elucidating the diversity and plant growth promoting

ISSN : 0972-995X (Print), 2321-7960 (Online)

National Journal of Life Science, Vol. 10(2) 2013 : 219-227

ELUCIDATING THE DIVERSITY AND PLANT GROWTH PROMOTING ATTRIBUTES OF WHEAT (TRITICUM AESTIVUM) ASSOCIATED ACIDOTOLERANT BACTERIA FROM SOUTHERN HILLS ZONE OF INDIA PRIYANKA VERMA1, AJAR NATH YADAV1, SUFIA KHANNAM KAZY2, ANIL KUMAR SAXENA1 and ARCHNA SUMAN1* 1

Division of Microbiology, Indian Agricultural Research Institute, New Delhi. Department of Biotechnology, National Institute of Technology, Durgapur.

2

ABSTRACT : To elucidate the bio-diversity of plant growth promoting wheat associated acidotolerant bacteria from Southern hills zone of India. In this study, the diversity and phylogenetic relationships among plant growth-promoting (PGP) bacteria isolated from the wheat phyllosphere, rhizosphere and endophytic growing in acidic soils of Tamilnadu (pH 3.8-6.8) were investigated. Bacterial diversity was analysed through amplified ribosomal DNA restriction analysis (ARDRA) using three restriction enzymes Alu I, Hae III and Msp I which led to the grouping of 135 isolates into 33 clusters at >75% similarity index. 16S rRNA gene based phylogenetic analysis, revealed that isolates belonged to 4 phyla namely actinobacteria (4%), bacteroidetes (4%), firmicutes (52%) and proteobacteria (40%) with 33 distinct species of 13 genera. In vitro for plant growth promotion (PGP) traits were done at different pH which included solubilization of phosphorus, potassium and zinc, production of ammonia, hydrogen cyanide, indole-3-acetic acid and siderophore, nitrogen fixation, 1-aminocyclopropane-1-carboxylate deaminase activity and bio-control against Fusarium graminerum, Rhizoctonia solani and Macrophomina phaseoli. Acidotolerant isolates may have application as inoculants plant growth promoting and bio-control agents in crops growing under acidic condition. Key words : Diversity, Acidotolerant bacteria, 16S rRNA gene, PGPB.

INTRODUCTION Wheat (Triticum aestivum L.) is one of the most important cereals world-wide and it is grown in temperate climate. The southern hills zone comprises of western hill regions of Tamilnadu. The zone is characterized by acidic condition and longer growing season. The wheat associated bacterial diversity inhabiting acid tolerance has been extensively investigated in the past few years with a focus on culture dependent techniques. Many acidotolerant bacterial genera have been reported from acidic environments including Azotobacter, Bacillus, Flavobacterium, Pseudomonas and Serratia (Yadav et al.,2011 and Yadav et al.,2013). Among the dominant bacterial forms in Indian rhizospheric soils, Bacillus, Enterobacter and Pseudomonas (Yadav et al.,2013), Lysinibacillus and Methylobacterium (Holland & Polacco, 1994 and Wellner et al.,2011) have been recovered. Quantitative and qualitative variations in these traits allow for these bacteria to inhabit diverse niches in agro-ecosystem. Wheat associated microbial diversity is considered important for maintaining for the sustain ability of agriculture production systems. Epiphytic, endophytic and rhizospheric bacteria are associated with wheat crops which play important role in plant growth promotion.

and Meena et al.,2012). Rhizospheric bacteria have the ability to attach to the root surfaces (rhizoplane) allowing these to derive maximum benefit from root exudates. Endophytic bacteria live in plant tissues without causing substantive harm to the host. Endophytic bacteria exist within the living tissues of most plant species in form of symbiotic to slightly pathogenic. Bacterial endophytes have been recovered from a variety of plants including rice, tomato, sweet corn, citrus and potato (Ulrich et al.,2008). Very few studies have done for isolation of bacterial endophytes from wheat (Zinniel et al.,2002 and Coombs & Franco,2003). Some endophytes reported from wheat were Achromobacter, Microbiospora, Micrococcus, Micromomospora, Mycobacterium, Nocardioides, Pantoea, Planobispora, Planomonospora, Planomonospora, Pseudomonas, Rhodococcus, Stenotrophomonoas, Streptomyces and Thermomonospora (Zinniel et al.,2002 and Coombs & Franco,2003).

The phyllosphere is common niche for synergism between bacteria and plant. Microorganisms on leaf surfaces are said to be extremophiles as they tolerate high temperature (40-55°C) and UV radiation in day time while cool temperatures (5-10°C) in night. Many bacteria such as Pseudomonas and Methylobacterium have been reported in the phyllosphere (Holland & Polacco,1994; Wellner et al.,2011

Epiphytic, endophytic and rhizospheric bacteria have been shown to promote plant growth. Plant growth promoting (PGP) bacteria may promote growth directly, e.g. by fixation of atmospheric nitrogen, solubilization of minerals such as phosphorus, potassium and zinc, production of siderophores and plant growth hormones such cytokinins, auxins and gibberellins (Glick et al.,1999). Some bacteria support plant growth indirectly, via production of antagonistic substances by inducing resistance against plant pathogens. PGPB are used as biological control agents for the suppression of soil borne pathogens. PGPB may be use as bioinoculants so that it fits into a long-term sustainable agricultural system. A number of bacterial species associated with the plant rhizosphere belonging to genera Azospirillum, Arthrobacter, Acinetobacter, Bacillus, Burkholderia, Enterobacter,

*Corresponding author (email : [email protected])

Received 28.08.2013

Accepted 10.11.2013

8.8 9.3

NA

7.1 5.8

KB

5.4 4.5

AMS

25(18+7)

5.9 8.5

JN

Phyllosphere

11.1 7.8

TSA

3.1 3.7

R2A 5.0 2.7

NA 2.7 2.3

T3A 2.5 3.7

KB 4.3 3.7

R2A

3.4 4.8

AMS

78(37+41)

4.2 3.5

JN

Rhizosphere 4.7 3.6

SEA

3.1 4.4

TSA

3.7 1.7

PM

Total viable count (cfu g-1 sediment or ml-1 water x 106) on different media

M# - Total morphotypes from phyllosphere, rhizoshpere and endophytic bacteria from wheat var. (HD2833+HW3094).

M#

HD2833 HW3094

Wheat var.

Table. 2 Total viable count of bacteria samples from Southern hills zone of India.

*Soil extract : 250 g soil sampling site + 1 L H2O; Autoclave and filter.

13.

10. 11. 12.

9.

8.

1.9 1.5

AM

1.1 2.89

LB

1.25 2.98

1.2 1.58

MDM YEM

32(19+13)

3.21 1.86

NA

Endophytic

Media and composition per liter

Nutrient agar (NA) : 5 g peptone; 5 g NaCl; 3 g beef extract; 18 g agar; pH -3-6±0.2. T3 agar : 3 g Tryptone; 2 g Tryptose; 1.5 g Yeast extract; 0.005 g MnCl2; 0.05 g Sodium Phosphate; 18 g agar; pH -3-6±0.2. Soil extract agar (SEA) : 2 g glucose; 1 g Yeast extract; 0.5 g K2HPO4; 100 ml Soil extract*; 18 g agar; pH -3-6±0.2. Tryptic soy agar (TSA) : 17 g tryptone; 3 g soya meal; 2.5 g dextrose; 5 g NaCl; 2.5 g K2HPO4; 18 g agar; pH -3-6±0.2. King's B agar (KB) : 20 g Protease Peptone; 1.5 g K2HPO4; 1.5 g MgSO4.7H2O; 10 ml Glycerol; 18 g agar; pH 3-6±0.2. Jensen's agar (JA) : 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; 18 g agar; pH 3-6±0.2. R2A 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 3-6±0.2. Ammonium minerals salt (AMS) : 0.70 g K2HPO4; 0.54 g KH2PO4; 1.00 g MgSO47H2O; 0.20 g CaCl2. 2H2O; 4.00 mg FeSO4.7H2O; 0.50 g NH4Cl; 100 µg ZnSO4. 7H2O; 30 µg MnCl2.4H2O; 300 µg H3BO3; 10 µg CuCl2. 2H2O; 200 µg CoCl2. 6H2O; 20 µg NiCl2. 6H2O; 60 µg Na2MoO4. 2H2O; 18 g agar; pH -3-6±0.2 Pikovskaya's medium (PM) : 10 g glucose; 0.5 g yeast extract; 0.5 g (NH4) 2SO4; 0.2 g KCl; 0.2 g NaCl; 0.1 g MgSO4.7H2O; 5.0 gm Ca3 (PO4) 2; traces FeSO4. 7H2O; traces MnSO4.7H2O; 18 g agar; pH -3-6±0.2. Aleksandrov's medium (AM) : 5 g Sucrose; 2 g Na2 HPO4; 0.5 g MgSO. 7H2O; 0.1 g CaCO3; trace FeCl3; 0.2 mg H2 MoO4. H2O; 5 g potassium alumina silicate, 18 g agar; pH -3-6±0.2. Luria Bertani media (LB) : 10 g Casein acid hydralysate; 5 g Yeast extract; 10 g NaCl; 18 g agar; pH -3-6±0.2. Modified Dobereiner medium (MDM) : 10 g Sucrose; 5 g Malic acid; 0.2 g K2HPO4. H2O; 0.4 g KH2PO4. H2O; 0.1 g NaCl; 0.01 g FeCl3; 0.002 g Na2MoO4; 0.2 g MgSO4.7H2O; 0.02 g CaCl2. H2O; 18 g agar; pH 3-6±0.2. Yeast extract mannitol agar (YEMA) : 1 g yeast extract; 10 g Mannitol, 0.5 g K2HPO4. H2O; 0.002 g MgSO4. 7H2O 0.1 g NaCl; 18 g Agar; pH 3-6±0.2.

S.

1. 2. 3. 4. 5. 6. 7.

Table. 1 The different media used in this study for isolation of wheat associated bacteria.

220 VERMA et al.

DIVERSITY AND PGP ATTRIBUTES OF WHEAT ASSOCIATED BACTERIA Flavobacterium, Methylobacterium, Pseudomonas, Rhizobium and Serratia (Lavania et al.,2006; Yadav et al., 2011; Meena et al.,2012 and Yadav et al.,2013). The present study attempted to decipher the culturable bacterial diversity associated with wheat from Southern hills zone, Tamilnadu, employing different media, followed by screening of isolates for pH tolerance. ARDRA analysis was done for phylogenetic clustering of the acidotolerant isolates. Sequencing the 16S rRNA gene of representative acidotolerant strains was undertaken for identification. Representative strains from each operational taxonomical unit (OTUs) were screen in vitro for plant growth promotion at low pH.

MATERIAL AND METHODS Samples collection : Wheat plants with rhizospheric soil were collected from southern hills zone (11°22' 12'' N : 76°48' 00''E) of India. The Southern hills zone is characterized by acidic soil (3-6 pH). A total 16 samples of two wheat varieties viz., HD2833 and HW3094 were collected from different part of southern hills zone of India including IARI Regional Station (Coordinates ranging from 28.08°N and 77.12°E). Samples were collected in sterile polythene bags labelled, transported on ice and processes immediately.

Enumeration of wheat associated bacteria : The culturable bacteria from rhizospheric soil were isolated through enrichment using the standard serial dilution plating technique. The thirteen different media were used to isolates acidotolerant bacteria (Table.1). Endophytic bacteria were isolated using method described by Conn and Franco (2004). Epiphytic bacteria were isolated form plant leaves by imprint method described by Holland (Holland and Polacco,1994). The plates were incubated at 30°C and the population was counted after 3-7 days. Colonies that appeared were purified by repeated restreaking to obtain isolated colonies using respective medium plates. The pure cultures were maintained at 4°C as slant and glycerol stock (25%) at -80°C for further use. All the isolates were screened in triplicates for acid tolerance by incubating the culture spot inoculated plates at different pH tolerance was carried out by spot inoculating the cultures onto nutrient agar medium with pH ranging from 3 to 11, with an increment of one. PCR amplification of 16S rDNA and amplified rDNA restriction analysis (ARDRA) : Genomic DNA was extracted by the method as earlier described by Kumar et al. (2013). The amount of DNA extracted was electrophoresed on 0.8% agarose gel. Amplification of 16S rDNA was done by using t h e u n i v e r s a l p r i m e r s p A ( 5 ' A G A G T T T G AT C C T G G C T C A G - 3 ' ) a n d p H ( 5 ' AAGGAGGTGATCCAGCCGCA-3') (Kumar et al.,2013). The PCR amplified 16S rDNA were purified by QIA quick PCR product purification kit (Qiagen). 100 ng purified PCR products were digested separately with three restriction endonucleases Alu I, Hae III and Msp I (GeNei) in 25 µl reaction volumes, using the manufacturer's recommended buffer and temperature. The clustering analysis was 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 and dendrogram was constructed using the UPGMA method (Nei and Li,1979). 16S rDNA Sequencing and phylogenetic analysis : PCR amplified 16S rRNA genes were purified and sequenced at SCI

Proteobacteria, 40 %

Physico-chemical properties of samples : The pH and conductivity of the samples was recorded on sampling site. Soil sample analyzed for soil organic carbon according to Walkley and Black's rapid titration method (Walkley and Black,1934) Total nitrogen (%) was analyzed using kjeldahl's procedure by N-analyzer UDK-149, velp scientifica srl., Italy. Soil organic matter was determined by the loss of ignition method. Exchangeable cations (Ca, K, Mg and Na) were extracted with 1 M ammonium acetate (pH 7.0) and Ca, K and Na contents were determined with an atomic absorption spectrophotometer. Available phosphorus was determined by the Bray II method (Bray and Kurtz,1945). Soil analysis was done at Division of Soil Sciences, IARI, New Delhi, India.

221

Fig. 2 Abundance of different bacteria, a distribution of phylum and group in the samples surveyed.

222

VERMA et al.

Fig. 1 Phylogenetic tree showing the relationship among 33 bacterial isolates, 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 with algorithm using MEGA4 software (Tamura et al.,2007). One thousand bootstrap replicates were performed. Bootstrap values are indicated on the branches. The tree was rooted using Halococcus sp. (JX428954) as the outgroup

DIVERSITY AND PGP ATTRIBUTES OF WHEAT ASSOCIATED BACTERIA Genome Chennai, India. Bacterial isolates were identified based on percentage of 16S rRNA gene sequence similarity (>97%) with that of a prototype strain sequence in the GenBank database using BLASTn program. The phylogenetic tree was constructed on the aligned datasets using the neighbour-joining method (Saitou and Nei,1987) implemented in the program MEGA 4.0.2 (Tamura et al.,2007). Bootstrap analysis was performed as described by Felsentein (1981) on 1000 random samples taken from the multiple alignments. The partial 16S rDNA sequences were submitted to NCBI Gen Bank and accession numbers were assigned from KF054946 to KF054975. In vitro screening of isolates for PGP traits : Representative isolates from each cluster were screened for PGP attributes initially as qualitative estimation for in vitro production of ammonia (Cappucino and Sherman,1992), siderophore (Schwyn and Neilands,1987), HCN (Bakker and Schippers,1987), gibberllic acid (Brown and Burlingham,1968) and indole-3acetic acid (Bric et al.,1991). The isolates were screened for solubilization of phosphorus (Pikovskaya,1948), potassium (Hu et al.,2006) and zinc (Saravanan et al.,2004). The ability to fix nitrogen was evaluated using semi-solid nitrogen-free NFb medium (Dobereiner et al.,1996). The bacterial strains were screened for their ability to utilize the 1Aminocyclopropane-1-carboxylate (ACC) as sole nitrogen source, a trait that is consequence of the activity of the enzyme ACC deaminase (Jacobson et al.,1994). All assays were done in triplicate at 3, 5 and 7 pH. In vitro antagonistic activity of bacterial isolates was evaluated against three fungal pathogens Fusarium graminerum, Rhizoctonia solani and Macrophomina phaseoli according to the method described by Sijam and Dikin (2005).

RESULTS AND DISCUSSION Enumeration and characterization of wheat associated bacteria : A total of 135 bacterial strains were isolated and maintained; 74 (18 epiphytic, 37 rhizospheric and 19 endophytic) and 61 (7 epiphytic, 41 rhizospheric and 13 endophytic) from wheat var. HD2833 and HW 3094 respectively. Significant variations were observed among the culturable bacterial population (CFU) of each sample on different growth media. The abundance of bacteria in the samples varied from 11.1 x 104 to 5 x 106 and 3.21 x 104 to 5.44 x 106 for wheat var. HD2833 and HW 3094 respectively. The population of bacteria varied from 11.1 x 106 to 5.0 x106, 3.2 x 104 to 9.3 x 106, 4.8 x 106 to 3.0 x 106 for isolation sources of phyllosphere, rhizosphere and endophytic respectively (Table.2) The pure colonies obtained from each sample on different media were isolated based on colony morphology and cultural characteristics. Out of 33 representatives strains, 12 and

223

21 could tolerates 3-5, 5-8 respectively. Bacterial isolates were tolerance to NaCl varied from 3 to 10 % NaCl (w/v). Out of 33 representative 33 were tolerant to 3 % NaCl, while 25 and 12 isolates could tolerate 5 and 10% NaCl respectively. Isolates could grow in the temperature ranges 30°C-50°C. Physico-chemical properties of samples : Physical and chemical characteristics of the soil varied considerably amongst the samples in which pH values were highly variable from 3.8 to 6.8 (Table.3). Total nitrogen content in dry weight was very low 0.7% and 1.1% on average from HW3094 and HD2833 respectively. Organic carbon content was significantly higher, achieving of 4.39% organic matter in a HD2833 and 4.13% of HW3094 soil samples (Table.3). PCR amplification of 16S rDNA and ARDRA : PCR amplification of 16S rDNA followed by ARDRA with three restriction endonucleases was carried out to look for the species variation among the morphotypes selected. The 16S rDNA amplicons were digested with restriction enzymes, which generated profiles having 3 to 7 fragments ranging in size from 100 to 860 base pairs. ARDRA results revealed that among the restriction endonucleases, Alu I was more discriminatory as compared to Msp I and Hae III. A combined dendrogram was constructed for each sampling site to determine the percent similarity among the isolates. At a level of 75% similarity the isolates were grouped into 33 clusters. 16S rRNA gene sequencing and phylogenetic analysis : A total of 33 strains were selected based on the restriction pattern generated by the 16S rRNA gene sequences and the sequence data was analyzed by BLAST and the nearest match from GenBank data was reported. Sequences were deposited in the GenBank. DNA sequencing and phylogenetic analysis revealed that all the showed >97-100% isolates similarity with the sequences within the GenBank. The phylogenetic tree of 33 identified bacteria was constructed to determine their affiliations (Fig.1). Analysis of the 16S rRNA sequences revealed that isolates belonged to 4 phyla namely actinobacteria (4%), bacteroidetes (4%), firmicutes (52%) and proteobacteria (40%) with 13 genera with 33 distinct species. The proteobacteria were most pre-dominant phylum followed by firmicutes. Out of the 33 OTUs, 13 strains belonged to the phylum proteobacteria. Among phylum proteobacteria, 7 strains Azotobacter tropicalis, Enterobacter ludwigii, Pseudomonas chlororaphis, Pseudomonas fluorescens, Pseudomonas rhodesiae, Serratia marcescens and Variovorax dokdonensis were belonged to the class -proteobacteria. Two strains Variovorax paradoxusv and Variovorax soli belonged to class -proteobacteria and three strains Methylobacterium sp., Methylobacterium phyllosphaerae and Methylobacterium radiotolerans belonged to the class -proteobacteria. The

Table. 3 Physico-chemical properties of soil. Wheat HD2833 HW3094

pH

EC

%OC

Avail. N

Avail. P

Avail K

Zinc

Exch. Na

Exch. Ca

Exch. Mg

3.8-6.6 4.2-6.8

46.8 48.1

4.3 4.1

177 219

13.7 12.2

1086 1936

1.50 1.55

51.83 53.21

16.93 17.06

10.58 16.36

Variovorax soli Bacillus thuringiensis Variovorax paradoxusv Lysinibacillus fusiformis Bacillus atrophaeus Bacillus rigui Variovorax dokdonensis Pseudomonas rhodesiae Bacillus barbaricus Pseudomonas chlororaphis Lysinibacillus sphaericus Enterobacter ludwigii Bacillus nanhaiensis Pseudomonas fluorescens Bacillus solisalsi Flavobacterium sp. Bacillus cereus Pseudomonas rhodesiae Bacillus nealsonii Bacillus circulans Methylobacterium sp. Methylobacterium phyllosphaerae Serratia marcescens Bacillus pumilus Planococcus salinarum Planomicrobium sp. Bacillus aerophilus Lysinibacillus fusiformis Azotobacter tropicalis Micrococcus luteus Staphylococcus devriesei Staphylococcus epidermidis Methylobacterium radiotolerans

IARI-THD-1 IARI-THD-2 IARI-THD-3 IARI-THD-4 IARI-THD-5 IARI-THD-6 IARI-THD-7 IARI-THD-11 IARI-THD-12 IARI-THD-13 IARI-THD-16 IARI-THD-17 IARI-THD-20 IARI-THD-21 IARI-THD-24 IARI-THD-25 IARI-THD-26 IARI-THD-28 IARI-THD-30 IARI-THD-32 IARI-THD-35 IARI-THD-43 IARI-THW-5 IARI-THW-8 IARI-THW-9 IARI-THW-15 IARI-THW-17 IARI-THW-20 IARI-THW-22 IARI-THW-25 IARI-THW-27 IARI-THW-28 IARI-THW-31

21.52±1.3 8.41±0.8 45.92±1.9 10.43±0.8 4.47±0.6 108.12±1.2 16.54±0.9 3.6.35±1.0 46.64±1.2 6.72±2.7 8.90±1.0 46.52±1.2 426.44±1.5 9.94±1.0 21.35±1.0

PO4

3.3±1.2 1.2±1.0 1.1±1.0 1.2±0.9 -

1.3±1.0 1.6±1.0 1.4±1.3 -

K#

Solubilization

5±0.9 6±1.2 4±1.5 4±1.0 6±1.1 5±1.2 6±1.1 3±1.0 2±1.3 5±1.2 6±1.2 7±0.8 -

Zn# 2±0.5 3±1.2 8±1.3 2±0.3 5±0.5 2±1.0 3±1.2 5±1.3 3±1.1 1±1.0 5±0.8 3±1.2 2±0.8 2±1.1 5±1.0 5±1.1 -

Sidero#

Production

# Numerical values are mean ± SD of three independent observations; P-solubilization (µg mg-1day-1) * None - weak + moderate ++strong +++; IAA (µg mg-1 protein day-1); Gibberllic acid (GA); Biocontrol (BC)

Nearest phylogenetic relative

Strain number

115.7±3.9 46.13±0.5 32.72±0.2 24.28±2.6 72.14±1.4 222.29±2. 227.3±1.9 22.80±1.9 32.20±1.2

IAA#

+ -

+ + + + + +

GA*

Other activities

+ + + + + + + +

HCN*

Table. 4 Identification and characterization of the bacterial isolates from Southern hills zone of India.

+ + + + + + + -

N2 Fix* + + + + + + + + + + + + -

BC* 5-8 5-8 5-8 5-8 3-5 3-5 5-8 3-5 3-5 3-5 5-8 5-8 5-8 3-5 3-5 5-8 5-8 5-8 3-5 5-8 3-5 5-8 3-5 5-8 5-8 5-8 3-5 5-8 3-5 5-8 5-8 5-8 5-8

pH tolerance

224 VERMA et al.

DIVERSITY AND PGP ATTRIBUTES OF WHEAT ASSOCIATED BACTERIA one-one strains belonged to the phylum actinobacteria and bacteroidetes represented by Micrococcus luteus and Flavobacterium sp. Out of the 33 OTUs, 17 strains belonged to phylum firmicutes were grouped into seven clusters of three families of bacilli namely Bacillaceae (13 strains Bacillus aerophilus, Bacillus atrophaeus, Bacillus barbaricus, Bacillus cereus, Bacillus circulans, Bacillus nanhaiensis, Bacillus nealsonii, Bacillus pumilus, Bacillus rigui, Bacillus solisalsi, Bacillus thuringiensis, Lysinibacillus fusiformis and Lysinibacillus sphaericus), Planococcaceae (two strain Planococcus salinaru and Planomicrobium sp.) and Staphylococcaceae (two strain Staphylococcus devriesei and Staphylococcus epidermidis). Overall Micrococcus from actinobacteria, Bacillus from firmicutes and Pseudomonas from proteobateria were the most frequently recovered genera (Table.4). Comparative analysis of bacteria associated with wheat : The majority of bacteria from two wheat var. HD2833 and HW3094 belong to proteobacteria followed by firmicutes, actinobacteria and bacteroidetes. Out of 135, 74 isolates from wheat var. HD2833 which included 18, 37 and 19 isolate from phyllosphere, rhizosphere and endophytic respectively while 61 isolates from wheat var. HW3094 which included 7, 41 and 13 isolates from phyllosphere rhizosphere and endophytes. There were obvious differences among the phyllosphere, rhizosphere and endophytic bacterial communities. Lower bacterial diversity was observed in the endophytes compared to phyllosphere and the rhizosphere. Pink-pigmented facultative methylotrophs (PPFMs) Methylobacterium phyllosphaerae, Methylobacterium radiotolerans and Methylobacterium sp. were most dominant in phyllosphere. Variovorax and Methylobacterium were isolated from phyllosphere only. Enterobacter ludwigii, Flavobacterium sp., Micrococcus luteus, Pseudomonas rhodesiae, Pseudomonas chlororaphis, Pseudomonas rhodesiae and Staphylococcus epidermidis, were isolated from endophytic only. Staphylococcus devriesei is common bacteria isolated from rhizosphere as well as from endophytes. Azotobacter tropicalis, Bacillus aerophilus, Bacillus atrophaeus, Bacillus barbaricus, Bacillus cereus, Bacillus circulans, Bacillus nanhaiensis, Bacillus nealsonii, Bacillus pumilus, Bacillus rigui, Bacillus solisalsi, Bacillus thuringiensis, Lysinibacillus fusiformis, Lysinibacillus sphaericus, Pseudomonas fluorescens, Planococcus salinarum, Planomicrobium sp., Serratia marcescens, Staphylococcus devriesei and Staphylococcus epidermidis were isolates from rhizosphere. Plant growth promoting (PGP) attributes : Out of 33 representatives, 15, 11 and 6 strains were positive for solubilisation of phosphorus, zinc and potassium respectively (Table.4). Further zinc solubilizing bacteria (ZSB) were screened using different inorganic zinc compounds. Out of 33 representatives, 14, 13 and 11 strains solubilised zinc carbonate, zinc oxide and zinc phosphate respectively. Of 33 representatives 16 strains produced siderophore and 7 strains indole-3-acetic acid and 3 strains produced gibberellic acid while only one strain (IARI-THW-8) produced ammonia (Table.4). ACC deaminase activity was showing by 2 strains (IARI-THW-8

225

and IARI-THD-26) while 7 isolates were showing N2 fixation confirms by acetylene reduction assay (ARA). Among plant growth promoting activities, siderophore producing strains were highest (17%) when compared to P-solubilization (16%), Bio-control (13%), Zinc solubilization (12%), IAA production (9%), ammonia (11%), HCN production (9%), Nitrogen fixation and GA (8%), K-solubilization (6%), ACC deaminase (2%) and ammonia production (1%). Isolate solublized highest amount of phosphorus IARI-THW-25 (426±1.5µg mg-1day-1) followed by IARI-THD-28 (108±1.2 µg mg-1day-1) while Isolate IARI-THD-35, solublized lowest amount of phosphorus (3.6±1.0 µg mg-1day-1). Isolate IARITHW-22 showed highest IAA production (227.3±1.9µg mg-1 protein day-1) followed IARI-THW-20 (222.29±2.4µg mg-1 protein day-1) while Isolate IARI-THW-28 show lowest production of IAA (22.80±1.9µg mg-1 protein day-1). Highest solubilization of potassium by isolates IARI-THD-26 (3.3± 1.2 mm) while lowest by IARI-THW-8 (1.1±1.0 mm). Isolate IARI-THW-28 show highest zinc solubilization (7±0.8 mm) while isolate IARI-THW-9 show lowest zinc solubilization (2±1.3 mm) (Table.4). For bio-control all isolates were tested for the production of siderophores, HCN and Triple-culture assays. A high number of the antagonistic isolates 16 and 8 were positive for production of siderophores and HCN respectively. Of 33 stains, 12 strains were anatagonastic against Fusarium graminerum, Rhizoctonia solani and Aspergillus fumigatus (Table.4). Microbial diversity is fundamental for the maintenance and conservation of global genetic resources. The phyllosphere, rhizosphere and endophytic are a hot spot of bacterial diversity, harbours bacterial flora whose diversity is mainly expressed in terms of functions adaptation which help in plant growth. Our present study includes the diversity of bacterial communities associated with the phyllosphere, rhizosphere and endophytic of two wheat var. HD2833 and HW3094 growing in acidic climate of southern hills zone of India. A total of 135 wheat associated bacterial isolates obtained from two var. of wheat. There is few study which consider to isolates only one type of bacterial association (epiphytic or endophytic or rhizosphere) with wheat. We isolate all types of bacterial association with wheat as epiphytic, endophytic and rhizospheric. Sequencing of 16S rRNA gene of the representative isolates from 33 clusters identified as plant growth promoting bacteria, which were taken up for phylogenetic analysis. Partial sequencing of the smaller subunit of 16S rRNA gene assigned all the 33 isolates to different species, grouped in 4 phyla, actinobacteria, bacteroidetes, firmicutes and proteobacteria (Fig.2). In our study proteobacteria were most dominant phylum. Of 33 representative strains, 3 strains with distinct genera Methylobacterium belong to alphaproteobacteria. Two strains Variovorax belong to betaproteobacteria while most predominant class of proteobacteria is gamma-proteobacteria consisting 5 genera with 7 distinct species Azotobacter tropicalis, Enterobacter ludwigii, Pseudomonas chlororaphis, Pseudomonas fluorescens, Pseudomonas rhodesiae, Serratia marcescens

VERMA et al.

226 and Variovorax dokdonensis (Table.4). Second dominant phylum is firmicutes having 17 strains with 5 genera Bacillus, Lysinibacillus, Planococcus, Planomicrobium and Staphylococcus. The genera Micrococcus and Flavobacterium were belonged to from the phylum actinobacteria and bacteroidetes respectively. In our present study we have isolated wheat associated bacteria form two var. growing at acidic condition soil in Southern hills zone of India. From the phyllosphere a total of 25 bacterial isolates, 18 from var. HD2833 and 7 from var. HW3094 were isolated. On basic of 16S rRNA sequencing these epiphytic bacteria are Methylobacterium radiotolerans, Methylobacterium phyllosphaerae, Methylobacterium sp., Variovorax dokdonensis, Variovorax paradoxusv and Variovorax soli. Some of above already reported as epiphytic bacteria. Pink-pigmented facultative methylotrophs (PPFMs) Methylobacterium radiotolerans, Methylobacterium phyllosphaerae and Methylobacterium sp. are a physiologically and taxonomically diverse group of bacteria with prominent plant growth promoting attributes (Meena et al.,2012). The genus Methylobacterium is among the commonly recorded leaf epiphytes and represents abundant and stable members of the phyllosphere community of a wide range of crop plants (Holland & Polacco,1994 and Meena et al.,2012). PPFMs are reported to influence the seed germination and growth of many crop plants by producing plant growth regulators like cytokinins and indole-3-acetic acid (Meena et al., 2012 and Ivanova et al.,2001). On the basic of 16S rRNA sequencing these bacteria were identified as 33 distinct species of 13 genera Azotobacter, Bacillus, Enterobacter, Flavobacterium, Lysinibacillus, Methylobacterium, Micrococcus, Planococcus, Planomicrobium, Pseudomonas, Serratia, Staphylococcus and Variovorax. Most of plant growth promoting bacteria were already reported (Coombs & Franco,2003; Kim et al.,2011; Meena et al.,2012 and Yadav et al.,2013). Among PGPR, Members of Bacillus and Bacillus derived genera (BBDG) are ubiquitous bacteria that include both free-living PGPR and pathogenic species. PGPR belonging to BBDG have been reported to enhance the growth of several plants such as wheat, tomato, sugar beet, sorghum and peanut. A next to BBDG, another group of PGPR belonged to the genus Pseudomonas (Yadav et al.,2013). Of the Pseudomonas species identified as PGPR in our study are identified as Pseudomonas chlororaphis, Pseudomonas fluorescens and Pseudomonas rhodesiae. Previously it is reported that Pseudomonas PGPR are highly resistant to various environmental stresses (Paul and Nair,2008). Production of ACC deaminase by Pseudomonas fluorescens increases the resistance of plants to salt stress (Sandhya et al.,2010). Microbes associated with plants can be harmful and beneficial. Plant growth promoting bacteria (PGPB) promote growth directly by nitrogen fixation, solubilization of phosphorus and potassium, production of siderophores, ammonia, HCN and plant growth hormones (cytokinins, auxins and gibberellic acid) (Tilak et al.,2005). Many bacteria support

plant growth indirectly by improving growth restricting conditions via production of antagonistic substances. A number of bacterial species associated with the plant belonging to genera Azospirillum, Alcaligenes, Arthrobacter, Acinetobacter, B a c i l l u s , B u r k h o l d e r i a , E n t e ro b a c t e r, E r w i n i a , Flavobacterium, Pseudomonas, Rhizobium and Serratia are able to exert a beneficial effect on plant growth. In conclusion, utility of such acid tolerant bacterial strains in the context of acidic agro ecosystems is immense considering the unique crop growing situations and the climatic conditions of the acidic agricultural systems. Such systems require situation-specific microbial inoculants that withstand extremities of acid 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 acidic agro ecosystems, where acid 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 fertilisers. The strain reported in this study seems to be an ideal candidate for promotion as bioinoculants, due to its acid tolerance and multiple abilities of plant growth promotion traits.

ACKNOWLEDGMENTS The authors are grateful to the Division of Microbiology, Indian Agricultural Research Institute (IARI), New Delhi for providing the necessary facilities for undertaking this study. The financial support from Department of Biotechnology (DBT), Ministry of Science and Technology under the project "Development of plant growth promoting microbial consortium for rice-wheat sugarcane cropping system" is greatfully acknowledged.

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