of 5–9, could tolerate 10 % NaCl, produce IAA; ammonia and exihibited ACC deaminase activity at low temperatures. A. altamirensis was first isolated from a subterranean environment, the Altamira Cave (Jurado et al. 2006) and later from Vetiver grass and rice rhizosphere (Bhromsiri and Bhromsiri 2010). It is a yellow coloured psychrotrophic bacterium that could tolerate 10 % NaCl and produced IAA and ammonia at low temperatures. B. terrae was first time isolated from an alkaline soil in Korea (Yoon et al. 2006) and not reported from any other ecological niche. It is a light yellow coloured psychrotolerant bacterium that could grow in a pH range of 6–11, tolerate 10 % NaCl and produced siderophore and ammonia at low temperatures. In the present study some novel species were also isolated showing BLAST similarity \96 % to sequences available in NCBI databases and needs to be further characterised. The population of psychrophilic and psychrotolerant bacteria in the samples of Leh Ladakh varied from


105

Fig. 4 The Venn diagram illustrates the number of PGPR showing the PGP traits of phosphate solubilization, ACC deaminase activity, siderophore production and indole-3-acetic acid production

Fig. 3 a Rarefaction curves of observed OTUs in the water/sediment samples from Leh Ladakh b biplot showing relationship between different sampling sites and temperature, pH and conductivity; c principal coordinate analysis of the diversity indices (H) of the16S rRNA PCR-ARDRA profiles of the six sites in relation to 16S rRNA gene sequences, Component 1 and component 2 accounted for 54.71 % and for 15.45 % of the total variation, respectively

9.12 9 105 to 1.65 9 106 cfu g-1 sediment or mL-1 water. Bacterial abundance in cold desert of Leh Ladakh (low temperature and high altitude 4,050–5,395 m) was 10–100 folds higher to that on other high mountains. Bacterial abundance reported from surface snow on Mount Sonnblick (3,106 m, Austria) ranged from 9.5 9 103 to 1.3 9 104 cells/mL (Sattler et al. 2001), in surface snow on Mt. Everest (6,600 and 8,000 m) from 2.11 9 104 to

9.44 9 104 cells/mL (Liu et al. 2009). Microbial abundance in snow cover has been reported to range from 103 to 105 per ml in melted snow and exhibit variations with altitude and latitude. Although the bacterial abundance in snow on Leh Ladakh was similar or higher to other high altitude and low temperatures regions in the world; but the community structure was different. The remarkable differences in bacterial community structure among the habitats have been earlier reported to be most likely due to post-deposition changes in bacterial abundance during the acclimation processes (Liu et al. 2009). A total of 325 bacteria were recovered from six sites in Leh Ladakh region. 16S rDNA-ARDRA analysis grouped all the isolates into 175 groups. Partial sequencing of the smaller subunit of 16S rRNA gene assigned all the 175 groups to 57 distinct species, grouped into 4 phyla viz. Actinobacteria (19 %), Bacteroidetes (3 %), Firmicutes (54 %) and Proteobacteria (28 %) (Fig. 5a). Among the six sites analysed, highest diversity was found in Pangong Lake and all the four phyla were represented at this site (Fig. 5b). The other five sites showed the presence of only members of Actinobacteria, Proteobacteria and Firmicutes. The diversity at Pangong Lake could be attributed to cumulative influence of low temperature, high altitude and high salt concentration (25 ppt). Out of 325 isolates from cold desert of Leh Ladakh, 175 belonged to phylum Firmicutes. The dominance of Firmicutes has been reported among the cultured isolates from permafrost sample of the Canadian high Arctic (Steven et al. 2007). Among family Bacillaceae (112 isolates), genus Bacillus (91 isolates) was

123


Fig. 5 Abundance of different bacteria; a distribution of phylum and group in the samples surveyed; b distribution of total bacteria in six sampling sites

most dominant followed by its derived genera Lysinibacillus (18 isolates) and Pontibacillus (3 isolates). However earlier reports suggests low representation of Bacillus and Bacillus derived genera (BBDG) at low temperature and high altitude sites (Kishore et al. 2010; Prasad et al. 2014; Sahay et al. 2013; Shivaji et al. 2011). Of the 175 isolates of Firmicutes, 9 strains—B. flexus (IARI-ABL-37), B. marisflavi (IARI-ABL-39), Bacillus licheniformis (IARIABL-38), Bacillus mojavensis (IARI-ABL-40), P. antarcticus (IARI-ABL-9), P. kocurii (IARI-ABL-3), Pontibacillus sp. (IARI-ABL-41), S. beijingensis (IARI-ABL-18) and Staphylococcus arlettae (IARI-ABL-33) from Pangong Lake exhibited optimum growth under cold and saline conditions, consistent with the properties of its habitat and its phylogeny is predictive of a cold active and halotolerant phenotype. Second dominant phylum was Proteobacteria (a, b and c-Proteobacteria) with Pseudomonas and Psychrobacter as the most dominant genera. These genera were already reported from cold environments (Liu et al. 2009; Shivaji et al. 2011). 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 plants. Though most research work conducted so far has largely focused on psychrophilic/

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psychrotolerant bacteria, it is a welcome sign that many agriculturally important microbes are being described from various parts of the earth. Microorganisms adapted to cold environment and possessing PGP attributes could be exploited to be used as inoculants for crops grown at high altitudes. Microorganisms play a central role in the natural P cycle. There are considerable populations of P-solubilizing bacteria in soil and in plant rhizospheres. It has been reported that indolic compounds have positive effect on root growth and cause rapid establishment of roots beneficial for young seedlings as it increase their capacity to obtain water and nutrients from their environment. It has been estimated that 80 % of bacteria isolated from the rhizosphere can produce the plant growth regulator IAA (Glick et al. 1999). Similar results were reported in the present study. Iron, is largely required by all living organisms for many activities. Microorganisms secrete iron-binding ligands (siderophores) that can bind ferric iron and make it available to the host microorganisms. In the present study 20 isolates were identified that could produce siderophore at low temperatures. Members of the Firmicutes (mainly members of families Bacillaceae and Paenibacillaceae) are widely used in agriculture as plant growth promoting and disease suppressing bacteria, besides their use in industry as a source of enzymes and in medicine. The importance of this group has led to several studies targeting their phenotypic and genotypic diversity in different ecological niches (Yadav et al. 2011), in food and industry waste. Plant growth promotion by bacteria is a well established and complex phenomenon, and is often attributed to multiple PGP traits exhibited by the associated bacterium. Hill and mountain agro systems require situation-specific microbial inoculants that withstand extremities of cold and retain their functional traits for plant growth promotion. The plant growth promotion potential of the bacterial strain dealt in this study requires further evaluation and validation before its use as a bioinoculants in the hill and mountain agro ecosystems, where temperature is a major determinant of plant and microbial activity. The selection of native functional plant growth promoting microorganisms is a mandatory step for reducing the use of energy intensive chemical fertilizers. 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. Ethical standard The experiments undertaken comply with the current laws of India, the country where the investigation was undertaken.


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