Azospirillum Isolates Research Articles

Comparative effectiveness of different isolates of Azospirillum on nitrogen fixation and yield and yield attributing characters of tomato in Chhattisgarh

Among the total six isolates, isolates LAIS-21, LAIK-06 and LAIJ-04 were more potential regarding nitrogen fixation and fixed 19.2 mg, 14.9 mg and 11.0 mg N g-1 malate, respectively. Out of these three isolates, isolate LAIS-21 recorded higher plant height (45.5 cm), number of fruits per plant (36.2), fruit diameter (4.8 cm), fruit weight (57.9), root length (10.3 cm), fresh root weight (23.7 g), yield per plant (2.1 kg) and N content in leaf (58.8 g kg-1), in comparison to other isolates, and also recorded 31.8, 54.7, 23.7, 9.2, 60.9, 23.7, 110.0, 34.6% increase in aforementioned parameters over control under green house conditions. Isolate LAIS-21 also recorded highest yield (808.3 q ha-1) of tomato under field conditions with 92.4% increase over control. Key words: Azospirillum, nitrogen fixation, tomato, yield and yield attributing characters.

Influence of Diazotrophic Bacteria on Antioxidant Enzymes and Some Biochemical Characteristics of Soybean Subjected to Water Stress

Drought stress is an abiotic stress that imposes serious constraints on plants. The present investigation was carried out to determine the inter-relationship between some physiological attributes of soybeans affected by drought stress and pure isolates of Azotobacter and Azospirillum. Drought stress and bacterial application increased catalase and glutathione peroxidase activity, whereas drought stress increased superoxide dismutase activity during the pod-filling stage. Abscisic acid and proline levels increased due to drought stress and bacterial application during the flowering stage, whereas total plant nitrogen was enhanced under well-watered conditions when plants were inoculated with bacteria. The close relationship between enzyme activity and drought stress with bacteria indicated that antioxidant enzymes play an important role in alleviating the detrimental effects of water stress. In addition, the enhancement of abscisic acid and proline could be positively linked with drought stress, and drought-induced abscisic acid could induce proline accumulation and the expression of antioxidant enzyme genes.

EVALUATION OF SELECTED Azospirillum SP. ISOLATES FOR IAA PRODUCTION AND THEIR POTENTIAL IMPACT ON IMPROVING GROWTH, YIELD AND FRUIT QUALITY OF 'ANNA' APPLE TREES

Foliar spray with the diluted PGPR(plant growth promoting rhizobacteria) cultures had utmost importance due to its content and release of stimulants, nutrients, antibiotics, biocides and siderofores. Potentiality of these microorganisms, in plant rhizosphere, in activation and improving of plant growth as will as increasing plant tolerance to different plant biotic and abiotic stresses have been proved. However, microbial types and even microbial strains varied for their potentiality to adapt, inhabit and release of stimulants and phytochemicals. A number of Azospirillum sp. isolates were isolated from phyllosphere of apple, orange, lemon, mango and guava trees, grown at El-Bostan area, Egypt. These isolates were used in spray of apple trees with the dose of 20 and 40 L/feddan compared to water spray control. Foliar spray of apple trees with all Azospirillum isolates notably induced the plant growth and increased fruit yield, but did not significantly affected fruit quality. However, the used isolates largely varied in their efficiency and potentiality. A3 isolate was the superior followed by A7 which attained the highest increases in shoot length, shoot diameter, leaf area, leaf dry weight leaf content of chlorophyll a, b and total chlorophyll as will as fruit yield over those of control treatment. But, the quality parameters, i. e., SSC (soluble solids content), acidity, firmness and color did not show consistent significant variations. The treatments of spray with Azospirillum isolates attained high increase in net return (L E/fed), the spray with the treatments of A7D (isolated from mango phyllosphere and used with the rate of 40 L/fed.) and A7R, which isolated from mango phyllosphere and used with the rate of 20L / fed.) were the superior treatments which achieved increases in net return over that of control (water spray) by 6120 and 6200 L E/fed respectively, followed by A3R (isolated from phyllosphere of apple and applied with 20 L/fed.) which gave 5595 L E/fed. It is noted that the microbial culture dose of 40 L/fed had no significant differences than the application of 20 L/fed. Therefore, we recommend the spray with an efficient Azospirillum isolate like A3 or A7 with the dose of 20 L/fed, and it is also of importance to condense studies on isolation and evaluation of these microorganisms to select the most efficient strains for use, as inoculants, in spray of apple trees.

Population dynamics and identification of efficient strains of Azospirillum in maize ecosystems of Bihar (India)

Information on inoculum load and diversity of native microbial community is an important prerequisite for crop management of microbial origin. Azospirillum has a proven role in benefiting the maize (Zea mays) crop in terms of nutrient (nitrogen) supply as well as plant growth enhancement. Bihar state has highest average national maize productivity although fertilizer consumption is minimum, indicating richness of Azospirillum both in terms of population and diversity in soils. An experiment was planned to generate basic information on Azospirillum population variation in maize soils under different agricultural practices and soil types of Bihar, to identify suitable agricultural practices supporting the target microorganism and efficient Azospirillum strain(s). No tillage, growing traditional maize cultivar, land use history (diara soil having history of maize cultivation), soil organic carbon (>1%) and intercrop with oat supported prevalence of Azospirillum in maize rhizosphere. Native Azospirillum population varied from 1 million to 1 billion/g soil under diverse agricultural practices and soil types. Such richness, however, does not necessarily mean that artificial inoculation of Azospirillum is not required in Bihar soils as 92% of Azospirillum isolates (50 isolates) were poor in nitrogen-fixing ability and 88% were poor on IAA production. Efficient strains of Azospirillum based on growth (three), acetylene reduction assay (three), IAA production (three), broad range of pH (two) and temperature tolerance were identified. The findings suggested that maize crop in Bihar should be inoculated in universal mode rather than site-specific mode.

A rapid and simple PCR method for identifying isolates of the genus Azospirillum within populations of rhizosphere bacteria

To develop a rapid and simple genus-specific polymerase chain reaction (PCR) method for detecting and identifying isolates of the genus Azospirillum which is well-recognized as plant growth-promoting rhizobacterium. Nine pairs of PCR primers were designed based on the Azospirillum 16S rRNA, ipdC, nifA and nifH genes to assess their genus specificity by testing against 12 Azospirillum (from seven species) and 15 non-Azospirillum reference strains, as compared with the fAZO/rAZO pair reported by Baudoin et al. (J Appl Microbiol, 108, 2010, 25). Among the primer pairs assessed, the Az16S-A pair designed on the 16S rRNA gene sequence showed the highest genus specificity: it successfully yielded a single amplicon of the expected size in all the 12 Azospirillum strains and for a close relative, Rhodocista centenaria. The PCR with the Az16S-A primers generated a detectable amount of the amplicon from ≥10³ CFU ml⁻¹ of Azospirillum cell suspensions even in the presence of contaminants and accurately discriminated Azospirillum and non-Azospirillum species in both 35 Azospirillum-like and 70 unknown isolates from plant roots and rhizosphere soils. We developed a rapid and simple PCR method for detecting and identifying Azospirillum isolates within populations of rhizosphere bacteria. The method developed would serve as a useful tool for isolating a variety of indigenous Azospirillum bacteria from agricultural samples.

Rapid detection and identification of the free-living nitrogen fixing genus Azospirillum by 16S rRNA-gene-targeted genus-specific primers

The modern agricultural practice utilizing plant growth promoting rhizobacteria (PGPR) has brought great benefits in the promotion of crop growth. Among PGPR, Azospirillum is considered as an important genus which is not only closely-associated with plants but also shows potential in the degradation of organic contaminants. However, lack of media for selective isolation or techniques for specific detection or identification limit the exploration of these rhizobacteria. This motivated us to design a genus-specific oligonucleotide primer pair which could assist in rapid detection of species of the genus Azospirillum by means of PCR-specific amplification. The sensitivity and specificity of the newly designed primer pair Azo494-F/Azo756-R were tested against 12 Azospirillum type strains and other closely-related genera. The Azospirillum-specific 16S rRNA gene fragment (263bp) was successfully amplified for all the reference Azospirillum species with the designed primer pair. No amplification was noted for closely-related species from other genera. The genus specificity was validated with 18 strains including environmental isolates. Interestingly, two strains assigned earlier as Azospirillum amazonense (DSM 2787(T)) and Azospirillum irakense (DSM 11586(T)) failed to produce an Azospirillum-specific fragment with this primer pair. Further phylogenetic analysis of these two isolates based on 16S rRNA gene sequences shows that these two strains might belong to other genera rather than Azospirillum. Preliminary screening of isolates and soil samples with the Azospirillum-specific primers was successful in terms of the rapid detection of Azospirillum isolates. By using real-time PCR analysis the minimum limit of Azospirillum detection was 10(2) CFUg(-1) in the seeded soil sample. The newly designed primers can be used to study the diversity of Azospirillum in ecosystems and aid in the exploration of novel species.

The Genetic Diversity of Culturable Nitrogen-Fixing Bacteria in the Rhizosphere of Wheat

A total of 17 culturable nitrogen-fixing bacterial strains associated with the roots of wheat growing in different regions of Greece were isolated and characterized for plant-growth-promoting traits such as auxin production and phosphate solubilization. The phylogenetic position of the isolates was first assessed by the analysis of the PCR-amplified 16S rRNA gene. The comparative sequence analysis and phylogenetic analysis based on 16S rRNA gene sequences show that the isolates recovered in this study are grouped with Azospirillum brasilense, Azospirillum zeae, and Pseudomonas stutzeri. The diazotrophic nature of all isolates was confirmed by amplification of partial nifH gene sequences. The phylogenetic tree based on nifH gene sequences is consistent with 16S rRNA gene phylogeny. The isolates belonging to Azospirillum species were further characterized by examining the partial dnaK gene phylogenetic tree. Furthermore, it was demonstrated that the ipdC gene was present in all Azospirillum isolates, suggesting that auxin is mainly synthesized via the indole-3-pyruvate pathway. Although members of P. stutzeri and A. zeae are known diazotrophic bacteria, to the best of our knowledge, this is the first report of isolation and characterization of strains belonging to these bacterial genera associated with wheat.

Azospirillum strains isolated from roots and rhizosphere soil of wheat (Triticum aestivum L.) grown under different soil moisture conditions

The present study deals with the isolation and characterization of Azospirillum strains isolated from roots and rhizosphere soil of wheat (at tillering and anthesis stages) plants grown under different moisture regimes in the field and in pots. The survival of Azospirillum isolates from plants of irrigated field and those from well-watered pots was higher than that of Azospirillum strains isolated from roots and rhizosphere soils of plants grown under arid and semiarid (14–8% soil moisture) field conditions and under water-stressed (8% soil moisture) conditions in pots. On the basis of carbon/nitrogen source utilization, the Azospirillum strains isolated from wheat under field and pot conditions were grouped in three groups. The unweighted pair group method with arithmetic means cluster analysis based on random amplification of polymorphic DNA showed that two groups of Azospirillum were similar. The strains isolated from plants (at tillering stage) grown under low moisture conditions either in pots or in field were genetically similar to strains isolated from plants grown under well-watered conditions in both pots and field. Inoculation of wheat with isolates from water-stressed plants induced tolerance to water stress in inoculated plants. Isolates from water-stressed conditions exhibited lower production of indole acetic acid, gibberellic acid, and trans zeatin riboside but a higher production of abscisic acid.

PRODUCTION OF SOME BIODEGRADABLE POLYMERS BY SOME BACTERIAL ISOLATES

Several bacterial isolates were isolated from Dakahlia and Damitta Governorates. The isolates were purified, identified and tested for PHB production. The tested isolates were 17 isolates of Azotobacter chroococcum, 8 isolates of Azospirillum lipoferum, 22 isolates of Alcaligenes eutrophus, 3 isolates of A. latus, 3isolates of Bacillus subtilis, 2isolates of B. cereus, 5 isolates of B. megaterium, 2 isolates of B. coagulans, 3 isolates of B. polymexa, 4 isolates of B. thuringiensis, 2 isolates of Pseudomonas fluorescens, 2 isolates of P. putida, 2 isolates of P. alcaligenes, 2 isolates of P. aeruginosa, 4 isolates of Rhizobium leguminosarim, 2 isolates of R. meliloti, one isolate of R. japonicum and 12 isolates of Streptomyces albus.Also, the occurrence of alginate in different isolates of Azotobacter and Pseudomonas were investigated. All isolated strains proved to be PHB producers. The amount of PHB produced by Bacillus isolates varied from 0.02 to 0.16 g/l, while by Azospirillum isolates the PHB amounts ranged from 0.12 to 0.58 g/l. Using Rhizobium isolates, the highest amount of PHB (0.36g/l) was produced by R. leguminasarim No 3 , while the lowest amount (0.06 g/l) was obtained by R. leguminosarim No 2. Usig two media, Alcaligenus eutrophus No. 20, seemed to be the most active PHB producer among all Alcaligenes isolates (0.52 g/l). By Pseudomonas isolates, Pseudomonas fluorescens No. 2 produced 0.30g/L of PHB while P. aeruginosa No. 2 produced 0.10 g/L. Generally the isolate of Azotobacter chroococcum No. 16 showed the highest values. The cell dry weight, PHB concentration and the yield of PHB were 2.28 g/l, 0.78 g/l, 34.21%, respectively. And the highest amount of alginate concentration (g/l) was 0.44 by Azotobacter chroococcum No. 14.

Potential allelopathic effects of sunflower (Helianthus annuus L.) on microorganisms

Potential allelopathic effects of sunflower (Helianthus annuus L.) cultivar Hysun 38 on Rhizobium, Azospirillum, and phosphate-solubilizing bacteria (PSB) were studied by applying oxidase, QTS tests and Gram staining. Microorganisms for the control group were isolated from soil sampled before sowing sunflower. A commonly observed effect of allelochemicals was a decrease, compared to control, in the number of colonies of the three groups of microorganisms, isolated from the roots of sunflower in the case of Azospirillum and PSB and from the rhizosphere of sunflower in the case of Rhizobium. Isolates of Azospirillum and PSB from the rhizosphere of sunflower differed in their response: the number of colonies of Azospirillum remained the same as that in control but that of PSB was greater than that in control. QTS tests showed a range of responses, which varied with the group of microorganisms as well as with the habitat from which they were collected, namely roots of sunflower, rhizosphere of sunflower, and control. Allelopathy also influenced the results of Gram staining test but not those of the oxidase test.

Natural occurrence of Azospirillum brasilense in strawberry plants

Azospirillum species are free-living nitrogen-fixing bacteria commonly found in soil and in association with roots of different plant species. For their capacity to stimulate growth they are known as plant growth-promoting bacteria (PGPB). In this work, we demonstrate the natural occurrence and colonization of different parts of strawberry plants by Azospirillum brasilense in the cropping area of Tucuman, Argentina. Although bacteria isolations were carried out from two strawberry cultivars, e.g., Camarosa and Pajaro, attempts were successful only with the cultivar Camarosa. Whereas different strains of Azospirillum were isolated from the root surface and inner tissues of roots and stolons of the cultivar Camarosa, we have not obtained Azospirillum isolates from the cultivar Pajaro. After microbiological and molecular characterization (ARDRA) we determined that the isolates belonged to the species A. brasilense. All isolates showed to have the capacity to fix nitrogen, to produce siderophores and indoles. Local isolates exhibited different yields of indoles production when growing in N-free NFb semisolid media supplemented or not with tryptophan (0.1 mg ml−1). This is the first report on the natural occurrence of A. brasilense in strawberry plants, especially colonizing inner tissues of stolons, as well as roots. The local isolates showed three important characteristics within the PGPB group: N2-fixation, siderophores, and indoles production.

Isolation and characterization of Azotobacter and Azospirillum strains from the sugarcane rhizosphere

Bacteria with the ability to grow on nitrogen-free media and with nitrogenase activity under aerobic or microaerobic conditions were isolated from sugarcane roots collected from four different agricultural locations in Granada (Spain). Isolates were Gram negative rods and were identified as Azotobacter chroococcum and Azospirillum brasilense. Our results suggest that Azotobacter isolates do not have a particular affinity for sugarcane rhizospheres and that, on the contrary, Azospirillum isolates show specific association and perhaps endophytic colonization of sugarcane. However, obligate endophytes (Gluconacetobacter diazotrophicus) were not found in the apoplastic fluid of the stems and macerates extracts of sugarcane tissues with the procedure applied. Population of this microorganism might be in low number in the Spanish sugarcane varieties studied which is also discussed.

Impact of the use of biofertilizers on cotton ( Gossypium hirsutum ) crop under irrigated agro-ecosystem

High nitrogen fixing, phosphate solubilizing, phytohormones producing isolates of Azotobacter, Azospirillum, Acetobacter and Pseudomonas were used as inoculants for cotton. Important cultures were selected on the basis of their effect on root/shoot length and chemotactic behaviour. Selected bioinoculants were earlier tested for their beneficial properties like nitrogen fixation (ARA), ammonia excretion, IAA production etc. These bio-inoculants were further tested for phosphate solubilization property. Various chosen strains were tested with Desi (HD 123) and American (H 1098) cotton under field conditions (as for wheat). Plant height and boll weight were determined at the time of harvesting whereas survival rate of inoculated bacteria was determined after 30, 80 and 130 days respectively. In the year 2000–01, on the basis of boll number and boll weight plant−1 AVK 51 (36; 76.2 g plant−1), HT 57 (27; 56.9 g plant−1), AC18 (33; 61.5 g plant−1), Ala 27 (36; 61.4 g plant−1) and Pseudomonas (34; 71.3 g plant−1) were identified as significant both for American and desi cotton varieties. Highest survival rate was observed with Mac 68 (33.4 × 105) followed by HT54 (31.5 × 105) after 30 days of sowing, which decreased after 80 days and remained constant up to 130 days. This trend was observed with all the cultures. Similar results were observed in 2001–02. 25 kg ha−1 N saving was observed with A. chroococcum (AVK51) bioinoculant for cotton crop.

Perchlorate-reducing microorganisms isolated from contaminated sites.

An extensive microcosm survey of perchlorate-contaminated sites was undertaken to assess the ability of indigenous microorganisms to degrade perchlorate. Samples from 12 contaminated sites and from one pristine location were analysed. Perchlorate was degraded to below detection limit in all electron donor-amended microcosms. Perchlorate-reducing microorganisms (PRMs) were numerous at most of these sites. Sixteen distinct PRMs were isolated that were phylogenetically related to either Dechloromonas in the Beta Proteobacteria (9/16 isolates) or to Azospirillum in the Alpha Proteobacteria (7/16 isolates). The majority of previously isolated PRMs are in the Beta Proteobacteria related to Dechloromonas or Dechlorosoma. This study indicates that PRMs of the genus Azospirillum may be more prevalent at contaminated sites than the current record of isolates suggests. Cell yields, electron donor to perchlorate ratios and maximum specific growth rates were similar among the isolates and similar to the few previously published values. However, the Monod half-saturation constants for perchlorate for the two Azospirillum isolates characterized were lower than those measured for other genera, suggesting that they may be more effective at low concentrations of perchlorate. These results extend the current understanding of PRMs from diverse environments and provide added confidence that microbial perchlorate reduction is ubiquitous, even at highly contaminated sites, and can be harnessed effectively for bioremediation.

Phylogenetic and physiological characterization of indigenous azospirillum isolates in Thailand

We isolated indigenous Azospirillum bacteria in Thailand and analyzed the physiological characteristics and phylogenetic properties of the 16S rDNA sequences. Azospirillum bacteria were isolated from the rhizosphere soil and surface-sterilized root samples of nonleguminous plants, such as sugarcane, vetiver grass, wild rice, sweet potato, maize, and cultivated rice, grown in Thailand. Nitrogenase activity evaluated by the acetylene reduction activity (ARA) and indole-3-acetic acid (IAA) production enabled to classify the Thai isolates into two groups: Group I, which showed a high lAA production, and Group II, which showed a low ARA and lAA production. Phylogenetic analysis of the 16S rDNA sequences revealed that the 12 isolates in Group I were closest to Azospirillum largimobile, with 97 to 99% homology values, and they were also close to Azospirillum brasilense. The 16S rDNA sequence of TS18 in Group II was distant from all seven known species in the genus Azospirillum, indicating that TS18 may belong to a new species of Azospirillum. Analyses of other physiological characteristics, such as colony morphology, biotin requirement, carbon utilization, and acid formation, also revealed the existence of specificity and diversity among the Thai Azospirillum isolates.

The effect of combined Azospirillum inoculation and nitrogen fertilizer on plant growth promotion and yield response of the blanket flower Gaillardia pulchella

The investigation was carried out to examine the performance of Azospirillum isolates on growth and N uptake of Gaillardia pulchella with two nitrogen levels. Azospirillum strain OAD-2 inoculation significantly increased plant height, number of leaves per plant, branches per plant and total dry mass accumulation in G. pulchella than other inoculations and/or uninoculated control. The application of 150 kg N ha −1 further enhanced the above plant growth parameters significantly over application of 112 kg N ha −1. The N uptake of G. pulchella was enhanced due to Azospirillum strain OAD-2 inoculation, which was further augmented in the presence of nitrogen. The combination of Azospirillum strain OAD-2 and 150 kg N ha −1 showed the highest N uptake at 120 DAT. Plant growth and yield parameters also increased when inoculated with the non-diazotrophic, but efficient IAA producing, Azospirillum strain OAD-57. From this investigation, it can be concluded that Azospirillum strains OAD-2 and OAD-11 can play an important role in the N nutrition of G. pulchella.

Isolation and selection of indigenous Azospirillum spp. from a subtropical island, and effect of inoculation on growth of lowland rice under several levels of N application

Thirty-five Azospirillum strains (13 strains from plant roots and 22 strains from soils) were isolated from Ishigaki island, Japan, which has a subtropical climate. These strains were different from each other according to polymerase-chain-reaction band patterns obtained by using a random primer (OPT-08). Two Azospirillum strains (AZ43 and AZ92-2) were also examined for use in further experiments. Inoculation of lowland rice with these strains enhanced early growth of rice to various degrees. Inoculation of strains VIII.P1-2, AZ92-2, V.S2-2, and V.P5 in sterilized soil yielded higher shoot dry weights than the application of 90 μg N g–1 soil without inoculation. Only inoculation with strains AZ92-2 and VIII.P1-2 caused higher N uptake than the application of 90 μg N g–1 soil. Three strains were selected for the next experiment based on the results of their effect on the early growth of rice. An investigation was conducted to determine the ability of two indigenous Azospirillum strains (V.S2-2 and VIII.P1-2) and one stock strain (AZ92-2) to promote growth and nutrient-uptake of lowland rice in unsterilized soil under several levels of N application (0, 80, 160, and 240 mg N pot–1). Inoculation with these strains without N application increased shoot dry weight by 12–15% compared to the uninoculated treatment. Inoculation with Azospirillum V.S2-2 together with the application of 160 mg N pot–1 resulted in a shoot dry weight as high as that obtained in the treatment with 240 mg N pot–1 without inoculation. Thus, in this former case, the amount of N applied could be reduced by 80 mg pot–1 due to the effect of the microbial inoculum without a significant change in the high, targeted, yield.

Variation in Nitrogen Fixing Ability among Natural Isolates of Azospirillum

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.

Isolation of Azospirillum spp. from natural soils by immunomagnetic separation

An immunomagnetic separation (IMS) technique was developed to isolate the nitrogen-fixing bacterium, Azospirillum, from soil. Samples were reacted with polyclonal antibodies (pAb) raised in rabbits against whole-cell antigens of a mixture of five strains of Azospirillum brasilense. Following reaction with pAb, samples were treated with immunomagnetic beads (IMB: magnetic beads coated with anti-rabbit IgG antibodies), to allow capture of antibody-coated Azospirillum cells. The IMB were trapped by means of a magnet to permit washing for removal of unbound bacteria and were plated onto Congo Red agar (CRA) to allow growth of azospirilla. The method was optimized by incubating and washing samples in a 10 ml glass test-tube instead of a 1 ml plastic Eppendorf tube and by incubating the CRA plates in plastic bags to control the composition of the gas phase and moisture. Azospirillum spp. were isolated from the rhizospheres of wheat plants grown in soil from seven different regions in New South Wales (NSW). The IMS procedure is more efficient than culturing in nitrogen-free semi-solid medium (Nfb) for detecting and counting azospirilla in natural soils.

Compost extract added to microcosms may simulate community-level controls on soil microorganisms involved in element cycling

Interactions among populations of soil microorganisms might alter soil microenvironments sufficiently to allow populations collectively to inhabit sites which individually they could not inhabit. We tested the hypothesis that soluble microbial products in soil microenvironments mediate commensalistic interactions among populations involved in N 2-fixation, denitrification, sulfate reduction and P solubilization. We measured the growth of bacteria in microcosms amended with sterile compost extract. Of the 7 Azospirillum isolates tested, 3 fixed more N 2 when amended with 50 μg compost extract-C ml −1 medium. Fixation of N 2, by one isolate amended with NH 4Cl or compost extract decreased with increasing concentrations (0.15–15 μg N ml −1 medium) of NH 4Cl, but not with increasing concentrations of compost extract. Optical density of cultures of Bacillus sp. increased 6-fold with addition of 11 μg compost extract-C ml −1 medium under denitrifying conditions. Adding 6 μg compost extract-C ml −1 medium stimulated the growth of all 10 sulfate-reducing enrichment cultures, and three did not grow without compost extract. Addition of 10 μg compost extract-C ml −1 medium, however, increased microbial-P in only one of the 10 cultures in P-limiting medium. This evidence is consistent with the hypothesis that exchange of growth factors among populations in microenvironments contributes to control of microorganisms involved in element cycling.