Molecular characterization of diazotrophic bacteria isolated from rhizosphere of wheat cropping system from central plain region of Punjab

electrical conductivity (EC), 0.26 0.77% organic carbon (OC), 14 -119 ppm (ammonical N) and 28 -119 ppm (nitrate N). Variable diazotrophic population was obtained on eight different nitrogen free media. Diazotrophic count was found to be positively affected by OC; whereas, it was negatively affected by pH, EC, ammonical and nitrate nitrogen. A total of 169 diazotrophs were isolated and characterized using cultural, morphological and biochemical techniques and tentatively identified as diverse genera of Pseudomonas sp., Bacillus sp., Azotobacter sp., Rhizobium sp., Azospirillum sp., Beijerinckia sp. and Derxia sp. Using molecular techniques, sixty seven isolates were found to be positive for amplification of nif H. Based on unweighted pair group method with arithmetic mean (UPGMA) clustering, dendrogram was obtained and the representative cultures were identified as Xanthomonas sp., Beijerinckia indica, Flavobacterium johnsoniae, Pseudoxanthomonas suwonensis, Lysinibacillus sphaericus, Stenotrophomonas maltophilia and Pseudomonas aeruginosa.


INTRODUCTION
Soil is a dynamic, living matrix that is an essential part of the terrestrial ecosystem.Soil microorganisms promote physicochemical changes in the soil, such as the stabilization of soil organic matter, nitrogen fixation and other alterations in soil properties necessary for plant growth.Moreover, soil microorganisms are sensitive biomarkers available and most useful for studying microbial diversity in different ecosystem.Bacteria are the most dominant group of microorganisms in the soil and usually equal to half of the microbial biomass in the soil.In view of its global significance in agriculture production and human health, wheat agro ecosystem has been studied extensively from the point of view of bacterial diversity during the last two decades.
Rhizosphere is the region around the root and it is the most active site of microbial activity.Free-living diazotrophs have the potential to improve plant productivity and reduce the use of synthetic fertilizers, thereby sustaining agricultural production (Roesch et al., 2008).Wheat is a major cereal grown in Punjab and is mainly practiced as rice-wheat, cotton-wheat and sugarcanewheat cropping system in which rice wheat is the most common.Therefore, microbial diversity need to be explored from such agro-ecosystems so as to maintain soil and plant health.Now-a-days a variety of molecular based methods are used for characterization and identification of bacteria.These includes amplified ribosomal DNA restriction analysis (ARDRA) (Zhang et al., 2011), random amplified polymorphic DNA (RAPD) (Sikora et al., 1997), repetitive extragenic palindromic elements (REP) (Naik et al., 2008) and 16-23S intergenic spacer (Li et al., 2011).Keeping all these points in view, the objective of the present study was to study the diazotrophic diversity in rhizospheric soil of wheat-based cropping systems of Punjab to ascertain soil health and fertility.

Collection of soil samples
Punjab is situated in the northwest of India.It is bordered by Pakistan in the West, Jammu and Kashmir in the North, Himachal Pradesh in North-east and Haryana and Rajasthan in South.Globally, Punjab is situated at 73° 55'' E to 76° 50'' E longitude and 29° 30'' N to 32° 32'' N latitude.The total area of Punjab is 50362 square kilometers which is 1.54% of the country's total geographical area.The soil samples were collected from wheat based cropping systems viz.rice-wheat, sugarcane-wheat and cotton-wheat cropping systems to analyze diazotrophic diversity from central plain region of Punjab (Table 1).The soil sampling sites were geo-referenced and the rhizospheric soil samples were collected (from different locations in a field and then making a composite sample) from 0-15 cm depth at 45 DAS by carefully uprooting the plants.

Soil physicochemical properties
The soil samples were analyzed for various physicochemical properties viz: soil texture, pH by using potentiometric method (Jackson, 1973), electrical conductivity by solubridge method (Richard, 1954) and organic carbon (OC) was determined using rapid titration method (Walkley and Blacks, 1934).The mineral nitrogen content (both ammonical and nitrate N) of the soil was determined by modified Kjeldahl method (Page et al., 1982).

Isolation of diazotrophic bacteria
The isolation of various diazotrophic bacteria was done using eight different nitrogen free media viz.Jensen's, Burks, nitrogen free agar (NFA), nitrogen deficient medium for Derxia, Beijerinckia, Klebsiella and Enterobacter, LGI medium and Dobereiner's medium (as provided by the manufacturer).Ten grams of the fresh soil was transferred to Erlenmeyer flask (150 ml) containing 90 ml sterile distilled water and was shaken at 120 rpm for 15 min.Serial dilution Pathania et al. 863 spread plate method was used for the isolation of diazotrophic bacteria using 10 -4 and 10 -5 dilutions in triplicates.The Petri plates were incubated for 4-7 days at 28°C.Colonies, which appeared to be morphologically different, were isolated and sub-cultured.

Cultural and morphological characterization
The cultural characterization of the diazotrophic isolates was done on the basis of colony characteristics like colour, pigment, shape, size, diameter, margin, elevation and texture of colony on solid nitrogen free medium.The bacteria were morphologically identified based on Gram's, endospore and metachromatic staining.The motility of the isolates was tested using semi-solid motility test medium.

Biochemical characterization of diazotrophs
The isolates were characterized using standard biochemical methods as given in the Bergey's manual of systematic bacteriology (Brenner et al., 2005).Biochemical tests like nitrate reduction, oxidase, catalase, urea hydrolysis, gelatin liquefaction, starch hydrolysis, citrate utilization, methyl red, Voges Proskauer, indole production, TSI test and H2S production were performed.

Molecular characterization of diazotrophic isolates
The diazotrophic potential of isolates was assessed by amplification of nif H (Ozawa et al., 2003) and amplification of 16S rDNA was done using universal primer pair: Forward: 5'-AGAGTTTGATCCTGGCTCAG-3' and Reverse: 5'-ACGGCTACCTTGTTACGACTT-3' (Weisburg et al., 1991 ).The PCR reaction mixture for nif H and 16S rDNA consisted of a final concentration of 1x PCR product, 1.5 mM MgCl2, 100 µM dNTP's, 0.25 µM forward and reverse primers, 1 U taq polymerase and 100 ng of template DNA.The thermocycling conditions consisted of initial denaturation at 95°C for 5 min, denaturation at 95°C for 50 s, annealing at 62 (nif H) and 63°C (16S rDNA) for 1 min, elongation at 72°C for 1 min followed by 28 cycles and final extension at 72°C for 10 min with a hold at 4°C.All the nif H positive isolates were subjected to restriction analysis of 16S rDNA using three restriction enzymes Hae III, Rsa I and Taq I.The restriction product was run on 1.2% agarose gel containing ethidium bromide (0.5 µg/ml) at 60 V for 4-5 h.The banding patterns were scored in a binary matrix and the data were analyzed using NTSys software (Rohlf, 1998).The UPGMA clustering resulted in the formation of dendrogram and the representative isolates having multiple plant growth promoting traits were identified using partial sequencing of 16S rDNA.The sequences were aligned using BLAST (NCBI) version 2 (Altschul et al., 1990) and the related sequences were used for the preparation of phylogenetic tree (Tamura et al., 2007).

Soil physicochemical properties
Majority of soil samples exhibited loam texture followed by silt loam, sandy loam, loamy sand and clay loam textural classes.The pH of the soil samples ranged from 5.9 -8.7.A maximum value of pH (8.7) was recorded in the soil sample No. 7 and minimum value of pH (5.9) was obtained from the soil sample No. 1.The electrical conductivity of the soil samples ranged from 0.13 -0.51 dSm -1 .Maximum (0.51 dSm -1 ) EC was obtained from soil sample 19 and minimum value (0.13 dSm -1 ) was obtained from the soil sample 23.The organic carbon of the soil samples varied from 0.26 to 0.79%.The OC of the soil sample 8 was found to be maximum (0.79%) whereas, minimum (0.26 %) was obtained from the soil sample 1.
The ammonical nitrogen of the soil samples varied from 14 -119 ppm.The ammonical nitrogen of the soil sample 12 was found to be maximum (119 ppm) whereas, minimum (14 ppm) was obtained from the soil samples 14 and 16.The nitrate nitrogen of the soil samples varied from 28 -119 ppm.The nitrate nitrogen of the soil sample 9 was found to be maximum (119 ppm) and minimum (28 ppm) was obtained in the soil sample 12 (Table 1 and Figure 1).

Cultural, morphological and biochemical characterization
A total of 169 diazotrophic cultures were isolated from 26 soil samples.The morphotypes were selected on the basis of color, shape, size, margin and texture of colonies obtained on nitrogen free media.A total of thirty eight different morphotypes were obtained on eight different types of nitrogen free media (Table 3).The cultural characteristics of morphotypes varied from transparent opaque, transparent, peach/pink transparent, white, cream, yellow, light yellow, brown and orange.Shape of the colonies varied from round to polymorphic, margin from entire to irregular and texture from mucoid to nonmucoid.Majority of isolates ( 22) had transparent, round, small, medium, entire and mucoid texture followed by transparent opaque, round, medium, entire and mucoid morphotypes (20).Morphological characterization of the isolates revealed that majority of isolates were Gram negative rods followed by Gram positive rods, some were coccobacilli while a few were found to be spirillum.Majority of isolates were non endospore formers, while some were positive for endospore formation.Majority of isolates were found to be negative for metachromatic staining.Most of the isolates were found to be motile, while some were found to be nonmotile.Majority of the isolates were found to be positive for oxidase, catalase, citrate, urease, while variable reactions were obtained for MR, VP, indole, gelatin, nitrate reduction and TSIA test.Majority of isolates were negative for pectin, gelatin and H 2 S production. Majority

Molecular characterization
The initial screening of the isolates for diazotrophy was done based on amplification of nif H. Sixty seven isolates were found to be positive for presence of nif H and were by Rsa I (Figure 2).The dendrogram obtained from 67 isolates was clustered into six major groups at 52% similarity.Among these six groups, two were main groups as group I and group II, and other groups as III, IV, V and VI were separate lineages (Figure 3a).The group I was further divided into two subgroups, subgroup 'a' and subgroup 'b' at 63% similarity.The subgroup 'b' was further divided into group 'b1' and 'b2' at 73% similarity.

DISCUSSION
Different textural classes were obtained in the present study and the abundance of these textural classes from central plain region has also been studied by Dahiya (1990).Among the soil properties, soil pH is one of the strong determinants of microbial community structure in soil.For majority of the samples, the values of pH were in normal range which is very important for the growth of bacteria in the rhizospheric region.The electrical conductivity represents the extent of salts present in a soil and the values of EC reported in the present study were under normal range and are in congruence with the results obtained by Jha (2007), who reported variations in EC of soil samples collected from  different agroclimatic regions of Punjab (Table 1).Soils with high clay and organic matter usually have higher nutrient concentrations because there is more total surface area for nutrients to attach (Aimrun et al., 2009).In the present study, different values of percent organic carbon were obtained which is supported by the observations made by Sahoo et al. (2009), who predicted that the range of variation in organic carbon content may be due to soil texture, sediment quality, nature of vegetation, rate of accumulation of dead and decayed part and animal mate-rials.N is one of the major nutrients required for the nutrition of plants however, high values of both ammonical and nitrate nitrogen results in adverse effects on bacterial diversity (Somani, 2005).The microbial diversity is influenced by both environment and choice of enrichment media (Bhromsiri and Bhromsiri, 2010).The highest diazotrophic count was obtained on Burk's medium followed by Jensen's medium whereas, minimum count was obtained on Beijerinckia medium (Table 2).This could be due to preferable source of carbon in Burk's and Jensen's medium whereas, the pH of Beijerinckia medium was low, which might have favoured the growth of bacteria which could tolerate only low pH.In the present study, all the isolates were able to grow and subculture on nitrogen free media.Similar results were obtained by Sgroy et al. (2009), who assessed the nitrogen fixing capability of various isolates based on their ability to grow on N deficient medium and all the isolates were able to grow on nitrogen free medium.Naher et al. (2009) found that the soil diazotrophic populations of seven soil types from Malaysia ranged from 2.3 × 10 4 to 2.2 × 10 6 cfu g -1 soils.Islam et al. (2010) observed that the diazotrophic bacterial counts in Korean soil on four N free media varied from 2.4 log CFU g -1 in JNFb medium to 7.5 log CFU g -1 in BAz medium.The variation in the number of morphotypes obtained from each soil samples may be due to the favorable pH, low EC, ammonical and nitrate nitrogen and high organic carbon which favours the growth of bacteria (Table 3).Moreover, the variation in morphotypes could be attributed to the stage of plant growth at the time of sampling as maximum diversity was obtained from the soil samples collected after 45 days of sowing.The bacterial count is affected by the pH, EC, OC, ammonical and nitrate nitrogen.The bacterial count is related positively with organic carbon whereas, EC, ammonical and nitrate nitrogen had a negative effect on the bacterial count.The bacterial count is found to be maximum at optimum pH and decreases below and above optimum pH (Figure 1a).Organic carbon can serve as an important tool in determining the status of food available to microbes and indicates the extent of fertility for the sustainance of microbes.The microbial load is found to be higher when the organic carbon in the sediments is higher (Sahoo et al., 2009).Naher et al. (2009) reported that the variation in the diazotroph population could be attributed to differences in the soil chemical properties.The diazotrophic count of soil sample number 7 from central plain region was low inspite of having normal EC, medium OC (Figure 1b) and low levels of ammonical and nitrate nitrogen (Figure 1c).The low count can be related with the pH of the soil samples which was slightly towards alkaline range.Maximum diazotrophic count was obtained in the soil samples with normal pH, low EC, high OC and low values of ammonical and nitrate nitrogen (Figure 1).
Based on conventional characterization, the isolates were identified as belonging to the genera Pseudomonas sp., Bacillus sp., Azotobacter sp., Rhizobium sp., Azospirillum sp., Beijerinckia sp., and Derxia sp.The identification of bacteria based on their cultural, morphological and biochemical characteristics has been done by various researchers for different genera such as Azotobacter (Bhatia et al., 2009), Pseudomonas (Selvakumar et al., 2009), Bacillus (Jadhav et al., 2010), Rhizobium (Ghosh et al., 2008), Beijerinckia and Derxia (Lasker et al., 2010), Paeniacillus and Azospirillum (Azlin et al., 2005).Joshi and Bhatt (2011) characterized bacteria using biochemical characterization and identified isolates as Bacillus sp. as the most dominant genera followed by Pseudomonas sp., Serratia sp., Flavobacterium sp., Micrococcus sp., Klebsiella sp., Azotobacter sp., Enterobacter sp., Xanthomonas sp., Staphylococcus sp. and Micrococcus sp.Gram negative bacteria are more predominant than the Gram positive bacteria in the rhizosphere.In the present study also, majority of bacteria were Gram negative with the predominance of Pseudomonas followed by Bacillus and these results find support from the observations made by Bowen and Foster (1978), who stated that the abundance of Pseudomonas in the rhizosphere might be due to the existence of more favorable environmental conditions for their growth.Lilinares et al. (1994) concluded that the predominance of Bacillus could be due to its ability to efficiently use nutrients provided by the plants in the form of exudates and produce substances that inhibit the growth of other microorganisms in their vicinity.
Molecular characterization based on amplification of 16S rDNA revealed the presence of Xanthomonas sp., B. indica, F. johnsoniae, P. suwonensis, L. sphaericus, S. maltophilia and P. aeruginosa.Variation of 16S rDNA allows the inference of the phylogenetic relationship among taxonomically related as well as distinct organisms (Espinosa-Victoria et al., 2009).Based on ARDRA (Figure 2), different groups were obtained in the dendrogram (Figure 3a) and these results find support from the observations made by Chowdhury et al. (2007) who divided the bacteria into various groups based on ARDRA pattern and one representative from each of the 19 groups was selected for cloning and sequencing of 16S rRNA gene so as to compare sequence similarity.Park et al. (2005) revealed that apart from normally encountered rhizosphere microflora Azospirillum, Azotobacter, Herbaspirillum, Klebsiella sp., other species such as S. maltophilia, B. fusiformis and P. fluorescence were encountered.The existence of diverse genera of diazotrophic bacteria have been reported by several researchers (Sgroy et al., 2009;Bhromsiri and Bhromsiri, 2010;Vendan et al., 2010;Palaniappan et al., 2010).In the present study, the bacterial cultures belonged to different groups of alpha proteobacteria, beta proteobacteria and firmicutes (Figure 3b) all these groups of bacteria have already been reported by several researchers (Bharathkumar et al., 2008;Islam et al., 2010).The future prospects include screening different isolates having multiple functional characteristics as plant growth promoting agents and their studies under glass house and field conditions.The exceptionally potential isolates can be further be used and recommended as biofertilizers for different crops.

Figure 1 .
Figure 1.Effect of a) pH, b) EC and OC and c) ammonical and nitrate nitrogen on diazotrophic count on Jensen's media.

Table 1 .
Soil sample collection sites and physicochemical properties of soil samples.

Table 2 .
Diazotrophic bacterial count of soil samples on different nitrogen free media.