Vol. 8(9), pp. 862-871, 26 February, 2014

DOI: 10.5897/AJMR2013.5948

ISSN 1996-0808 ©2014 Academic Journals

http://www.academicjournals.org/AJMR

African Journal of Microbiology Research

Full Length Research Paper

Molecular characterization of diazotrophic bacteria isolated from rhizosphere of wheat cropping system

from central plain region of Punjab

Neemisha Pathania1, S. K. Gosal1*, G. S. Saroa2 and Yogesh Vikal3

1Department of Microbiology,

Punjab Agricultural University, Ludhiana, Punjab, 141004, India.

1Soil Science Department, Punjab Agricultural University, Ludhiana, Punjab, 141004, India.

3School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana, Punjab, 141004, India.

Accepted 24 September, 2013

Soil is a hot spot for microbial diversity, however, the excessive use of agrochemicals have reduced natural microflora of soil. Soil samples were collected from central plain region of Punjab and geo-referenced. Physicochemical properties of the soil samples ranged from 5.9-8.7 (pH), 0.13-0.51 dSm

-1

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. Key words: Biochemical characterization, diazotrophic count, nitrogen fixing bacteria, physicochemical properties, nif H, 16S rDNA.

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.

*Corresponding author. E-mail: skgosal@rediffmail.com. Tel: +91-161-2401960. Ext: 330. Fax: +91-161-2400945.

Rhizosphere is the region around the root and it is the most active site of microbial activity. Free-living dia-zotrophs 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 sugarcane-wheat 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 iden-tification 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. MATERIALS AND METHODS 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).

RESULTS

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

864 Afr. J. Microbiol. Res.

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

Location Texture pH

EC

(dSm-1)

OC

content

(%)

Mineral nitrogen content

(ppm)

Longitude Latitude Ammonical Nitrate

75°48'34.329'' 30°45'15.045'' Sandy Loam 5.9 0.14 0.26 70 90

75°51'53.327'' 31°57'12.181'' Sandy Loam 7.1 0.36 0.75 56 35

75°49'59.935'' 30°44'37.127'' Loamy Sand 6.5 0.17 0.47 70 84

75°45'40.752'' 30°45'30.425'' Loamy Sand 7.1 0.29 0.31 70 90

76°12'25.942'' 30°50'26.679'' Loam 7.7 0.42 0.68 68 56

75°53'07.321'' 30°31'49.252'' Loamy Sand 7.5 0.38 0.59 84 98

75°28'07.812'' 30°46'47.075'' Loam 8.7 0.28 0.67 63 70

75°40'16.518'' 30°48'30.166'' Loamy Sand 7.0 0.20 0.79 77 63

75°41'07.916'' 30°50'11.844'' Sandy Loam 7.6 0.34 0.70 84 119

75°28'07.812'' 30°46'47.075'' Loam 8.3 0.39 0.56 63 98

75°36'18.131'' 31°05'49.028'' Sandy Loam 7.7 0.27 0.47 28 49

75°20'27.771'' 31°34'15.043'' Silt Loam 7.9 0.42 0.56 119 28

74°40'20.859'' 31°42'43.826'' Silt Loam 7.6 0.21 0.32 112 42

75°13'42.375'' 31°33'06.346'' Silt Loam 7.3 0.40 0.69 14 98

74°41'29.121'' 31°21'10.358'' Loam 7.1 0.14 0.45 70 49

74°55'07.591'' 31°39'50.06'' Clay Loam 7.0 0.31 0.77 14 77

74°56'56.894'' 31°40'50.156'' Clay Loam 7.2 0.43 0.35 91 42

74°48'49.143'' 31°29'43.588'' Loam 7.5 0.32 0.53 56 35

74°38'20.393'' 31°11'51.161'' Loam 7.9 0.51 0.45 70 42

75°13'58.770'' 31°20'13.607'' Loam 7.0 0.38 0.61 112 84

75°48'28.762'' 31°18'33.879'' Clay Loam 7.6 0.41 0.53 47 77

75°48'38.784'' 31°21'26.215'' Loam 6.6 0.35 0.57 91 105

75°47'28.070'' 31°19'39.651'' Clay Loam 7.6 0.13 0.66 98 42

75°49'20.120'' 31°19'08.132'' Silt Loam 7.4 0.26 0.61 72 49

75°25'12.604'' 31°09'49.653'' Silt Loam 7.1 0.43 0.68 21 70

75°40'00.831'' 30°06'34.387'' Loam 7.0 0.31 0.53 91 58

EC: Electrical conductivity, OC: organic carbon.

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). Diazotrophic count on nitrogen free media The maximum count of diazotrophic bacteria from central

plain region on eight different media was 730 ×104 CFU

g-1

on Jensen’s N free media, 800 × 104 CFU g

-1 on Burks

N free media, 79 × 104 CFU g

-1 on nitrogen free agar

(NFA), 61 × 104 CFU g

-1 on Klebsiella Enterobacter

Nitrogen deficient media, 51 × 104

CFU g-1

on nitrogen deficient media for Derxia, 25×10

4 CFU g

-1 on nitrogen

deficient media for Beijerinckia, 57 × 104

CFU g-1

on Dobereiner’s medium and 52 × 10

4 CFU g

-1on LGI

medium. The maximum value of bacterial count (800 × 10

4 CFU g

-1) was observed on Burks’s medium. The

minimum count of diazotrophic bacteria among eight different media was 270 × 10

4 CFU g

-1 on Jensen’s N free

media, 250 × 104 CFU g

-1 on Burks N free media, 24 ×

104 CFU g

-1 on Nitrogen free agar (NFA), 22 × 10

4 CFU g

-

1 on Klebsiella Enterobacter Nitrogen deficient media, 2 ×

104

CFU g-1

on nitrogen deficient media for Derxia, 0.1×10

4 CFU g

-1 on nitrogen deficient media for

Beijerinckia, 20 × 104

CFU g-1

on Dobereiner’s medium and 25 × 10

4 CFU g

-1on LGI medium. The minimum value

of bacterial count (0.1 × 104 CFU g

-1) was obtained on

Pathania et al. 865

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(b)

(c)

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zotr

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zotr

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il)

Am

mo

nic

al a

nd

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rate

N

itro

gen

(pp

m)

Soil samples

Ammonical Nitrogen Nitrate Nitrogen

Figure 1. Effect of a) pH, b) EC and OC and c) ammonical and nitrate nitrogen on

diazotrophic count on Jensen’s media.

Beijerinckia nitrogen deficient medium (Table 2). 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

866 Afr. J. Microbiol. Res.

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

Nitrogen free media (×104CFU/g) Bacterial

isolates Jensen’s Burk’s NFA KE Derxia Beij Dob LGI

430 310 33 50 35 05 39 32 09

620 630 44 30 38 11 25 31 12

570 450 38 61 38 15 33 35 13

680 700 60 29 36 15 33 49 06

440 330 39 29 24 25 44 45 02

340 420 41 27 15 13 32 33 05

270 280 26 30 27 11 32 39 02

650 690 60 54 35 17 57 42 06

450 470 50 39 22 03 37 44 05

350 380 39 28 03 0.1 38 41 05

580 500 55 32 24 05 39 35 05

310 290 24 41 12 02 20 27 06

300 350 35 22 32 01 28 33 05

650 710 70 46 51 0.1 33 34 05

720 660 69 61 45 0.1 42 41 03

460 560 65 45 11 05 42 44 04

610 550 63 41 35 03 56 47 04

710 640 79 57 31 0.1 36 39 03

580 550 53 39 15 01 48 51 04

290 340 32 35 02 0.1 36 25 08

650 750 62 49 30 01 42 37 09

280 250 24 30 23 13 29 32 14

380 400 36 33 30 0.1 43 38 07

360 400 41 26 33 0.1 49 52 09

730 800 68 37 33 0.1 44 38 15

460 440 48 28 30 0.1 36 33 03

Total 169

NFA: Nitrogen free agar medium; KE: Klebsiella Enterobacter nitrogen deficient medium, Beij: Beijerinckia, Dob: Dobereiner’s.

from entire to irregular and texture from mucoid to non-mucoid. 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 non-motile. 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 H2S production.

Majority of Pseudomonas sp. were cream to off white in

colony colour, Gram negative non endospore forming rods, and positive for MR reaction, H2S production, urea hydrolysis, amylase production, catalase activity and citrate utilization whereas, negative tests were obtained for nitrate reduction, VP reaction and indole. The genus Bacillus exhibited cream/opaque colonies, Gram positive endospore forming rods positive for catalase, starch, urea, casein and gelatin hydrolysis tests. Rhizobium was characterized based on creamy colonies, Gram nega-tive and positive tests for catalase, VP, indole production, nitrate reduction whereas, negative test for citrate utilization. Azotobacter was identified on the basis of dew drop colonies, Gram negative rods with positive reactions for oxidase, catalase, urease and nitrate reduction. The genus Beijerinckia was identified based on their cultural characteristics as formation of brown pigment with aging, highly furrowed and mucoid colonies having positive oxidase, catalase, urease and nitrate reduction tests. Identification of genera Derxia was done on the basis

Pathania et al. 867

Table 3. Cultural characteristics (morphotypes) of various diazotrophic isolates.

Cultural characteristics No. of

isolates

Transparent slightly opaque, round, medium, entire margin, mucoid 20

Transparent, round, small, medium, entire margin, mucoid 22

Transparent, round, large, entire margin, mucoid 11

Transparent, polymorphic, small, entire margin, mucoid 05

Transparent, polymorphic, medium, entire margin, mucoid 06

Transparent, polymorphic, large, entire margin, mucoid 05

Transparent, polymorphic, small, irregular margin, mucoid 04

Transparent, polymorphic, medium, irregular margin, mucoid 03

Transparent, polymorphic, large, irregular margin, mucoid 02

Transparent, round, medium, Irregular margin 02

Peach/pink transparent, round, small/medium, entire, mucoid 03

Peach yellow, round, small, entire margin, non mucoid 03

Peach/pink cream round, small, entire, mucoid 06

White, round, small, entire margin, mucoid 03

White, round, medium, entire margin, Mucoid 06

White fragile, round, small/medium, entire /irregular margin, non mucoid 03

White/cream, round, small, entire margin, non mucoid 04

White/cream, round, medium, entire margin, non mucoid 02

White/cream, round, large, entire margin, non mucoid 01

White/cream transparent, round, small, entire margin, mucoid 03

White/cream transparent, round, medium, entire margin, mucoid 04

Cream/white transparent, round, small, entire margin, non mucoid 01

Cream/white transparent, round, medium, entire margin, non mucoid 03

Cream/white transparent, round, large, entire margin, non mucoid 02

Cream/white transparent, round, medium, entire margin, mucoid 07

Cream/white transparent, round, large, entire margin, mucoid 02

Cream, polymorphic, small, entire margin, mucoid 01

Cream, polymorphic, medium, entire margin, mucoid 01

Cream yellow, round, medium, entire margin, non mucoid/mucoid 01

Curdy, polymorphic, large, irregular margin, mucoid 04

Yellow transparent, round, small, entire margin, mucoid 01

Yellow transparent, round, medium, entire margin, mucoid 08

Yellow transparent, round, large, entire margin, mucoid 01

Light yellow, round, small/medium/large, entire margin, non mucoid 02

Light yellow, polymorphic, small/medium/ large, entire margin, mucoid 11

Yellow, round, small/medium, entire margin, mucoid/ non mucoid 02

Yellow brown, round, medium, entire margin, mucoid 01

Brown, polymorphic, large, entire margin, non mucoid 03

Total number of Isolates 169

of highly mucoid colonies having positive reaction for oxidase whereas, negative reactions for catalase, nitrate reduction and indole production. Azospirillum was identified mainly based on their growth on Dobereiner’s medium and highly motile spirillum shaped cells. Based on observations of cultural, morphological and bioche-mical characteristics, the isolates were tentatively identified as diverse genera of: Pseudomonas sp. (30%), Bacillus sp. (29%), Azotobacter sp. (24%), Rhizobium sp.

(9%), Azospirillum sp. (3%), Beijerinckia sp. (2%), and Derxia sp. (1%). 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

868 Afr. J. Microbiol. Res.

Figure 1

Figure 2

a)

b)

c)

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

29

1632 bp

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

29

1632 bp

1632 bp

Figure 2. Restriction analysis of diazotrophic isolates with a) Hae III, b) Taq I and c) Rsa I.

assigned numbers CPR1-CPR67. The digestion carried with restriction enzymes resulted in different fingerprinting profiles and the number of fragments formed after restriction varied from 2-5 by Hae III, 2-6 by Taq I and 2-5 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. The group II was further divided into two subgroup ‘a’ and subgroup ‘b’ at 64 and 66% similarity, respectively. The representative cultures were identified as Xanthomonas sp., Beijerinckia indica, Flavobacterium johnsoniae, Pseudoxanthomonas suwonensis, Lysinibacillus sphaericus, Stenotrophomonas maltophilia, and

Pseudomonas aeruginosa (Figure 3b). 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

Pathania et al. 869

(a)

(b)

Similarity Coefficient

0.15 0.36 0.57 0.79 1.00

CPR1 CPR7 CPR11 CPR17 CPR21 CPR42 CPR8 CPR19 CPR59 CPR32 CPR13 CPR12 CPR18 CPR2 CPR3 CPR46 CPR31 CPR55 CPR54 CPR38 CPR63 CPR47 CPR48 CPR6 CPR15 CPR25 CPR30 CPR50 CPR44 CPR56 CPR66 CPR37 CPR62 CPR26 CPR36 CPR52 CPR67 CPR49 CPR4 CPR27 CPR16 CPR23 CPR39 CPR24 CPR40 CPR28 CPR57 CPR58 CPR5 CPR51 CPR29 CPR10 CPR9 CPR45 CPR61 CPR14 CPR22 CPR43 CPR33 CPR20 CPR64 CPR34 CPR60 CPR35 CPR41 CPR53 CPR65

Figure 3. a) Dendrogram based on UPGMA clustering of diazotrophic isolates; b) Phylogenetic tree of

diazotrophic isolates.

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-

870 Afr. J. Microbiol. Res. 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 environ-ment 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 attri-buted 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, morpholo-gical 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 pro-moting 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. REFERENCES

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