Isolation and Characterisation of Diazotrophic Bacteria from Rhizosphere of different Rice Cultivars of South Assam, India

Free living heterotrophic bacteria were isolated from the rhizosphere of 10 local and cultivated varieties of rice grown in Karimganj district of South Assam. Among the 25 isolates, 11 isolates withplant growth promoting activity were identified based on phenotypic and 16S rDNA sequence analysis. The strains were identified as Shingomonasa zotifigens, Pseudomonas putida, Stenotrophomonas maltophila, Acinetobacter radioresistance, Alkaligenes faecalis, Enterobacter cloaceae subsp. dissolvens, Pantoea agglomerans, Klebsiella pneumoneae, Achromobacter xyloxidans, Herbispirillum rubrisubalbicans and Herbispirillum sp. The efficient strains are isolated from the local varieties of rice plant. The isolate KR-23 (Sphingomonas azotifigens) was a novel bacteria reported for the first time as nitrogen fixing bacteria from India. The nitrogen fixing ability along with IAA production, ACC deaminase activity and P-solubilisation by the bacteria has shown their potential for plant-growth-promoting rhizobacteria. KR-6 (Stenotrophomon asmaltophila) and KR-7(Herbispirillum rubrisubalbicans) have been reported earlier as plant pathogens but they have shown a high potential for nitrogen fixing and auxin producing activity in the present study.

1990) and competitive suppression of plant phytopathogens (Glick, 1995, Park et al., 2004) are the beneficial effects of these microbes on plant.The agricultural soil of South Assam experience heavy annual rainfall, resulting in leaching of nutrients causing economic loss to the farmers (Anonymous, 2007).The use of bio-fertilisers may be an alternative to the chemical fertiliser for achieving sustainable rice farming while improving productivity in rice-agro ecosystem.
The objective of the present study was to isolate, identify and molecular characterisation of diazotrophic bacteria from the rhizosphere of native local varieties of rice from different microhabitats of South Assam, India and characterise them for nitrogen fixation and plant growth promoting activities.

Soil sample collection and isolation of diazotrophs
Ten native rice cultivars grown under different microhabitats in Karimganj districts of South Assam were randomly selected.Riceplant were uprooted from a depth of 0-5cm for rhizospheric soil sampling.The samples were then immediately placed in sterilized plastic packs and sealed brought to the laboratory and stored at a temperature of 4 o C. Ten grams of the rhizospheric soil sample was transferred to a 250ml of Erlenmeyer flask containing 90ml of sterile distilled water and shaken (120rpm) for 30mins.Serial dilution was made and 0.1ml aliquots (10 3 -10 5 ) were spread on plates containing Burk's N-free medium.The plates were incubated for 7 days at 30 0 C. Morphologically different colonies appearing on the medium were isolated and sub-cultured for further analysis.

Phenotypic characterisation of the bacterial isolates
Physiological and biochemical characters of the bacterial isolates were examined according to the methods described in Bergey's Manual of Systematic Bacteriology (Holt et al., 1994).Colour, pigment, form, diameter, elevation, margin, surface, opacity and texture of the colonies of respective isolates were observed.Motility and morphology were examined with the help of phase contrast microscopy.The gram reaction was performed as per standard protocol.Plate assays were performed for determining the starch hydrolysis, carbon source utilisation of the isolates.MR-VP test, indole test, urea hydrolysis test were also performed.

16S rDNA gene amplification and sequencing
Genomic DNA was obtained by using

Acetylene reduction assay (ARA)
Nitrogenase activity of bacterial strains was determined in semisolid nitrogen free malate medium (by using acetylene reduction assay (ARA; HARDY et al. 1973).Pure bacterial colonies were inoculated on to NFM medium in McCartine vials of 10 ml capacity and incubated at 28 ± 2 °C for 48 h.Acetylene gas (10% vol/vol) was injected to the vials.After incubation for 16 h at 28 ± 2 °C, gas samples (100 ìl) were analyzed on a Hewlett Packard gas chromatograph (Model HP 6890, USA) using Porapak-N column and H2 flame ionization detector.After completion of ARA the cell were predigested by adding 10% SDS and sonicated briefly.Protein concentration in the resulting mixture of suspension was determined by Lowry's method. (Lowry et al.,1951).

Indole-3-acetic acid production
IAA production by the isolates was determined by growing the isolates in Burk's medium supplemented with L-tryptophan (100mg/l) at 30 0 C. The supernatant of the culture fluid was obtained by centrifuging the stationary phase cultures at 10,000rpm for 15min and the pH was adjusted to 2.8 with 1N HCL.The auxins from the acidified cultures extracted with the equal volumes of ethyl acetate (Park et al., 2004;Tienet al., 1979) was evaporated to dryness and re-suspended in 4ml of ethanol.Analysis of the samples was done by HPLC (Water series 474, USA) using UV detector on a Nova-Pak 5-ODS C-18 column.Methanol: acetic acid: water (30:1:70 v/v/v) was used as mobile phase at a rate of 0.6mlmin -1 (Rasulet al., 1998).Pure indole-3-acetic acid (Sigma USA) was used as standard.The IAA of the samples was quantified by comparing the retention times, peak areas, and UV absorbance spectra with those of the standard using a Millenium 32 Login software with an interface (Waters, USA) attached with computer.

Phosphorous solubilisation
Test for P-solubilisation was done following Goldstein (1986).The appearance of clearing zone around bacterial colonies after 96h of growth at 30 0 C was used as indicator for Psolubilisation.Plates inoculated with heat killed cells served as control.

ACC deaminase activity
ACC deaminase activity was determined following the method described by Glick et al., 1995.For this 1 µl of each LB pure bacterial culture was inoculated into agar plates containing NFb or NFb-ACC modified by addition of 1-aminocyclopropane-1-carboxylate (5.0 gl -1 ) as unique nitrogen source.Plates were incubated at 28 0 C and observed daily for colony formation for upto 4 days.Colonies were re-inoculated and incubated in the same experimental conditions.Newly colonies formed in NFb with addition of ACC were considered positive for ACC deaminase activity.

Statistical analysis
Results of the measurements were subjected to analysis of variance (ANOVA) using SAS package, Version 8.2 (SAS, 2001).

RESULTS
The isolation and identification of diverse groups of diazotrophs has paved the way to decrease the costs and use of inorganic N-fertiliser as well as minimises the risk of pollution of agroecosystem from the application of chemical fertilisers.Previous researches on isolation of nitrogen fixing bacteria have revealed a broad diversity of diazotrophs associated with different crop rhizosphere (Vessey, 2003).Diverse free living or associative N 2 -fixing microorganisms (aerobes, facultative anaerobes, heterotrophs, phototrophs) grew in wetland rice fields and contributed to soil N. The location, variety of rice plant, soil type and ARA, IAA activity and ACC deaminase activity of different isolates from rhizosphere soil has beenshown in Table 1.Total 25 free living nitrogen fixing bacterial isolates were isolated on Burk's nitrogen free media.The isolates were then purified and sub-cultured on solid nitrogen free medium.
The ability to reduce acetylene was an indicator of nitrogen fixing potential and was specific for monitoring functional nitrogen fixing potential (Andrade et al., 1997).Out of 25 isolates, 11 were selected for their efficiency in nitrogen fixing, having ARA activity , IAA activity and ACC deaminase activity.Among all the strains KR-23 exhibited highest nitrogenise activity (300 nmolC 2 H 4 h -1 mg -1 protein).
Biochemical characterisation of the strains showed that all the strains were gram -ive, motile rods and having varying metabolic activities.The biochemical characteristics of all the isolates are given in Table 2.
From 16S rDNA sequence analysis of the strains it was observed that ninety nine percent sequence identity was observed between the 16S rDNA sequence of KR-4 with Pseudomonas putida; KR-23 and KR-5 with Spingomonas azotifigens; KR-6 with Stenotrophomonas maltophila; KR-16 with Herbispirillum sp.KR-7 with Herbispirillum

DISCUSSION
Plant growth promoting rhizobacteria offer an environmentally sustainable approach to increase crop production and health.The IAA production and ACC deaminase activity of the microbes help in the stimulation of growth and pathogenesis of the plants.In this study all the 11 strains produced considerable amount of IAA and are also capable of ACC deaminase activity which can be comparable to the earlier studies on various bacteria by different workers (Malik et al., 1997;Sucksstorff and Berg, 2003;Park et al., 2004;Xieet al., 2006).The amount of ARA and IAA activity and the capability of ACC deaminase activity is shown in Table 1.Among all the isolates Sphingomonas azotifigens, Pseudomonas putida, Pantoeaagglo merans, Enterobacter cloaceae sub sp.dissolvens, Klebsiella pneumoneae and Herbispirillum sp can solubilise phosphorous.
This study reports the isolation and characterisation of the strains of S.azotifigens, S. maltophila, P. putida, Herbispirillum sp., H. rubrisubalbicans, P.agglomerans, A.xyloxidans, A radioresistance, A. faecalis, E. cloaceae subsp.dissolvens and K. Pneumoneaefrom the inorganic fertiliser rich rhizosphere soil of rice agro-ecosystem of South Assam confirming their nitrogen fixing potential.
Sphingomonas azotifigens is reported from the rice fields of South Assam for the first time in India in our study.All the 11 isolates are capable of IAA production and ACC deaminaseactivity, among them some are capable of P-solubilisation.The isolates Pseudomonas putida, Sphingomonas azotifigens, Herbispirillum sp and has been regarded as PGPR in earlier studies (Gholami, 2009;Park et al., 2004;Baldaniet al., 2009), but Stenotrophomonasmaltophila and Herbispirillum rubrisubalbicans, as producer of IAA and capable of ACC deaminase is also reported first time in our study.These two bacteria are reported as pathogenic and endophytes in many earlier study (Reinhardt et al.,2008;Denton et al.1999; Hale  et al., 1972; Gillis et al., 1990).An increased knowledge of the role of these isolates in plant growth promotion under pot culture as well as field condition is required to prove them as plant growth promoting rhizobacteria.