South Wales, Australia, using an immunomagnetic separation procedure which does not select strains according to their nitrogen-fixing ability. By combining ...
MICROBIAL ECOLOGY Microb Ecol (1998) 36:193–201 © 1998 Springer-Verlag New York Inc.
Variation in Nitrogen Fixing Ability among Natural Isolates of Azospirillum S.O. Han, P.B. New Department of Microbiology, Faculty of Agriculture, University of Sydney, New South Wales 2006, Australia Received: 23 July 1997; Accepted: 4 December 1997
A
B S T R A C T
A total of 285 strains of Azospirillum were isolated from soils from seven geographic regions in New South Wales, Australia, using an immunomagnetic separation procedure which does not select strains according to their nitrogen-fixing ability. By combining amplification and restriction analysis of 16S rDNA (ARDRA) patterns with serological, morphological and biochemical results, we found that almost all isolates were A. brasilense and A. lipoferum. There was wide variation in the nitrogenase (acetylene reduction) activity of isolates grown in nitrogen-free, semisolid medium, with differences in average activities between regions. Isolates with zero or negligible nitrogenase activity were found in samples from only two regions, one of which had two out of 26 strains with no activity. Representative isolates, having the highest, the lowest, and intermediate nitrogen fixation rates for each site, were used to inoculate the roots of wheat plants in a model system. Most of the isolates, in association with wheat roots, reduced between 1 and 5 nmol C2H4 ⭈ mg dry root−1 ⭈ day−1, but certain strains gave considerably higher activities. The rank order of nitrogen fixation activity on wheat roots did not correlate well with that of nitrogen fixation in pure culture; some strains that fixed nitrogen vigorously in pure culture had low rates of fixation on roots, and vice versa. This inconsistency could not be explained by variations in the root colonizing ability of different strains. However, isolates of A. lipoferum had a higher average nitrogenase activity than A. brasilense, both in Nfb medium and in association with wheat roots. The majority of the most active nitrogen fixers were A. lipoferum. When wheat plants were inoculated with mixtures of two or four strains, nitrogen fixation rates were generally between the rates for the component strains when inoculated individually. There was no benefit from using mixtures of different strains.
Introduction Bacteria of the genus Azospirillum are widely distributed in soil and are associated with the roots of forage grasses, ceCorrespondence to: P.B. New
reals, and non-gramineous plants [4]. Nitrogen fixation by Azospirillum has been of interest for many years; beneficial responses of crops to inoculation with this bacterium have been reported [5]. While there is growing evidence that most yield increases are due primarily to hormone production by the bacteria, there is still the possibility that azospirilla con-
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tribute some fixed nitrogen to inoculated crops [4]. However, there is no information available on the variation in nitrogen-fixing ability of Azospirillum strains in natural soils. Other studies on the physiology and genetics of nitrogen fixation have been confined to only a few ‘‘typical’’ strains [27, 29]. This study investigates the variation in nitrogen-fixing rates among members of natural populations, using Azospirillum strains isolated by immunomagnetic separation (IMS) [16], a method which does not bias selection towards strains with high nitrogen-fixing ability.
S.O. Han, P.B. New ing both local and overseas isolates), and with none of 9 strains of soil bacteria belonging to Agrobacterium, Alcaligenes, Azotobacter, Bacillus, Citrobacter, Corynebacterium, Pseudomonas, Rhizobium, and Sphingomonas species. In the IMS procedure, aliquots of soil samples were incubated with pAb, then treated with immunomagnetic beads (IMB: Dynal AS, Oslo, Norway) coated with anti-rabbit IgG antibodies. The IMB with attached cells of Azospirillum were trapped by a magnet and plated on Congo Red differential medium (CRA) [24]. Isolates were characterized by a combination of serological tests (rocket immunoelectrophoresis [28], agglutination, and fluorescent antibody staining), cell morphology (wet mount and Gram stain), and biochemical properties (catalase, oxidase, and cultural properties in Nfb semi-solid medium [19]). Amplification and restriction analysis of 16S rDNA (ARDRA) was also performed [3, 15].
Materials and Methods Isolations Soils sampled from seven sites in New South Wales, Australia (Fig. 1), between 1993 and 1995 were placed in pots and sown with wheat (Triticum aestivum L. cv. Morocco). Pots were maintained with adequate water in a glasshouse at 25°C. Azospirillum strains were isolated from the rhizospheres of the plants by IMS, using a polyclonal antiserum (pAb) prepared against a mixture of five local strains of Azospirillum brasilense [16]. The antiserum cross-reacted with all strains of azospirilla tested (18 of A. brasilense, 8 of A. lipoferum, 1 of A. amazonense, and 1 of A. halopraeferens, compris-
Bacterial Strains and Growth Conditions Bacterial strains obtained from culture collections are listed in Table 1. Azotobacter chroococcum was grown on Winogradsky agar [8]; all other cultures were maintained on peptone–yeast-extract agar or broth (PYE; composition in g ⭈ L−1: peptone 10, yeast extract 5, NaCl 5, pH 7.2).
N2 Fixation by Pure Cultures A standardized procedure was used for acetylene reduction assays. Bacteria were grown to exponential phase (∼36 h) at 30°C, in PYE medium, on a rotary shaker (150 rpm; 2.5 cm radius of rotation), and harvested by centrifugation (2800 × g, 30 min). After washing twice by centrifugation and resuspension in sterile saline (0.85 g ⭈ L−1 NaCl), the bacteria were resuspended in the original volume of sterile, distilled water. Aliquots (0.1 mL) were used to inoculate 10 ml Nfb medium in 28 ml, narrow-neck, McCartney (NN) bottles. Following incubation (unshaken) for 48 h at 30°C, the bottles were sealed with rubber serum stoppers (Thomas, USA). The gas phase in the head space was replaced with a mixture of acetylene, air, and nitrogen (10:10:80, v/v), giving a reduced partial pressure of oxygen (∼2%). Cultures were incubated for 20–24 h. Ethylene production was then measured by gas chromatography. After completion of the acetylene reduction assay, the bacterial cells were evenly mixed through the Nfb medium using a vortex mixer. The protein concentration was then determined by a modified Lowry method [20], using bovine serum albumin as standard.
Plant Studies
Fig. 1. Map indicating field sites from which samples were obtained. NSW State capital city Sydney (S) and the regional centers (north to south) Narrabri (N), Tamworth (T), Breeza (B), Warren (W), Dubbo (D), Condobolin (Cb), and Camden (C) are shown.
Wheat seeds (Triticum aestivum L. cv. Morocco) were disinfected by soaking them in 70% ethanol for 1 min, followed by 1% sodium hypochlorite (w/v) for 20 min. They were then rinsed six times with sterile, distilled water. Seeds were pregerminated for 2 d on water agar plates and checked for microbial contamination. After germination, seedlings were planted, one or two per bottle, at a depth of
Variation in Nitrogen Fixing Ability of Azospirillum Table 1. Bacterial strains Species Azospirillum brasilense
Azospirillum lipoferum Azospirillum amazonense Azospirillum halopraeferens Agrobacterium tumefaciens Alcaligenes eutrophus Azotobacter chroococcum Bacillus sp. Citrobacter freundii Conglomeromonas parooensis Corynebacterium sp. Pseudomonas putida Sphingomonas paucimobilis
Strain Sp7T Fp10* 576 582 586 592 593 Sp59b 635 LMG 7108T 24 JMP134 IP2
Source UQM 1774,a [26] P. Dartb
This laboratory, [22]
ATCC 29707c This laboratory
609 611
LMGd A. Kerre J.M. Pembertonf Institut Pasteur, Paris, France This laboratory This laboratory
ACM 2042 634 PaW85
L.I. Sly,g [9] This laboratory V.M. Kõivh
Sp1443
J.O. Ka,i [17]
195 was replaced with the acetylene/air/nitrogen mixture, and acetylene reduction was determined after 44–48 h incubation, in the dark, at 30°C. In preparation for ethylene analysis, the bottles were thoroughly shaken; the gas phase was mixed, using a 20 ml syringe in order to equilibrate the head space with soil-trapped ethylene. Bottles without acetylene were also included for the assay of endogenous ethylene production. Viable counts of bacteria on roots were determined by the Miles and Misra [21] method after completion of the acetylene reduction assay. Wheat plants were carefully removed from the vermiculite and shaken gently to remove most of the vermiculite attached to the roots. Roots were cut off, added to 10 ml FS1⁄5, vigorously mixed for 30 sec (vortex mixer), and drops of tenfold dilutions were added to CRA. Dry weights of roots were determined after drying at 80°C.
a
University of Queensland Culture Collection, Qld, Australia Dr. P. Dart, Research School of Biological Sciences, Australian National University, Canberra, Australia c The American Type Culture Collection (ATCC), Rockville, MD, USA d Laboratorium Microbiologie Rijksuniversiteit, Gent, Belgium e Prof. A. Kerr, Waite Agricultural Research Institute, University of Adelaide, S.A., Australia f Dr. J.M. Pemberton, Dept. of Microbiology, University of Queensland, Australia g Dr. L.I. Sly, University of Queensland Culture Collection, Qld, Australia h V.M. Kõiv, Institute of Molecular and Cell Biology, University of Tartu, Estonia i Dr. J.O. Ka, Dept. of Agricultural Biology, Seoul National University, Seoul, Korea * Sp7 nifA nalr strr T Type strains b
0.5 cm, in 4 g sterile vermiculite (Kenyan exfoliated vermiculite, average particle diam >4 mm) contained in 50 ml NN bottles (Fig. 2). The vermiculite moisture content was made equivalent to field capacity (3.4 ml ⭈ g−1 dry wt vermiculite) with 1⁄5-strength Fåhraeus solution (FS1⁄5) [13]. After plants had grown for 3–4 d, the vermiculite in each bottle was inoculated with 1 ml of a bacterial suspension containing about 106 cells ⭈ ml−1. Inocula were produced by growing isolates in NfbN medium at 30°C {Nfb medium without agar plus (NH4)2SO4, 0.1 ⭈ g L−1} until late exponential phase (∼40 h). The cultures were washed twice by centrifugation and resuspended in the original volume of FS1⁄5. The bacterial suspension was autoclaved for treating control plants. Plants were grown in a light chamber, with a light–dark cycle of 14 h (27°C)/10 h (22°C). Each bottle was weighed every 2–4 d and evaporative losses were replaced with sterile, distilled water. After 14 days’ growth, the bottles were sealed (B, Fig. 2), the gas phase
Fig. 2. Schematic of an inoculated plant bottle with two wheat seedlings. (A), Growth stage; (B), Sealed for acetylene reduction assay.
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Table 2. Properties of soil samples Source
Textural class
pH
Crop history
Sampling date
Number of isolates tested
Average N2ase activitya
Breeza Camden Condobolin Dubbo Narrabri Tamworth Warren
sandy clay sandy loam sandy clay loam loamy sand sandy clay loam clay clay
7.2 6.5 4.9 5.0 7.4 7.0 7.6
corn/sorghum grass pasture wheat wheat wheat/cotton wheat wheat/cotton/bean
Sept. 1993 Sept. 1995 Nov. 1993 Jan. 1994 May 1994 Aug. 1995 Aug. 1995
48 40 5 24 44 30 94
62.1 (3.7) 65.8 (4.4) 41.5 (7.2) 12.5 (2.4) 57.9 (3.0) 79.9 (5.1) 30.1 (1.7)
a
Mean of all isolates from a site (nmol C2H4 ⭈ mg protein−1 ⭈ h−1) (±standard error of mean)
Acetylene Reduction Assays (ARA) The amount of ethylene was determined by injecting 0.2 ml gas samples into a gas chromatograph (Varian model 3700) equipped with a Porapak T column (1.83 m × 3 mm) and a flame-ionization detector [23].
Results Isolations Azospirillum spp. were isolated by IMS from the rhizosphere of wheat plants grown in soil from seven regions of NSW (Fig. 1). Isolation data for the different soil samples are given in Table 2. All strains were identified as belonging to the genus Azospirillum by a combination of serological tests, biochemical properties, and cell morphology; representative isolates from each soil were further identified by ARDRA.
Identification by ARDRA 16S rDNA was amplified from 39 representative isolates having the highest, lowest, and intermediate nitrogen fixation rates in pure culture for each of six sites. DNA was also prepared from known strains representing four species of Azospirillum (A. brasilense, A. lipoferum, A. amazonense, and A. halopraeferens) and nine species of other soil bacteria (Table 1). Restriction endonuclease AluI was chosen to cut the amplified DNA on the basis of its previous use [15] to distinguish Azospirillum species. The ARDRA patterns of known members of the four Azospirillum species were typical for each species and distinct from those of the other soil bacteria (Fig. 3). With two exceptions, all representative isolates produced ARDRA patterns typical of either A. brasilense or A. lipoferum (Fig. 3). The remaining two strains, C6539 and W4002, could not be distinguished from the other isolates by
cell or colony morphology, serology, or biochemical reactions, but could not be allocated to the four tested Azospirillum species on the basis of their ARDRA patterns. Isolates from three of the six geographical regions each belonged to a single species: Breeza—A. brasilense, Narrabri—A. brasilense, and Tamworth—A. lipoferum. The majority of the isolates from Camden were A. lipoferum (5 of 6); the majority from Dubbo were A. brasilense (7 of 9); Warren strains were equally divided between both species (3 A. lipoferum, 4 A. brasilense). There was no significant correlation between the proportions of different species and the soil textural class or the soil pH of the sampling site (Spearman’s rank correlation test).
Nitrogen Fixation in Pure Cultures Standard conditions that were optimal for all the first strains tested (all Breeza, Dubbo and Narrabri isolates) were chosen to compare nitrogen fixation rates in pure cultures. It is possible that the conditions were suboptimal for some strains. Although 48 h cultures of most strains would be in the stationary phase, pre-incubation for 48 h gave higher rates of acetylene reduction and we observed much less variation in growth between strains than after 24 h preincubation. There was a wide range of variation in nitrogenase activity among the 285 different isolates tested (0.0 to 154.9 nmol C2H4 ⭈ mg protein−1 ⭈ h−1), with differences between regions in average activities of isolates (12.5 to 79.9 nmol C2H4 ⭈ mg protein−1 ⭈ h−1) (Figs. 4 and 5). Only two regions, Dubbo and Warren, had any isolates with zero or negligible nitrogenase activity. Isolates from Dubbo had the lowest average nitrogenase activity (12.5 nmol C2H4 ⭈ mg protein−1 ⭈ h−1); 4 out of 26 isolates had negligible activity, and two were completely unable to reduce acetylene. Dubbo isolates also exhibited the lowest variation in nitrogenase
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Fig. 3. ARDRA patterns following agarose gel electrophoresis of amplified 16S rDNA of 39 Azospirillum isolates digested with restriction endonuclease AluI. (A), known strains: A. brasilense Lanes 1–18, strains Sp7, 576, 582, 586, 592 and 593, A. lipoferum Sp59b, A. amazonense 635, A. halopraeferens LMG 7108, Agrobacterium tumefaciens 24, Alcaligenes eutrophus JMP134, Azotobacter chroococcum IP2, Bacillus sp. 609, Citrobacter freundii 611, Conglomeromonas parooensis ACM 2042, Corynebacterium sp. 634, Pseudomonas putida PaW85 and Sphingomonas paucimobilis Sp1443, respectively. (B), isolates of Azospirillum from different regions: Lanes 1–5, Breeza strains B1007, B1301, B1406, B1420 and B1426; lanes 6–11, Camden strains C6501, C6505, C6510, C6521, C6530 and C6539; lanes 12–20, Dubbo strains D0601, D0606, D0613, D06S2, D08S3, D1103, D1201, D1204 and D2001; lanes 21– 26, Narrabri strains N1607, N1609, N1621, N1623, N1804 and N1904; lanes 27–31, Tamworth strains T6208, T6209, T6223, T6224 and T6230; lanes 32–39, Warren strains W4002, W4008, W4021, W4043, W4058, W4068, W4079 and W4099 respectively. Numbers indicate the size of some bands of the molecular mass markers (DNA molecular weight marker VIII, Boehringer, Mannheim).
activity compared with isolates from other regions (Table 2). However, the highest average nitrogenase activity (79.9 nmol C2H4 ⭈ mg protein−1 ⭈ h−1) in Nfb medium was obtained for the Tamworth isolates. They also had one of the highest variations. The strain with the highest nitrogen fixation rate was isolated from this soil. This strain (A. lipoferum T6208) grows poorly on CRA and in Nfb medium, and probably would not have been isolated by conventional methods.
Nitrogen Fixation in Association with Wheat The nitrogenase activity in association with wheat roots was investigated using most of the strains identified by ARDRA. These were representative isolates having the highest, lowest, and intermediate nitrogen fixation rates in pure culture for each of six sampling sites. It was found that the association
reduced acetylene only under microaerobic conditions. Acetylene reduction was detected after 48 h of incubation, and was strictly dependent on live bacteria. The nitrogenasenegative mutant, Fp10, showed no activity in the association. Endogenous production of ethylene was also negligible. For most strains, the acetylene-reducing activity of the association was between 1 and 5 nmol C2H4 ⭈ mg dry root−1 ⭈ day−1, but certain strains showed considerably higher activities (Fig. 5). The rank order of nitrogen fixation activity on wheat roots did not correlate well with that of nitrogen fixation in pure culture (Spearman’s rank correlation coefficient = 0.202, p = 0.26). In some cases, strains that had high rates of nitrogen fixation in pure culture had low rates of fixation on roots, and vice versa. For example, the nitrogenase activity of strain W4021 was very low in pure culture (0.2 nmol C2H4
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nmol C2H4 ⭈ mg dry root−1 ⭈ day−1) (Fig. 6). A high nitrogen fixation rate in pure culture (>110 nmol C2H4 ⭈ mg protein−1 ⭈ h−1) was achieved by 24% of identified isolates of A. lipoferum, but not by any identified A. brasilense. Effect of Mixed Inoculations
Fig. 4. Individual isolates of Azospirillum arranged in order of nitrogenase activity in pure culture. Values are the means of three replicates (pooled standard error of the means, 18.0 nmol C2H4 ⭈ mg protein−1 ⭈ h−1). The isolates were obtained from soil from seven locations: B, Breeza; C, Camden; Cb, Condobolin; D, Dubbo; N, Narrabri; T, Tamworth; W, Warren. Strain Fp10, a nitrogenase negative mutant of A. brasilense Sp7, showed no ethylene formation in the medium.
⭈ mg protein−1 ⭈ h−1), but on wheat roots it was at least as high as the most active strains from four other sites. The inconsistency between results of nitrogenase activity in pure culture and those on wheat roots could not be explained by variations in the root-colonizing ability of different strains. For most strains, populations colonizing the rhizosphere lay within the range 0.75–2.3 × 108 cfu ⭈ mg dry root−1 (Fig. 5). Rhizosphere nitrogenase activity per cfu was not correlated with nitrogenase activity in pure culture (Spearman’s rank correlation coefficient = 0.084, p = 0.64). The best strain from Warren, A. lipoferum W4079, exhibited the highest nitrogenase activity on wheat roots and was the best rhizosphere colonizer. The next highest nitrogen fixation rates were on wheat roots in conjunction with isolates from Tamworth having the highest and intermediate rates in pure culture; these strains had average root-colonizing ability. Nitrogen Fixing Ability of A. brasilense and A. lipoferum Isolates of A. lipoferum exhibited a higher average nitrogenase activity compared to A. brasilense, both in Nfb medium (67.6 compared with 39.2 nmol C2H4 ⭈ mg protein−1 ⭈ h−1) and in association with wheat roots (4.3 compared with 2.0
In order to determine the effect of using mixed inocula, wheat seedlings were inoculated with the same total number of cells (∼106 ⭈ bottle−1) of four strains applied either in pairs (in a 1:1 ratio, in all combinations), all together (in a ratio of 1:1:1:1) or individually. All but two combinations gave nitrogen fixation rates that were intermediate between the rates for the component strains when inoculated individually (Table 3). In the combination D06S2 + W4021, the rate was less than that of the least active strain (D06S2), though not significantly so. The other exception was the combination W21 + W79, for which the rate was higher than that of either strain. The difference from the more active strain (W79), however, was not significant. The use of combined inocula gave no significant improvements in numbers of azospirilla colonizing the wheat roots. Concentrations were the same as those achieved by the component strains when inoculated alone, or intermediate between them (Table 3).
Discussion Following Do¨bereiner et al.’s [12] first report on the ecology of Azospirillum, all natural isolates of Azospirillum have been reported as able to fix nitrogen. Our studies using IMS have, for the first time, made it possible to study nitrogen fixation rates in natural populations of azospirilla without bias towards those strains which grow and fix nitrogen more vigorously in laboratory culture media. We have found substantial variation in the nitrogen fixation rate. Although most Azospirillum strains were able to fix nitrogen, the rate of fixation varied among the members of the population at each sampling site (Fig. 4). Large differences in nitrogenase activity were also observed between different regions. The Tamworth isolates exhibited the highest, and the Dubbo isolates the lowest, average nitrogenase activity. Only 2 out of 285 isolates lacked detectable nitrogenase activity (Fig. 4). These, plus several isolates with almost negligible activity, were concentrated in only two of the seven regions sampled. One was the Dubbo region, where they were recovered at high frequency. Thus, it appears that the proportion of nonnitrogen-fixing Azospirillum strains is very low, or zero, in most soil ecosystems.
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Fig. 5. Comparison of nitrogen fixation rates (acetylene reduction) by representative isolates, both in pure culture (A) and in association with roots of wheat seedlings (B). Bacterial colonization of wheat roots (C) is also shown. The isolates are grouped according to isolation site (B, Breeza; C, Camden; D, Dubbo; N, Narrabri; T, Tamworth; W, Warren), and are arranged within sites in order of nitrogenase activity in pure culture. Values are the means of at least three replicates. Error bars represent the standard errors of the means.
Assuming that there was no inherent bias toward one or another species in the IMS isolation technique, an assumption that seems reasonable given the broad specificity of pAb for all species of Azospirillum, there was no relationship between species distribution and soil textural class or pH. However, it was noticeable that the strains with the lowest nitrogen fixation rates (Dubbo isolates) were associated with a light-textured, low pH soil (Table 2). This soil, near the lower limit of pH for growth and nitrogen fixation by most Azospirillum strains [22], has presumably selected specialized strains with reduced nitrogen-fixing ability. The differences between the regions in nitrogen-fixing ability may be related to geographic variables, including soil type. Survival of inoculated A. brasilense in soil has previously been shown to be unaffected by pH, but to be positively correlated with percentage of clay, and negatively correlated with percentage of
sand [7]. Our results, however, show no obvious effect of soil texture or pH on the species distribution of Azospirillum. To assess the potential of Azospirillum as a crop inoculant, it is important to determine the extent to which the bacteria become established in the rhizosphere, and how well they fix nitrogen in that environment. Many investigators have attempted to quantify the colonization of host roots by specific strains of azospirilla [1, 6, 18, 25], but none has previously studied the variation of root colonization and nitrogen-fixing ability in a range of representative natural isolates. The results from inoculation of wheat plants under controlled conditions indicate that the nitrogenase activity of the Azospirillum–plant association is dependent on the isolate used, but cannot be predicted from rates of nitrogen fixation in pure culture. Some strains exhibited poor acetylene reduction on wheat roots, despite having performed
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Table 3. Effect of single, dual, or mixed inoculations of four strains of Azospirillum on nitrogenase activity and root colonization of wheat seedlings Inculated bacteria
Speciesa
Nitrogenase activityb
Root colonizationc
D06S2 T6208 W4021 W4079 D06S2 + T6208 D06S2 + W4021 D06S2 + W4079 T6208 + W4021 T6208 + W4079 W4021 + W4079 All 4 strains
bra lip bra lip bra + lip bra + bra bra + lip lip + bra lip + lip bra + lip 2 × (bra + lip)
0.34 (0.03)ab 1.00 (0.02)ef 0.66 (0.17)cde 0.87 (0.06)def 0.91 (0.10)def 0.14 (0.04)a 0.55 (0.07)bc 0.48 (0.05)bc 0.88 (0.05)def 1.06 (0.08)f 0.66 (0.03)cd
0.35 (0.04)abcd 0.48 (0.04)bcde 0.51 (0.08)bcde 0.59 (0.09)de 0.10 (0.04)a 0.58 (0.07)cde 0.76 (0.03)e 0.27 (0.02)ab 0.61 (0.12)de 0.51 (0.03)cde 0.28 (0.03)abc
a
bra, A. brasilense; lip, A. lipoferum Log10(1 + nmol C2H4 ⭈ mg dry root−1 ⭈ day−1) c Log10(1 + cfu ⭈ mg dry root−1 × 10−6) Values are the means of three replicates (standard errors of the means in brackets); values in the same column which share the same letter in the superscripts are not significantly different (p > 0.05), according to Tukey’s HSD method b
quite well in pure culture. The best of the isolates at fixing nitrogen in the wheat rhizosphere were from the Tamworth and Warren regions. These isolates also colonized the wheat roots efficiently. A. lipoferum strains T6208 and W4079 may be particularly suitable for inoculating wheat plants to improve yield in nitrogen deficient soils, especially those with a low proportion of good nitrogen-fixing strains.
In the experiment with mixed inocula, nitrogenase activity and root colonization were not significantly enhanced by combining two or more strains in the inoculum. Although a number of workers have reported stimulation of wheat growth using mixtures of Azospirillum strains [10, 14], there have been few plant inoculation studies involving comparisons between single and mixed strains of Azospirillum. None has involved measurement of nitrogen fixation rates. Mixtures of two [2] or four strains [11] caused increases in plant dry weight and total N content of wheat and foxtail millet, respectively, that were generally intermediate between those of the best and worst strains in the mixtures. Our results also suggest that there is no advantage in nitrogen-fixing ability to be gained from mixing Azospirillum strains.
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2. Fig. 6. Frequency distribution of nitrogen-fixing ability among representative isolates of A. brasilense (A and C) and A. lipoferum (B and D) in pure culture (A and B, nmol C 2 H 4 ⭈ mg protein−1 ⭈ h−1), and in association with roots of wheat seedlings (C and D, nmol C2H4 ⭈ mg root dry wt−1 ⭈ day−1). A, average nitrogenase activity ± standard error; N, total number of strains tested; curved line, normal curve fitted to the data {analyzed by the computer graphic statistics utility StatView娂 (BrainPower Inc., Calabasas, Calif. USA)}.
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