Gram-positive bacterial flora on the root surface of wheat (Triticum aestivum L.) grown under different soil conditions

We identified 108 Gram-positive bacterial strains isolated from the root surface of wheat grown under different soil conditions. The strains were divided into four groups based on morphological and physiological characteristics, but most appeared to be coryneform. The taxonomic position of the various groups was verified by the guanine+cytosine DNA contents of the strains. In general, the ranges of these values agreed with those described for the respective taxonomic positions in the literature, with a few exceptions. With soil improvement the distribution of the various groups on the root surface changed, with the coryneform group becoming dominant. This group was further divided into five subgroups, according to cell wall components, cellulose-decomposition, and morphological characteristics, and were identified to genus level. The distribution of these subgroups on the root surface of wheat did not alter with soil improvement. The genus Arthrobacter, the dominant subgroup, predominated in every plot.     

<Emphasis Type="Italic"> Gluconacetobacter diazotrophicus</Emphasis>: a natural endophytic diazotroph of Nile Delta sugarcane capable of establishing an endophytic association with wheat

Gluconacetobacter- like diazotrophs were encountered as dense populations inside the root and stem tissues of sugarcane cultivated in ancient agricultural fields of the Nile Delta. Counts of >10⁵ cells g^-1 were recorded in root and stem samples. The leaves contained a smaller population (<10³ g^-1). The typical dark-orange colonies which developed on LGIP agar plates were purified. Identification was performed with the API microtube systems: API 20E for Enterobacteriaceae and API 20NE for non-Enterobacteriaceae. API profiles of the local isolates were closely related to those of the type culture Gluconacetobacter diazotrophicus (ATTC 49037). The isolates successfully reduced C₂H₂ and produced appreciable amounts of ethylene in the presence of cane juice. This suggested that the local isolates are closely related to the type strain G. diazotrophicus. Wheat seedlings were inoculated with a number of isolates under gnotobiotic conditions. Both optical and scanning electron microscopy showed that endophytic Gluconacetobacter spp. were present in all the samples tested. They were observed in apparently intact and enlarged epidermal root cells, and also in stem tissues, indicating that the bacterium was able to migrate upward into the shoot tissues. Although Gluconacetobacter inoculation did not stimulate the growth of the cereal plant, the results obtained are particularly interesting because this bacterial species was capable of colonizing the internal tissues of wheat, not considered a natural host until now.     

Survival of bacteria introduced into soil by means of transport by Lumbricus rubellus

Four strains of bacteria, Rhizobium leguminosarum biovar trifolii, Pseudomonas fluorescens, Pseudomonas cepacia, and Flavobacterium sp., were introduced into loamy sand and then transported by earthworms of the species Lumbricus rubellus to uninoculated soil. Cell densities recovered from the earthworm gut and casts (both expressed per gram dry material) were significantly lower (up to 3 log units) than cell densities recovered from the inoculated soil. Total bacterial counts in casts were similar to those in the inoculated soil. In casts excreted into a sterile environment numbers of colony-forming units (CFU) increased, suggesting a favourable environment for growth. In casts excreted in a non-sterile environment, cell densities of introduced strains decreased. Casts therefore did not offer the introduced bacteria a protective micro-environment for survival in the bulk soil. Transport by worms of R. leguminosarum biovar trifolii and of P. fluorescens appeared to occur mostly by means of cast production; with the Flavobacterium sp. and P. cepacia a large proportion of the cells was possibly transported on the skin of earthworms.     

苹果树腐烂病拮抗细菌鉴定及其抑菌作用效果测定

为了开发一种高效低毒的苹果树腐烂病生防制剂,通过对峙培养法、形态学及分子生物学的方法进行了苹果树腐烂病菌拮抗菌的分离筛选及鉴定,采用离体枝条法测定了拮抗菌对苹果树腐烂病的防效,并采用显微观察和液体培养法分别研究了拮抗菌对苹果树腐烂病菌的抑菌机理和无菌滤液对苹果树腐烂病菌生长的影响。分离筛选结果表明,从甘肃省各苹果产区果园土壤和苹果树枝条上分离得到23株细菌,2株对苹果树腐烂病菌具有较好拮抗作用,分别为LZ-1201和TS-1203,其对苹果树腐烂病菌菌丝生长抑制率分别为79.00%和85.00%。鉴定结果表明,菌株LZ-1201和TS-1203分别为枯草芽孢杆菌(Bacillus subtilis)和解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。离体枝条防效测定表明,拮抗菌无菌滤液对苹果树腐烂病的防效随着稀释倍数增大而降低,原液防效最高,分别为74.43%和77.07%。抑菌作用机理结果表明,两株拮抗菌均可导致苹果树腐烂病菌丝膨大畸形、原生质浓缩、释放及溶解。拮抗菌无菌滤液对腐烂病菌生长的影响测定结果表明,无菌滤液对腐烂病菌分生孢子萌发和菌丝生长量均有显著抑制作用(P0.05),其无菌滤液稀释40倍时对腐烂病菌分生孢子萌发和菌丝生长量的抑制率均高于60%,表明该拮抗菌具有很好的生防潜力。     

Nodulation competition among Bradyrhizobium japonicum strains as influenced by rhizosphere bacteria and iron availability

Summary Bacteria isolated from the root zones of field-grown soybean plants [Glycine max (L.) Merr.] were examined in a series of glasshouse experiments for an ability to affect nodulation competition among three strains of Bradyrhizobium japonicum (USDA 31, USDA 110, and USDA 123). Inocula applied at planting contained competing strains of B. japonicum with or without one of eleven isolates of rhizosphere bacteria. Tap-root nodules were harvested 28 days after planting, and nodule occupancies were determined for the bradyrhizobia strains originally applied. Under conditions of low iron availability, five isolates (four Pseudomonas spp. plus one Serratia sp.) caused significant changes in nodule occupancy relative to the corresponding control which was not inoculated with rhizosphere bacteria. During subsequent glasshouse experiments designed to verify and further characterize these effects, three fluorescent Pseudomonas spp. consistently altered nodulation competition among certain combinations of bradyrhizobia strains when the rooting medium did not contain added iron. This alteration typically reflected enhanced nodulation by USDA 110. Two of these isolates produced similar, although less pronounced, effects when ferric hydroxide was added to the rooting medium. The results suggest that certain rhizosphere bacteria, particularly fluorescent Pseudomonas spp., can affect nodulation competition among strains of R. japonicum. An additional implication is that iron availability may be an important factor modifying interactions involving the soybean plant, B. japonicum, and associated microorganisms in the host rhizosphere.Paper No. 10648 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601, USA     

Effects of chitin amendment of soil on microorganisms,nematodes, and growth of white clover (Trifolium repens L.) and perennial ryegrass (Lolium perenne L.)

Effects of soil amendment with crabshell chitin on the growth of white clover (Trifolium repens L.) and perennial ryegrass (Lolium perenne L.), and on populations of soil bacteria, fungi, and plant-parasitic and free-living nematodes were investigated in a pot trial. Five soil samples were collected from Te Puke (Paengaroa Shallow Sand, a Typic Hapludand) and five from Hamilton (Bruntwood silt loam, an Aquic Hapludand), New Zealand. Subsamples of each soil were either amended with chitin or unamended and planted with white clover and ryegrass. The ryegrass shoot weight in amended soil was greater (P<0.01), most probably due to N mineralised from chitin. A significantly lower (P<0.01) root: shoot ratio of ryegrass in the amended soil also suggested improved N availability, and therefore less root mass was needed to support a given shoot mass. A reduction in nodulation was observed in 12-day-old white clover seedlings (P<0.05) and also in 6-week-old seedlings (P<0.01). The shoot weight of white clover was significantly lower (P<0.05) in amended soil, possibly due to phytotoxic effects of chitin. Chitin increased (P<0.01) the populations of bacteria and fungi by 13-fold and 2.5-fold, respectively. The cyst nematode of white clover, Heterodera trifolii, was significantly reduced in chitin-amended soil, possibly due to increased levels of chitinase produced by rhizosphere microorganisms. Two other plant-parasitic nematodes, Pratylenchus spp. and Tylenchus spp., were also reduced in ryegrass roots and in soil as a result of the chitin amendment. However, the total number of free-living nematodes increased 5.4-fold in amended soil.     

Effect of plant growth-promoting bacteria and soil compaction on barley seedling growth, nutrient uptake, soil properties and rhizosphere microflora

Inoculants are of great importance in sustainable and/or organic agriculture. In the present study, plant growth of barley (Hordeum vulgare) has been studied in sterile soil inoculated with four plant growth-promoting bacteria and mineral fertilizers at three different soil bulk densities and in three harvests of plants. Three bacterial species were isolated from the rhizosphere of barley and wheat. These bacteria fixed N₂, dissolved P and significantly increased growth of barley seedlings. Available phosphate in soil was significantly increased by seed inoculation of Bacillus M-13 and Bacillus RC01. Total culturable bacteria, fungi and P-solubilizing bacteria count increased with time. Data suggest that seed inoculation of barley with Bacillus RC01, Bacillus RC02, Bacillus RC03 and Bacillus M-13 increased root weight by 16.7, 12.5, 8.9 and 12.5% as compared to the control (without bacteria inoculation and mineral fertilizers) and shoot weight by 34.7, 34.7, 28.6 and 32.7%, respectively. Bacterial inoculation gave increases of 20.3–25.7% over the control as compared with 18.9 and 35.1% total biomass weight increases by P and NP application. The concentration of N and P in soil was decreased by increasing soil compaction. In contrast to macronutrients, the concentration of Fe, Cu and Mn was lower in plants grown in the loosest soil. Soil compaction induced a limitation in root and shoot growth that was reflected by a decrease in the microbial population and activity. Our results show that bacterial population was stimulated by the decrease in soil bulk density. The results suggest that the N₂-fixing and P-solubilizing bacterial strains tested have a potential on plant growth activity of barley.     

Diversity of Paenibacillus polymyxa populations in the rhizosphereof wheat (Triticum durum) in Algerian soils

The diversity of Paenibacillus polymyxa populations associated with the rhizosphere of durum wheat was investigated in Algerian soils sampled in regions where wheat had been cultivated for 5 and 26 years (Hamiz, H5 and Z26), 70 years (Algiers, D70), and more than 2 000 years (Tiaret, T2000 and K2000). A total of 111 strains were isolated by immunotrapping and identified as P. polymyxa using an API50CHB kit and restriction analysis of the amplified 16S rDNA gene. The phenotypic characteristics of the P. polymyxa populations were compared and the strains found not to cluster according to their origin. The longer the history of wheat cultivation, the lower the phenotypic diversity and the higher the frequency of nitrogen-fixing strains. Population genetic diversity, evaluated by ERIC-PCR, showed the same trends as phenotypic diversity. The distribution of ERIC genotypes among the different populations studied were compared using Pearson’s Chi-squared test. The strains isolated in D70, Z26, and H5 derived from soil populations sharing the same genetic structure, but those isolated in T2000 and K2000 each stemmed from a population with a specific genetic structure. These data suggest that the genetic structure of P. polymyxa populations has been affected by long-term wheat cultivation.     

Fluctuations in bacterial populations on the root surface of wheat (Triticum aestivum L.) grown under different soil conditions

Summary Populations of several bacterial groups on the root surface of wheat and in root-free soil were investigated in volcanic ash soil and non-volcanic ash soil throughout a series of predetermined intervals. Over time, the populations changed similarly both on the root surface and in root-free soil. The numbers of total bacteria, fluorescent Pseudomonas spp., phosphate-solubilizing bacteria, and NH _inf+ ^sup4 -oxidizing bacteria, were consistently lower in the plots with volcanic ash soil than with nonvolcanic ash soil, but the numbers of cellulose-decomposing bacteria were opposite to those of the other groups. Superphosphate application improved the growth of wheat in the volvanic ash soil. It did not, however, bring about any significant changes in the bacterial populations among the volcanic ash soils supplemented with three different levels of superphosphate, though there were some variations with plant age.     

Biological control of Rhizoctonia sp. root rot of Casuarina equisetifolia seedlings by Frankia spp. strains

Seventy Frankia spp. strains (nodulating N₂-fixing actinomycetes) were isolated from root nodules of Casuarina equisetifolia from different localities of Tamil Nadu state, India. From these, four strains (UMCe12, UMCe23, UMCe35, and UMce55) were selected. Their potential use as biological control agents for Rhizoctonia solani root rot disease of C. equisetifolia seedlings and their relative efficiency in nodule production were investigated. Between the two inoculum broadcast systems tested, seed-coating with Frankia spp. cell suspension was superior to the soil application of cells as sand-vermiculite-basal ammonium propionate inoculum. UMCe12 was the promising strain, offering the highest level of disease protection (81.1%) and nodule production (88.1%) in the R. solani-infested soil, followed by UMCe23 (60.3 and 65.5% of disease protection and nodule production, respectively), UMCe55 (53.5 and 58.2%), and UMCe35 (45.4 and 44.5%). Further, a significant positive correlation was observed between the dose of Frankia spp. and efficiency in both disease control and nodule production.     

Chitinolytic and proteolytic activity of streptomycetes isolated from root-free soil,rhizosphere and mycorrhizosphere of pine (Pinus sylvestris L.)

Summary We tested 75 strains of Streptomyces spp. (25 taken from each environment of soil, rhizosphere, and mycorrhizosphere of pine, Pinus sylvestris L.) and all exhibited chitinolytic activity and hydrolysed gelatine and sodium caseinate in agar media. Enrichment of these media with glucose and NH₄NO₃ caused induction or stimulation of proteolytic Streptomyces spp. strains (80%) derived from root-free soil; inhibition of this activity was observed in most strains (92%) isolated from the root zone. The post-culture liquids of the rhizosphere strains cultured in the absence of glucose revealed a significantly higher proteolytic activity than those obtained from the root-free soil. The addition of glucose to the medium stimulated proteolytic activity in the post-culture broth of Streptomyces strains derived from soil and the mycorrhizosphere.     

Dual inoculation of pretransplant stage Oryza sativa L. plants with indigenous vesicular-arbuscular mycorrhizal fungi and fluorescent Pseudomonas spp.

Summary This study examined the response of rice (Oryza sativa L.) plants at the pretransplant/nursery stage to inoculation with vesicular-arbuscular mycorrhizal (VAM) fungi and fluorescent Pseudomonas spp., singly or in combination. The VAM fungi and fluorescent Pseudomonas spp. were isolated from the rhizosphere of rice plants. In the plants grown in soil inoculated with fluorescent Pseudomonas spp. alone, I found increases in shoot growth, and in root length and fine roots, and decreases in root growth, and P and N concentrations. In contrast, in the plants colonized by VAM fungi alone, the results were the reverse of those of the pseudomonad treatment. Dual inoculation of soil with VAM fungi and fluorescent Pseudomonas spp. yielded plants with the highest biomass and nutrient acquisition. In contrast, the plants of the control treatment had the lowest biomass and nutrient levels. The dual-inoculated plants had intermediate root and specific root lengths. The precentages of mycorrhizal colonization and colonized root lengths were significantly lower in the dual-inoculated treatment than the VAM fungal treatment. Inoculation of plants with fluorescent Pseudomonas spp. suppressed VAM fungal colonization and apparently reduced photosynthate loss to the mycorrhizal associates, which led to greater biomass and nutrient levels in dual-inoculated plants compared with plants inoculated with VAM fungi alone. Dual inoculation of seedlings with fluorescent Pseudomonas spp. and VAM fungi may be preferable to inoculation with VAM alone and may contribute to the successful establishment of these plants in the field.     

Use of soil solarization to control root rots in gerberas (Gerbera jamesonii)

Summary An investigation was conducted during the summer months of 1986–1987 and 1987–1988 in Western Australia to evaluate the effect of soil solarization on the control of root rot of gerbera an also on the microbial and nutrient status of the soil. Infested soil cores were sampled from a site where root-rot was a severe problem and were removed to a non-infested site where they were subjected to soil solarization or fumigation. Soil solarization resulted in reduced root rot (root disease index 28.6%) in comparison to the untreated control (52.0%) 8 months after planting. Plants in the fumigated plots had 15.8% less disease than those in solarized plots. Solarization increased the total numbers of bacteria and actinomycetes, and the proportion of bacteria and fungi antogonistic to Fusarium oxysporum, F. solani and Rhizoctonia solani. The proportion of actinomycetes antagonistic to these fungi, however, did not differ between solarized and control soil treatments. There was a significant reduction in disease in plants grown in infested fumigated soil to which a 10% concentration of solarized soil had been added, suggesting the development of microbial suppression in solarized soil. Phytophthora cryptogea was eradicated to 30 cm by solarization as well as by fumigation. Solarization eliminated R. solani but not F. oxysporum to a soil depth of 10 cm. Solarization increased the levels of NO _n3 ^– -N and NH₄ ⁺-N in soil, but did not affect the concentrations of PO₄ ^3–, K⁺, Fe²⁺, organic C and pH. Yield (as number of flowers per plant) was increased by soil solarization and by fumigation.Increased yields and decreased disease severity in the solarized plots could have been caused by (1) a reduction in the infectivity of the infested soils, (2) an increase in the suppressiveness of the soil, and (3) an increased available of plant nutrients.     

Cadmium-tolerant plant growth-promoting bacteria associated with the roots of Indian mustard (Brassica juncea L. Czern.)

Eleven cadmium-tolerant bacterial strains were isolated from the root zone of Indian mustard (Brassica juncea L. Czern.) seedlings grown in Cd-supplemented soils as well as sewage sludge and mining waste highly contaminated with Cd. The bacteria also showed increased tolerance to other metals including Zn, Cu, Ni and Co. The isolated strains included Variovorax paradoxus, Rhodococcus sp. and Flavobacterium sp., and were capable of stimulating root elongation of B. juncea seedlings either in the presence or absence of toxic Cd concentrations. Some of the strains produced indoles or siderophores, but none possessed C₂H₂-reduction activity. All the strains, except Flavobacterium sp. strain 5P-3, contained the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which hydrolyses ACC (the immediate precursor of plant hormone ethylene) to NH₃ and α-ketobutyrate. V. paradoxus utilized ACC as a sole source of N or energy. A positive correlation between the in vitro ACC deaminase activity of the bacteria and their stimulating effect on root elongation suggested that utilization of ACC is an important bacterial trait determining root growth promotion. The isolated bacteria offer promise as inoculants to improve growth of the metal accumulating plant B. juncea in the presence of toxic Cd concentrations and for the development of plant-inoculant systems useful for phytoremediation of polluted soils.     

Inoculation of field-grown wheat (Triticum aestivum) with Azospirillum spp. in Brazil

Summary Three field experiments with wheat were conducted in 1983, 1984, and 1985 in Terra Roxa soil in Paraná, the major Brazilian wheat-growing region, to study inoculation effects of various strains of Azospirillum brasilense and A. amazonense. In all three experiments inoculation with A. brasilense Sp 245 isolated from surface-sterilized wheat roots in Paraná produced the highest plant dry weights and highest N% in plant tops and grain. Grain yield increases with this strain were up to 31 % but were not significant. The application of 60 or 100 kg N ha^–1 to the controls increased N accumulation and produced yields less than inoculation with this strain. Another A. brasilense strain from surface-sterilized wheat roots (Sp 107st) also produced increased N assimilation at the lower N fertilizer level but reduced dry weights at the high N level, while strain Sp 7 + Cd reduced dry weights and N% in the straw at both N levels. The A. amazonense strain isolated from washed roots and a nitrate reductase negative mutant of strain Sp 245 were ineffective. Strains Sp 245 and Sp 107st showed the best establishment within roots while strain Cd established only in the soil.     

砒砂岩中植物促生芽孢杆菌的筛选及其对土壤的改良作用

[目的]筛选砒砂岩土壤中植物促生芽孢杆菌,为微生物强化植物改良砒砂岩土壤的科学设想提供资源和技术基础。[方法]以产生植物激素IAA(indole acetic acid)、铁载体和生物膜为筛选指标,从内蒙古砒砂岩区土壤和植物样品中筛选植物促生芽孢杆菌,采用盆栽试验探究植物促生芽孢杆菌改良砒砂岩土壤特性和促进苜蓿和黑麦草生长的作用。[结果]筛选到的12株芽孢杆菌产生IAA、铁载体和生物膜的能力不同,分别属于Bacillus halotolerans,B.atrophaeus,B.siamensis和B.zhangzhouensis种群。与对照相比,B.halotolerans P75能够显著增加砒砂岩土壤的有机质含量(24.7%)、速效磷含量(11.9%)和速效钾含量(21.0%)等养分指标,每1 g土壤可培养细菌达到7.4 lg CFU,土壤蔗糖酶活性显著增强(58.8%)。接种B.halotolerans P75后,在砒砂岩土壤中生长的苜蓿和黑麦草干重增加22.3%～81.5%,[结论]从生长于砒砂岩土壤的苜蓿根内筛选到B.halotolerans P75,可提高砒砂岩土壤肥力,促进苜蓿和黑麦草生长,具有强化植物改良砒砂岩土壤的潜力。     

Phosphatase activity in the rhizosphere and its relation to the depletion of soil organic phosphorus

Summary The distribution of phosphatase activity and of phosphate fractions of the soil in the proximity of roots was studied in order to evaluate the significance of phosphatases in P nutrition of various plants (Brassica oleracea, Allium cepa, Triticum aestivum, Trifolium alexandrinum). A considerable increase in both acid and alkaline phosphatase activity in all the four soil-root interfaces was observed. Maximum distances from the root surface at which activity increases were observed ranged from 2.0 mm to 3.1 mm for acid phosphatase and from 1.2 mm to 1.6 mm for alkaline phosphatase. The increase in phosphatase activity depended upon plant age, plant species and soil type. A significant correlation was noticed between the depletion of organic P and phosphatase activity in the rhizosphere soil of wheat (r = 0.99^**) and clover (r = 0.97^**). The maximum organic P depletion was 65% in clover and 86% in wheat, which was observed within a distance from the root of 0.8 mm in clover and 1.5 mm in wheat. Both the phosphatases in combination appear to be responsible for the depletion of organic P.     

Association of Azospirillum brasilense with pearl millet (Pennisetum americanum (L.) Leeke)

Summary Microscopic observations of the root system of pearl millet (Pennisetum americanum (L.) Leeke) var. BJ 104 after surface sterilization and incubation in phosphate malate triphenyl tetrazolium chloride (TTC) revealed extensive colonization by Azospirillum spp. when plants were grown in sterile, partially sterile and field conditions as evidenced by the TTC-reducing property of active cells of the bacterium. Quantitative studies showed the need to standardize the techniques further to ensure more precise monitoring of the bacteria in the rhizosphere, as large numbers of soil bacteria were found capable of growth on specific media, thus interfering with the plate counts. Seed inoculation with A. brasilense increased the mean grain yield of pearl millet under different agroclimatic conditions in India. The mean increase in grain yield due to inoculation over uninoculated controls was also noticed with graded levels of fertilizer nitrogen (urea). Inoculation alone contributed to increased nitrogen uptake of plants with varying levels of fertilizer nitrogen application under sandy loam soil conditions (pH 7.3). The effects of inoculation were more prominent under lower levels of nitrogen than at the higher levels. The root biomass under field conditions was increased with Azospirillum spp. inoculation at 10 and 20 kg N/ha than their corresponding uninoculated controls.     

Influence of irrigation on the distribution and control of the nematode Meloidogyne javanica by the biocontrol bacterium Pasteuria penetrans in the field

Pasteuria penetrans, a bacterial parasite of plant-parasitic nematodes, is used to control root-knot nematode Meloidogyne spp. populations in vegetable crops. But its efficiency is variable, mostly because of the patchy distribution of the bacteria in arable fields. As the infective P. penetrans are non-motile bacteria in soil, abiotic soil factors can affect the bacteria–nematode relationships. An epidemiological study, conducted in a vegetable field, showed that abiotic factors such as irrigation, soil water holding capacity and texture, affected the efficiency of P. penetrans. A correspondence analysis between these abiotic factors and the density of P. penetrans spores in the soil, and the proportion of Meloidogyne javanica juveniles infected by the bacteria, revealed that irrigation affected directly the distribution of the spores in soil pores related to their passive transport by water flow. Laboratory experiments conducted on the passive transport of spores confirmed that intensive irrigation leached the spores down the soil profile and decreased the percentage of infected Meloidogyne juveniles.     

Biological nitrogen fixation in Azospirillum strain-maize genotype associations as evaluated by the 15N isotope dilution technique

Few studies of the inoculation of cereal crops with N₂-fixing bacteria have included more than one or two plant genotypes. In a recent study performed in Argentina using 12 different maize genotypes, it was found in 2 consecutive field experiments that several of them responded consistently, either negatively or positively, to inoculation with a mixture of strains of Azospirillum spp. The present study in post was performed to investigate the effect of inoculation of individual strains (and a mixture) of Azospirillum spp., and their nitrate reductase negative (NR-) mutants, on the growth of four of these maize genotypes. Two of these genotypes were grown in ¹⁵N-labelled soil with the aim of quantifying any contributions of biological N₂ fixation. Two genotypes (Morgan 318 and Dekalb 4D-70) produced similar increases in grain yield when they were inoculated with a mixture of Azospirillum spp. strains or fertilized with the equivalent of 100 kg N ha^-1. The other genotypes (Dekalb 2F-11 and CMS 22) showed little response to inoculation or N fertilization. The Morgan 318 and Dekalb 4D-70 genotypes showed a large increase in total N accumulation, suggesting that the response was due to increased N acquisition, but not due to bacterial nitrate reductase as the NR- mutants generally caused plant responses similar to those of the parent strains. Despite problems with the stabilization of the ¹⁵N enrichment in the soil, the ¹⁵N isotope dilution results indicated that there were very significant biological nitrogen fixation (BNF) contributions to the Dekalb 4D-70 and CMS 22 maize genotypes.Dedicated to Professor J.C.G. Ottow on the occasion of his 60th birthday