Isolation and molecular characterisation of diazotrophic growth-promoting bacteria from wheat rhizospheric soils of Punjab.
Soil is a dynamic living matrix, and knowledge of its characteristics is essential to develop optimum land-use plans for maximising and sustaining agricultural production. Soil microbial diversity is an important index of agricultural productivity. The soil microorganisms play an important role in maintaining soil fertility and promoting plant health. The excessive use of agrochemicals has decreased the natural soil microflora, particularly the diazotrophs (Tilak et al. 2005). Among all types of microorganisms present in soil, bacteria are the most abundant. Among various beneficial bacteria, nitrogen fixers and phosphate solubilisers have an important role in plant growth promotion, as they act as biofertilisers. Nitrogen is the key element responsible for enhancing crop production, and plants cannot assimilate it unless it is reduced to ammonia by diazotrophic microorganisms (Galdagi et al. 2002). Biological nitrogen fixation is carried out by diazotrophs, which enable the reduction of atmospheric dinitrogen to ammonium by the nitrogenase enzyme complex.
A combination of phenotypic approaches and genotypic methods proves useful for studying microbial diversity within different genera (Nesme et al. 1995; Clerc et al. 1998). For the evaluation of nitrogen-fixing organisms in the environment, the nif H gene encoding nitrogenase reductase has been used to characterise the phylogenetic heterogeneity (Ueda et al. 1995). A variety of molecular methods are now used for identification of diazotrophs, including nif H gene amplification (Deslippe and Egger 2006) and 16S rDNA (Park et al. 2005) analysis. Molecular markers are considered more useful for the identification of bacterial isolates than are biochemical markers, as molecular markers are not influenced by environmental factors. Therefore, the present study was carried out to isolate diverse groups of potential diazotrophs from wheat rhizosphere for their characterisation using phenotypic and molecular techniques.
Materials and methods
Soil physicochemical properties Soil samples were collected from wheat rhizosphere soil of different wheat-based cropping systems (wheat-rice, wheatmaize, wheat--cotton) of Punjab. The soil sampling site was georeferenced. Plants were carefully uprooted to get the rhizospheric soil. Various physicochemical properties of the soil were determined, such as soil texture (hydrometer method), pH using potentiometric method (Jackson 1973), electrical conductivity (EC) (using Solubridge method; Richard 1954), and organic carbon (using rapid titration method; Walkley and Black 1934). The mineral nitrogen content of the soil was determined by modified Kjeldahl method (Page et al. 1982).
Isolation of diazotrophs
The serial dilution spread-plate technique was used to isolate pure cultures. Eight different nitrogen-free media (Jensen's medium; Burk's medium; nitrogen-free agar (NFA); medium for Klebsiella and Enterobacter (KE); medium for Derxia; medium for Beijerinckia; Dobereiner's medium; LGI) were used for maximum diversity. Pure colonies of bacterial cultures were isolated by subculturing on their respective media.
Morphological and biochemical characterisation Isolated bacterial cultures were characterised morphologically using the methods in Bergey's Manual of Systematic Bacteriology (Holt et al. 1994). Morphological (colour, texture, margins, shape of the colony, and motility) and physiological (Gram, capsule, metachromatic, and spore staining) characterisation was done using standard methods. Biochemical tests such as oxidase, catalase, gelatin liquefaction, urea hydrolysis, methyl red and Voges-Proskauer, starch hydrolysis, indole, nitrate and nitrite reduction, triple sugar iron test (TS1), and HzS production were also performed as per the standard test.
Diazotrophic potential was determined on the basis of nitrogenase activity, i.e. to reduce acetylene to ethylene. The method of Hardy et al. (1968) was used to measure the nitrogenase activity of the isolated cultures. Plant growth-promoting activity is enhanced by a variety of mechanisms such as production of ammonia (Lata and Saxena 2003), synthesis of phytohormones such as indole acetic acid (1AA), and solubilisation of phosphate. Production of IAA was estimated as described by Gordon and Weber (1951), and phosphate solubilisation was determined using the method of Edi-Promono et al. (1996).
Molecular characterisationIsolation of DNA
Total genomic DNA of each isolate was extracted from bacterial cultures grown in SOC broth until late exponential phase (109 cells/mL). To the pellet obtained, TE buffer (567 [micro]L), SDS (30 [micro]L, 10% w/v), and proteinase K (3 [micro]L, 20 mg/mL) was added and incubated for 1 h at 37[degrees]C. The samples were then incubated in extraction buffer of 100 [micro]L of 5 M NaC1 and 80 [micro]L of CTAB for 10min at 65[degrees]C. An equal volume of chloroform/ isoamyl alcohol was then added, mixed gently, and centrifuged (10 000G for 5 min). The upper aqueous layer was transferred to a fresh tube, and isopropanol (0.6 volume) was added and mixed gently until the appearance of a white, thread-like structure. The above solution was centrifuged at 10000G for 5min, supernatant was discarded, and the pellet was washed with 1 mL 70% ethanol, air-dried, and dissolved in 100 [micro]L of TE buffer.
Nif H and 16S rDNA amplification
Bacterial isolates were screened for the nitrogen-fixing capability by the presence of nif H genes responsible for nitrogen fixation. Screening for diazotrophs was done using two nif H primer pairs: nif H1 (forward: 5'-AAGGGCGGT ATCGGCAAGTC-3'; reverse: 5'-GCACGAAGTGGATCAG CTG-3') and ntfH2 (forward: 5'-TCTACGGAAAGGGCGGT ATCGG-3'; reverse: 5'GGCACGAAGTGGATC AGCTG-3'). Polymerase chain reaction (PCR) was conducted in a volume of 15 [micro]L for nif H containing 1 x Taq polymerase buffer (500 mM KC1, 15mM Mg[Cl.sub.2], and 100ram Tris-HC1, pH 9.0), 1.2mM Mg[Cl.sub.2], 0.2 mM dNTPs, 20 pmol of each primer, 1 unit of Taq DNA polymerasc, and 100 ng of template DNA. Finally, one drop of mineral oil was added to each of the reaction solutions. The thermo-cycling conditions for nif H consisted of an initial denaturation step at 95[degrees]C for 5 min followed by 28 amplification cycles of 95[degrees]C for 50 s, 62[degrees]C for 1 min, and 72[degrees]C for 1 min, and a final extension step of 72[degrees]C for 10min.
The PCR amplification of the 16S rDNA of nitrogenase-positive isolates was carried out using a primer pair of forward 5'-AGAGTTTGATCCTGGCTCAG-3' and reverse 5'-GGCTA CCTTGTTACGACTT-3'. The PCR reaction mixture of 50 [micro]L for 16S rDNA consisted of Taq polymerase buffer (1 x), Mg[Cl.sub.2] (1.2 mM), dNTPs (200 [micro]M), each primer (20pmol), Taq DNA polymerase (lU), and template DNA (100 ng). Finally, one drop of mineral oil was added to each of the reaction solutions. The thermo-cycling conditions for 16S rDNA consisted of an initial denaturation step at 95[degrees]C for 5 min; 28 amplification cycles of 95[degrees]C for 50 s, 63[degrees]C for 1 min, and 72[degrees]C for 1 min; and a final extension step of 72[degrees]C for 10 min. PCR products of 16S rDNA were subjected to restriction analysis using three restriction enzymes: HaeIII, Rsa l, and Taql. The restricted PCR products were run on 1.2% agarose gel.
The banding pattern was scored in the binary format and cluster analysis was performed on the basis of the average linkage method, known as the unweighted pair group method using arithmetic averages (UPGMA), by NTSYSpc 2.02 software (Rohlf 1998). Based on various groups obtained in the dendrogram, representative strains exhibiting potential functional traits were given for partial sequencing of 16S rDNA (Bangalore Genei, India) using the same forward and reverse primers as used for amplification, and related sequences obtained from the GenBank database National Centre for Biotechnology Information (NCBI) using BLAST version 2 (Altschul et al. 1990) were aligned and the consensus sequence was computed using tblastx software. The phylogenetic tree was prepared using CLUSTAL W, and the datasets were inferred by the neighbour-joining method of Saitou and Nei (1987).
Soil physicochemical properties
Texture of soil samples varied from loamy sand to sandy loam, pH range was 7.1-8.2, and EC 0.30-0.68 dS/m. Punjab soils are generally deficient in organic carbon, which ranged from 0.06 to 0.48%. Ammoniacal and nitrate nitrogen of soils are important factors which influence the diazotrophic count. In this study, ammoniacal and nitrate nitrogen of the soils ranged from 40 to 100 and 60 to 90 mg/kg, respectively (Table 1). The diazotrophic population was found to be different on the eight different media, with ranges 39-76 x 105cfu/g on Jensen's medium, 35-68 x [10.sup.5]cfu/g on Burk's medium, 30-42 x [10.sup.4]cfu/g on KE, 31-65 x [10.sup.4]cfu/g on NFA, 30-43 x [10.sup.4]cfu/g on LGI, 30-39 x [10.sup.4]cfu/g on Dobereiner's medium, 11 25 x [10.sup.3]cfu/g on the medium for Beijerinckia, and 17-35 x [10.sup.3]cfu/g on medium for Derxia. (Table 2).
Morphological and biochemical characterisation Morphological
On the basis of different morphological, physiological, and biochemical characterisation, 72 diazotrophic isolates were chosen for further study. The cultural characteristics of various isolates varied from transparent to translucent opaque, milky white to creamy, yellow, light pink coloured colonies. Bacterial colonies had differences in their shape on the medium as round, raised with smooth margins, protruded dome-shaped, and a few were of spreading type. Some colonies were mucoid. Most isolates were Gram-negative, motile, cell shapes varying from cocci to rods, and a few were spiral after staining. All isolates were positive for metachromatic granules and none of these showed capsule or endospore formation (Table 3).
The majority of isolates were positive for oxidase and catalase, except two isolates (SK01 and SK03) that were oxidase and catalase negative, respectively. All strains were found to be negative for methyl red and Voges-Proskauer, indole test, gelatin liquefaction, starch hydrolysis, and [H.sub.2]S production, except for SK01 and SK04 strains, which tested positive for gelatin liquefaction, and SK07 positive for methyl red. Only two strains, SK03 and SKI0, were found to utilise starch. The majority of isolates were positive for nitrate and nitrite reduction, urease production, and citrate utilisation, while two strains (SK07 and SK10) tested negative for nitrate and nitrite reduction, whereas SK02 and SK08 were negative for citrate utilisation. Six strains (SK01, SK02, SK03, SK07, SK08, SK09) exhibited variable results for the TSI agar medium test, thereby indicating their ability to ferment different sugars, i.e. sucrose, lactose, and glucose (Table 3).
The experiment on tolerance of variable concentrations of NaCl showed that all isolates were able to grow at 7% NaCl, few at 10% (SK04, SK08, SK10, and SK37), and only two isolates (SK04 and SK08) were able to tolerate 12.5% NaCl.
Different isolates were evaluated for functional characterisation using acetylene reduction assay (ARA) for nitrogen fixation activity, IAA production, phosphate solubilising, and ammonia production. Thirty-seven isolates were positive for ARA, among which 28 isolates exhibited nitrogenase activity ranging from 22.3 to 72.0 nmol [C.sub.2][H.sub.4]/h; the strain SK01 showed the highest nitrogenase activity (72.0 nmol [C.sub.2][H.sub.4]/h). The remaining isolates exhibited low nitrogenase activity. The production of IAA by diazotrophic isolates varied from 11.2 to 23.0[micro]g/mL; isolate SK01 showed maximum (23[micro]g/mL) 1AA production. All isolates produced ammonia in peptone water using Nessler's reagent and colour change in broth, from yellow to brown, which was indicative of ammonia production. From the identified diazotrophic isolates, only five isolates (SK01, SK04, SK07, SK10, SK37) showed the development of sharp phosphate solubilisation zones, ranging from 8 to 11 mm diameter on Pikovskaya's medium. (Table 4).
Molecular characterisation of diazotrophs
Of 72 bacterial isolates, 37 were screened for diazotrophy using nif H1 and nif H2 primers for nitrogenase enzyme. Among these, 32 isolates showed amplification of nif H product resulting in product of 610 bp size. However, a few isolates did not amplify mfH fragment, although they were able to grow on nitrogen-free media and possessed ARA activity. The amplification of diazotrophic isolates using 16S rDNA amplified a fragment of -1500bp. The amplified fragment tbllowed by digestion with three different enzymes, HaeIII, Taql, and Rsa L produced different fingerprint profiles for various isolates (Fig. 1).
The UPGMA ordered the isolates into two major groups and one separate lineage (Fig. 2). Group 1 was subgrouped into two clusters. Cluster I consisted of 12 isolates: SK04, SK11, SK12, SK19, SK20, SK09, SK22, SK21, SK13, SK26, SK08, and SK14. This group was further subclustered into two clusters at 57% similarity. Cluster 11 consists of six isolates (SK17, SK09, SK02, SK27, SK18, SK06), and the isolates were grouped at >85% similarity. Group ll consisted of 17 isolates (SK15, SK25, SK03, SK23, SK36, SK16, SK24, SK34, SK35, SK37, SK07, SK28, SK33, SK01, SK27, SK31, SK32) at 45% similarity and was subclustered into two clusters at 69% and 73% similarity coefficient, respectively. Two isolates, SK10 and SK30, were 100% identical to each other and formed a separate lineage in the dendrogram. Dice's similarity coefficient ranged from a high of 1.00 to a low of 0.45.
The phylogenetic tree of representative cultures is presented in Fig. 3. The identified cultures were found to be closely related to species of genus Stenotrophomonas maltophilia (SK01), Sphingomonas paucimobilis (SK02), Rhizobium larrymoorei (SK03), Agrobacterium tumefaciens (SK04), Stenotrophomonas sp. (SK07), Azotobacter sp. (SK08), Azospirillum sp. (SK09), Xanthomonas oryzae (SKI0), and Pseudomonas aeruginosa (SK37) with 86-99% similarity. The sequences were deposited to the National Bureau of Agriculturally Important Microoganisms, ICAR, UP, India, for accession numbers.
The present investigation was aimed at isolating potential diverse diazotrophic bacteria from wheat rhizosphere soil of the Punjab. The soil physicochemical properties influence the occurrence of microflora in the rhizospheric soil. High ammoniacal and nitrate nitrogen in the soil have negative effect on the diazotrophic population. However, organic carbon had a positive effect on the diazotrophic population. Several factors such as physicochemical properties of soil, stages of plant growth, and root exudates are reported to influence the microbial population and diversity of diazotrophs in the rhizosphere. On the other hand, some cropping systems can have different microbial diversity at different locations (Yohalem and Lorbeer 1994; Dalmastri et al. 1999; Vessey 2003). Microbial diversity is influenced by environmental conditions, soil types, and physicochemical properties, which vary from one place to another place (Grayston et al. 1998). The isolates selected in the present investigation belong to different regions and cropping systems, such as wheat-rice, wheat-maize, and wheat-cotton. However, diversity and efficiency of plant growth-promoting rhizobacteria of diazotrophs were found to be associated with cropping system and not with the location of isolation.
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The ability of diazotrophs to reduce acetylene to ethylene is an indirect measure to access the nitrogen-fixing potential of the isolates (Andrade et al. 1997). Some isolates did not show ARA activity although these isolates were able to grow and subculture luxuriantly on nitrogen-free media (Ozawa et al. 2003). Production of IAA is one of the key factors for stimulating growth of plants. The majority of diazotrophic isolates were capable of producing 1AA, which is known to have pronounced effects on plant growth and development (Glick 2005). In the present study, all nine strains were able to produce 11.2-23.0 [micro]g/ mL of IAA, which is comparable with earlier studies on various bacteria including Pseudomonas and Stenotrophomonas (Suckstorff and Berg 2003).
Different isolates showed variable phosphate solubilisation activity; however, a few isolates could not solubilise inorganic phosphorus. The solubilisation of rock phosphates has been reported to depend on their structural complexity and particle size, as well as on the nature and quantity of organic acids secreted by microorganisms (Gulati et al. 2008). Pseudomonas aeruginosa and Stenotrophomonas maltophilia are also regarded as good phosphate solubilisers and can be used as potential biofertiliser. Similarly, Agrobacterium tumefaciens, Xanthomonas oryzae, and Stenotrophomonas sp. were also capable of solubilising phosphate, which is similar to results obtained by Vessey (2003). Isolate SK01 (Stenotrophomonas maltophilia) was found to be functionally most promising with the highest value for all parameters studied. This isolate showed very good response as a potential plant growth-promoting rhizobacterium under pot conditions (data not shown), although it was isolated from soil with very low organic carbon (0.06%). Therefore, it may prove to be a potential plant growth-promoting bacterium.
Bacterial isolates were screened for the nitrogen-fixing capability by the presence of mfH gene, which encodes for nitrogenase reductase, a key component of the nitrogenase enzyme involved in nitrogen fixation. This enzyme is highly conserved in nature, which makes it an ideal molecular tool to determine the potential for biological nitrogen fixation in different environments (Zehr and Capone 1996). The nitrogenase iron protein gene n/fH is one of the oldest existing functional genes, and the relationships among bacteria based on sequence divergences of this gene have been reported to be in agreement with the phylogeny inferred from 16S rDNA gene sequences (Weisburg et al. 1991 ; Borneman et al. 1996). The ntfH product was 610 bp, in congruence with the results of Normand et al. (1988) and Flores-Mireles et al. (2007).
On the basis of morphological and biochemical characterisation, the isolates were identified as Azotobacter, Azospirillum, Stenotrophomonas, and Pseudomonas. In these soil samples, the Azotobacter population was the most dominant inhabitant, followed by Azospirillum, Stenotrophomonas, and Pseudomonas. Identification of isolates using molecular characterisation based on 16S rDNA partial sequencing revealed the isolates as belonging to different genera: Azotobacter sp., Azospirillum sp., Stenotrophomonas maltophilia, Sphingomonas paucimobilis, Rhizobium larrymoorei, Agrobacterium tumefaciens, Xanthomonas oryzae, and Pseudomonas aeruginosa. Various diazotrophs were isolatedfrom rice roots and its rhizosphere, and were found to belong to the genera Azospirillum, Azotobacter, Flavobacterium, Pseudomonas, Xanthomonas, and Zooglea (Malik et al. 1997). Diazotrophs belonging to the genera Bacillus, Azospirillum, Pseudomonas, and Flavobacterium predominate among the complete bacterial microflora of rice rhizosphere (Velazco et al. 1999).
In conclusion, soil types and conditions significantly influenced the diazotrophic populations. On the basis of biochemical identification, Azotobacter, Azospirillum, Pseudomonas, and Stenotrophomonas were the predominant diazotrophs. The presence of Pseudomonas and Sphingomonas in the rhizoplanes is well known, with reports that the plants may benefit from the association with both species by accelerating root growth and inhibiting growth of some soil pathogens (Adhikari et al. 2001). The majority of diazotrophic strains were capable of fixing atmospheric nitrogen, producing IAA, and some also had the ability to solubilise phosphorous. So, these results clearly indicate that apart from normally encountered rhizosphere microflora Azotobacter sp. and Azospirillum sp., other diazotrophs as Stenotrophomonas, Sphingomonas, Rhizobium, Agrobacterium, Xanthomonas, and Pseudomonas were also found. However, on the basis of molecular characterisation and partial sequencing of 16S rDNA, the isolates were identified as Azotobacter sp., Azospirillum sp., Stenotrophomonas maltophilia, Sphingomonas paucimobilis, Rhizobium larrymoorei, Agrobacterium tumefaciens, Xanthomonas oryzae, and Pseudomonas aeruginosa. Nitrogen fixation is an important process for which potential nitrogen fixers are required in the rhizosphere and can save up to 25-30% in nitrogenous chemical fertiliser. Biofertiliser applications become more relevant for obtaining enhanced production in less fertile soil systems where sufficient nutrients must be provided for vigorous growth and high outputs and where an emphasis on understanding soilplant-microbe interactions governing nutrient acquisition by plants plays a pivotal role. Thus, diazotrophs can be used as potential biofertilisers and can help in improving the soil health as well as plant growth promotion by making available major nutrients as nitrogen and growth-promoting substances.
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This work was supported by Indian Council of Agricultural Research under the AMAAS (Application of Microorganisms in Agriculture and Allied Sectors) project.
Received 9 June 2011, accepted 21 September 2011, published online 20 December 2011
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S.K. Gosal (A,E), G.S. Saroa (B), Y. Vikal (c), S.S. Cameotra (D), Neemisha Pathania (A), and A. Bhanot (A)
(A) Department of Microbiology, Agricultural University, Ludhiana--141 004, Punjab, India.
(B) Department of Soils, Agricultural University, Ludhiana--141 004, Punjab, India.
(C) School of Agricultural Biotechnology, Agricultural University, Ludhiana 141 004, Punjab, India.
(D) Institute of Microbial Technology, Chandigarh, India.
(E) Corresponding author. Email: firstname.lastname@example.org
Table 1. Cropping system, location, and physicochemical properties of soil samples Soil sample/ Latitude Longitude Texture cropping system l. Wheat-maize 31[degrees]45.062 75[degrees]44.681 Loamy sand 2. Wheat-rice 30[degrees]96.970 74[degrees]61.380 Sandy loam 3. Wheat maize 31[degrees]32.627 75[degrees]12.795 Loamy sand 4. Wheat rice 32[degrees]03.080 75[degrees]25.050 Sandy loam 5. Wheat-cotton 31[degrees]52.047 75[degrees]15.148 Sandy loam 6. Wheat-cotton 29[degrees]59.037 75[degrees]20.530 Sandy loam 7. Wheat rice 30[degrees]75.820 75[degrees]7_5.810 Loamy sand Soil sample/ pH EC OC N[H.sub.4.sup.+]-N cropping system (dS/m) (%) (mg/kg) l. Wheat-maize 7.9 0.44 0.06 70 2. Wheat-rice 8.2 0.30 0.13 40 3. Wheat maize 7.7 0.46 0.18 70 4. Wheat rice 7.7 0.64 0.42 100 5. Wheat-cotton 7.7 0.68 0.47 90 6. Wheat-cotton 7.3 0.37 0.48 90 7. Wheat rice 7.1 0.59 0.31 70 Soil sample/ N[O.sub.3] -N cropping system l. Wheat-maize 80 2. Wheat-rice 70 3. Wheat maize 80 4. Wheat rice 70 5. Wheat-cotton 80 6. Wheat-cotton 60 7. Wheat rice 90 Table 2. Diazotrophic populations (x[10.sup.4]cfu/g) from soil samples and different isolates on different nitrogen-deficient media Soil sample Jensen's Burk's KE NFA LGI Dobereiner's 1 530 500 30 47 30 32 2 610 570 34 52 39 35 3 390 350 38 40 36 38 4 400 370 31 32 41 38 5 590 520 36 31 32 30 6 760 680 42 65 43 39 7 570 530 39 49 37 32 Soil sample Beijerinckia Derxia Isolate no. 1 2.0 2.5 SKO1 2 1.5 2.3 SK02, SK08, SK09 3 1.1 1.7 SK03 4 1.7 2.9 SK04 5 1.4 2.2 SK07 6 2.5 3.5 SK10 7 1.8 1.9 SK37 Table 3. Cultural, morphological, and biochemical characteristics of diazotrophic isolates +, Growth; -, no growth; Al, alkaline; Ac, acidic; W, weak SK01 SK02 Cultural Transparent, Transparent, mucoid, mucoid, round, entire, round, entire, raised, large raised, large, dew drop Shape Rods Rods Motility Slow motile Motile Metachromatic + + Catalase test + + Oxidase test - + Methyl red test - Nitrate reduction + + Nitrite reduction + + Citrate utilisation + (W) - Gelatin liquefaction + - Urease production + + Starch hydrolysis - - TSI agar (48 h) Al slant, Al slant, Ac butt Ac butt (w) [H.sub.2]S - - production SK03 SK04 SK07 Cultural Light cream Transparent, Transparent, translucent, mucoid, non-mucoid, mucoid, round, round, highly round, entire, furrowed, sticky, flat, raised raised, large small Shape Spiral Cocci Cocci Motility Motile Motile Motile Metachromatic + + + Catalase test - + + Oxidase test + + + Methyl red test - - + Nitrate reduction + + - Nitrite reduction + + - Citrate utilisation + + + (W) Gelatin liquefaction - + - Urease production + + + Starch hydrolysis + TSI agar (48 h) Al slant, No change Al slant, Ac butt Ac butt [H.sub.2]S - - - production SK08 SK09 SK10 Cultural Transparent, White, non- Transparent, mucoid, mucoid, mucoid, entire, large, entire, small entire, large dew drop like dew drop like Shape Rods Spirillum Rods Motility Motile Motile Motile Metachromatic + + + Catalase test + + + Oxidase test + + + Methyl red test - Nitrate reduction + + - Nitrite reduction + + - Citrate utilisation - + (W) + Gelatin liquefaction - Urease production + + + Starch hydrolysis + TSI agar (48 h) Ac slant, Ac slant, No change Al butt Al butt [H.sub.2]S - - - production SK37 Cultural White, mucoid, entire, medium Shape Rods Motility Motile Metachromatic + Catalase test + Oxidase test + Methyl red test Nitrate reduction + Nitrite reduction + Citrate utilisation + Gelatin liquefaction Urease production + Starch hydrolysis TSI agar (48 h) No change [H.sub.2]S - production Table 4. Functional characteristics of different diazotrophic isolates +, Growth; -, no growth Isolate Acetylene IAA Ammonia Phosphate no. reduction assay production production solubilisation (nmol ([micro]g/mL) zone ethylene/h) (mm diam.) SKO1 72.0 23.0 + 11 SK02 30.1 16.0 + - SK03 22.3 11.2 + - SK04 52.2 13.0 + 9 SK07 39.4 18.4 + 9 SK08 40.8 22.8 + - SK09 35.7 21.3 + - SKlO 30.9 12.8 + 8 SK37 23.7 16.9 + 10