Growth Promotion of Maize (Zea mays L.) by Plant-Growth-Promoting Rhizobacteria under Field Conditions

Plant-growth promoting rhizobacteria (PGPR) play an important role in plant health and soil fertility. The experiment was conducted as factorial experiment with two factors of Azospirillum and Azotobacter. The bacterial strains were Azospirillum lipoferum s-21, A. brasilense DSM 1690, A. lipoferum DSM 1691, Azotobacter chroococcum s-5, and A. chroococcum DSM 2286. The results indicated that growth promotion by PGPR appears from early stages of growth, 45 days after inoculation (DAI). Beneficial effects of bacterial inoculation on ear growth were observed after 75 DAI. Inoculation with PGPR increased dry weights of leaf, stem, and grain and hence total biomass sampled at 90, 105, and 120 (harvest time) DAI. The greatest grain weight was produced by Azospirillum s-21 inoculation. Dual inoculation with Azotobacter s-5 + Azospirillum s-21 significantly increased total dry weight up to 115%. Results of this study showed that leaf area index and crop growth index were significantly affected by bacterial treatments.     

Potential for enhancement of root growth and nodulation of soybean co-inoculated with Azospirillum and Bradyrhizobium in laboratory systems

The potential enhancement of root growth and nodulation in vegetable soybean (AGS190) was studied with application of Azospirillum brasilense (Sp7) and A. lipoferum (CCM3863) co-inoculated with two Bradyrhizobium japonicum strains (TAL102 and UPMR48). Significant root growth stimulation and nodulation were observed in Azospirillum as well as during its co-inoculation with Bradyrhizobium. Nodule formation is linked with the initiation of new roots; nodules were almost absent even in Bradyrhizobium inoculated plant due to the absence of new roots development in clipped rooted seedlings. Total root length, root number, specific root length, root dry matter, root hair development and shoot dry matter were significantly increased by Azospirillum alone and its co-inoculum. Co-inoculated plants significantly influenced the number of nodules and its fresh weight. A. brasilense seemed to perform better in root growth and nodule development compared to A. lipoferum.     

Effects of Azospirillum brasilense on root morphology of common bean (Phaseolus vulgaris L.) under different water regimes

 The effects of inoculation with Azospirillum brasilense Cd on root morphology and growth of common bean (Phaseolus vulgaris L.) were studied under different growth systems and water regimes. The root systems were evaluated by image analysis. In a PVC-tube growth system, inoculation with A. brasilense at 10⁷ colony forming units (CFU) ml^–1 increased root length, root projection area, specific root length (m g^–1) and specific root area (cm² g^–1), as compared with non-inoculated controls, resulting in root systems with longer and thinner roots. Water stress induced similar root responses to those observed after inoculation with A. brasilense. No increase in plant biomass was observed in inoculated plants, suggesting that under the tested growth conditions, a relatively larger amount of resources is required for the maintenance of the thinner roots. In water-stressed potted plants, the effect of A. brasilense on tap root length was inoculum-concentration dependent. At 10⁷ CFU ml^–1 this effect was significant as compared to non-inoculated controls. In a pouch system without water stress, inoculation with A. brasilense at a concentration of 10⁵–10⁷ CFU ml^–1 2 days after germination resulted initially (2 days after inoculation) in an increase in root length (95%) and root fresh weight (66%), but reduced root diameter (20%), compared to controls. At this early stage of growth the distribution of root length among the different root diameter classes changed: the thinner-root classes had the largest proportion of longer roots. Received: 3 January 2000     

Differential colonization of Azospirillum lipoferum on roots of two varieties of rice (Oryza sativa L.)

Summary Seedlings of rice (IR42 and IR50) were aseptically dipped into Azospirillum lipoferum strain 34H suspension under dark, and the presence of bacteria on the differentiating regions of rice roots was observed by scanning electron microscopy. The bacterium did not colonize the root tips of IR42, while it colonized this region in the case of IR50, within 24 h after inoculation. In the early stages, most of the bacteria were embedded in the ruptured mucigel below the root cap cells of IR42. Mucigel was hardly detectable in IR50. While the root hair primordia of IR50 were colonized heavily with the bacterium within 24 h, the root hairs of IR42 were colonized 48 and 72 h after inoculation. This phenomenon in relation to plant varietal differences was discussed.     

Alleviation of noxious effects of cattle ranch composts on wheat seed germination by inoculation with Azospirillum spp.

Two commonly-used composts from dairy cow manure that are used to improve poor structure and fertility of desert soils have inhibitory effects on wheat seed germination, probably as a result of their high levels of humic acids. Inoculation of wheat seeds with two species of the plant growth-promoting bacteria Azospirillum brasilense Cd and A. lipoferum JA4 (separately) prior to sowing in these amended soils improved germination, similar to the natural level of germination of seeds in desert soil without compost amendment. Both compost amendments increased height of wheat seedlings in the range of 20–25%, increased shoot dry weight by 15–19%, but severely decreased (51–54% less) root dry weight. Inoculation of wheat seeds with A. brasilense Cd, but not with A. lipoferum JA4, significantly increased plant growth parameters (height, shoot and root dry weight) over control plants grown in soil-compost mixtures. This bacterial species could survive for a period of 20 days in compost humic acid solution, could increase its population when the humic acids served as the sole carbon source, and may change the composition of humic acids in which it grows. We suggest that inoculation with A. brasilense may alleviate noxious effects on germinating seeds caused by compost application by possibly transforming the composition of humic acids in the compost.     

Maize genotype effects on the response to Azospirillum inoculation

Two field experiments were carried out in Northern Argentina, during the 1989–1990 and 1990–1991 growing seasons, on Argentinian and Brazilian maize genotypes. The inoculant consisted of a mixture of four Azospirillum brasilense strain isolated from surface-sterilized maize roots in Argentina and three A. lipoferum strains isolated from surface-sterilized maize or sorghum roots in Brazil. Establishment of the inoculated strains was confirmed by the antibiotic resistance of the strains in the highest dilution vials. In all treatments, numbers of Azospirillum spp. were increased and the inoculated strains were found in the highest dilutions. While grain yields of the different genotypes varied between 1700 and 7300 kg ha^-1, total N accumulation was much less variable. Significant inoculation effects on total N accumulation and on grain yields were consistently negative with one Argentinian genotype and positive with four Argentinian and two Brazilian genotypes. Significant inoculation effects on leaf nitrate reductase activity at the flowering stage, observed in the range-55% to +176%, indicated the presence of various interactions between the plant NO _inf3 ^sup- metabolism and Azospirillum spp. Three Brazilian and one Argentinian maize genotype showed significant decreases in leaf nitrate reductase due to inoculation while four Argentinian genotypes showed significant increases in leaf nitrate reductase activity. The results of the present study, were consistent over the two field experiments and strongly indicate that more detailed plant genotype-Azospirillum spp. strain interaction studies, taking the entire N metabolism in the plant into account, are needed to allow better inoculation results of cereal crops.     

Effect of Azospirillum brasilense inoculation on root morphology and respiration in tomato seedlings

Summary The level of Azospirillum brasilense strain Cd colonization in the rhizosphere of some vegetables was 10⁴–10⁵ colony-forming units (CFU) per root of one plant in 2-week-old plants inoculated with 5 × 10⁸ Azospirillum cells. Significant increases in root length (35%) and in top (90%) and root (50%) dry weight and total leaf area (90%) were observed in 18-day-old inoculated tomato plants compared with non-inoculated controls. An inoculum concentration of 1 × 10⁸ to 5 × 10⁸ CFU/ml stimulated the appearance of root hairs. Large numbers of bacteria (1 × 10⁹ CFU/ml) caused asymmetrical growth of the root tip. In a petri dish system, Azospirillum (1 × 10⁸ CFU/ml) increased root dry weight (150%), protein content (20%), respiration rate per root (70%) and the specific activity of malate dehydrogenase (45%–65%) over non-inoculated controls. The specific respiration rate, expressed as micromol of O₂ per minute per milligram of dry weight of roots, was significantly lower in inoculated roots, suggesting that less energy was spent for accumulation of more dry material.     

Effect of inoculation with different strains of Azospirillum brasilense on cotton (Gossipium barbadense)

Summary The response of the cotton plant to inoculation with six strains of Azospirillum brasilense was investigated under subtropical conditions in Egypt. Azospirilla populations and activities were increased as a result of root inoculation with liquid inoculum of Azospirillum sp. Highest C₂H₂ — reduction activities on roots were obtained with strains S631 and Sp Br 14 (means of 216.85 and 209.50 nmol C₂H₄g^–1 root h^–1 respectively) while strain M4 gave the lowest activity (mean of 100.8 nmol C₂H₄g^–1 root h^–1). Statistical analysis showed that Azospirillum strains 5631, Sp Br 14, E15 and SC22 significantly increased the plant dry weight and nitrogen uptake while inoculation with strains M4 and SE had no significant effect in that respect.     

Effect of Azospirillum inoculants on arbuscular mycorrhiza establishment in wheat and maize plants

Plant growth-promoting rhizobacteria and arbuscular mycorrhizal (AM) fungi represent two main groups of beneficial microorganisms of the rhizosphere. The role of different strains of Azospirillum on AM fungi development was evaluated by measuring the percentage of AM colonisation of the root system in durum wheat and maize plants, grown under both greenhouse and field conditions. The effect of wild-type Azospirillum brasilense strain Sp245 and genetically modified (GM) derivatives overproducing indole-3-acetic acid was assessed at greenhouse level in (1) three different cultivars of durum wheat, in the presence of indigenous AM fungi and (2) maize plants artificially inoculated with Glomus mosseae and Glomus macrocarpum. In addition, the establishment of natural AM fungal symbiosis was evaluated using Azospirillum lipoferum CRT1 in maize plants at field level. Despite the stimulatory effect of the different Azospirillum inocula on root growth, no significant differences in AM colonisation were found, independently of the AM fungus involved, either in wheat or in maize plants. Similarly, GM A. brasilense, which strongly stimulates root development, did not affect AM formation. Although these results were obtained in conditions in which the mycorrhization rate was moderate (15–30%), overall considered they indicate that the use of wild-type or GM Azospirillum phytostimulators does not alter mycorrhization.     

Interactions of some nematicides with Azospirillum lipoferum and the growth of Zea maize

The possible interaction of four nematicides (Terbufos, Carbofuran, Fenamiphos, and Aldicarb) with Azospirillum lipoferum and growth of two Zea maize cultivars was studied in a greenhouse experiment. Application of nematicides, Fenamiphos in particular, resulted in higher plant length, dry matter production and N yield over the nematicide-untreated plants. Azospirillum spp. inoculation stimulated the growth of nematicide-treated Z. maize. Among the nematicides used, Carbofuran and Aldicarb inhibited the nitrogenase activity on plant roots more seriously than Fenamiphos and Terbufos. Generally, the inhibition percentages in acetylene reducing activity in soil of inoculated treatments were lower (14.4 - 61.8%) than those reported for the uninoculated ones (21.4 - 73.9%). Soil, irrespective of treatment, regained a part of its normal N₂-ase activity with time. Field concentrations of all nematicides showed different inhibitory effects on N₂-ase activity of Azospirillum spp. in culture medium, such effects increased with increased doses (10- and 100- fold) and incubation periods (10 days). The contribution of Azospirillum spp. to the N economy of soil treated with nematicides is discussed.     

N2-fixing bacteria in the rhizosphere: Quantification and hormonal effects on root development

The paper summarizes the results of a series of experiments on enumeration of N₂-fixing bacteria (diazotrophs) and hormonal effects of Azospirillum on root development. Numbers of N₂-fixing and N-heterotrophic bacteria were determined on the root (rhizoplane plus “inner” root surface) and in the rhizosphere soil (0–3 mm from the root surface) of Arrhenatherum elatius, other forage grasses and some herbaceous plant species. Pot experiments involved freshly collected soil from an unfertilized grassland area containing its natural population of N₂-fixing bacteria. The MPN (most probable number) of diazotrophs in relation to the MPN of the total bacterial population was always lower on the root than in the rhizosphere soil, suggesting that diazotrophs were not selectively advantaged at the root surface. Supply of mineral nitrogen (NH₄NO₃) decreased the proportion of N₂-fixing bacteria at the rhizoplane as well as in the rhizosphere soil. Similar results were obtained when N was supplied via the leaves. The data suggest that N₂-fixing bacteria in the rhizosphere are poor competitors once they loose their competitive advantage of binding dinitrogen. Correspondingly, the increase in the MPN of the diazotrophs found during plant development was interpreted as a result of decreased available combined N in the rhizosphere. The proportion of N₂-fixing bacteria relative to the total number of bacteria was generally below 1%. Considering the potential amount of substrate released from the roots and the substrate requirement of the bacterial population, N₂-fixation was considered insignificant for plant growth under the given conditions. For the investigations on possible beneficial effects on plant development by bacterial hormones, Azospirillum brasilense was chosen because evidence suggests that amongst the soil bacteria releasing hormones, especially IAA, certain strains of this species are more important than other bacteria. Application of A. brasilense Cd (ATCC 29710) onto the roots of young wheat plants grown in soil increased the number of lateral roots, the total root length and the number of root hairs. Similar results were obtained after application of IAA. This suggests that IAA is an important factor responsible for the effects observed after inoculation with A. brasilense. The increase in root surface may improve acquisition of nutrients and enhance growth of plants. Another hormonal effect of A. brasilense was an increase in nodulation of Medicago sativa grown on agar. Again pure IAA resulted in a similar increase in nodule number. Increases in nodule number were only in part associated with a change in root morphology. Therefore an effect of IAA on the plant immanent regulation system for nodulation is likely.     

Mitigation of salt stress in wheat seedlings by a <Emphasis Type="Italic">gfp</Emphasis>-tagged <Emphasis Type="Italic">Azospirillum lipoferum</Emphasis>

Root colonization and mitigation of NaCl stress on wheat seedlings were studied by inoculating seeds with Azospirillum lipoferum JA4ngfp15 tagged with the green fluorescent protein gene (gfp). Colonization of wheat roots under 80 and 160 mM NaCl stress was similar to root colonization with this bacterial species under non-saline conditions, that is, single cells and small aggregates were mainly located in the root hair zone. These salt concentrations had significant inhibitory effects on development of seedlings, but not on growth in culture of gfp-A. lipoferum JA4ngfp15. Reduced plant growth (height and dry weight of leaves and roots) under continuous irrigation with 160 mM NaCl was ameliorated by bacterial inoculation with gfp-A. lipoferum JA4ngfp15. Inoculation of plants subjected to continuous irrigation with 80 mM NaCl or to a single application of either NaCl concentration (80 or 160 mM NaCl) did not mitigate salt stress. This study indicates that, under high NaCl concentration, inoculation with modified A. lipoferum reduced the deleterious effects of NaCl; colonization patterns on roots were unaffected and the genetic marker did not induce undesirable effects on the interaction between the bacterium and the plants.     

Production of B-group vitamins by plant growth-promoting Pseudomonas fluorescens strain 267 and the importance of vitamins in the colonization and nodulation of red clover

 We have estimated the production of water-soluble B vitamins by plant growth-promoting rhizobacterium Pseudomonas fluorescens strain 267 in a minimal medium with different C sources and at different pH values. In the minimal medium, strain 267 produced large amounts of niacin (0.92 μg ml^–1) and pantothenic acid (0.75 μg ml^–1), but also other vitamins such as biotin, thiamine, cobalamine and pyridoxine. The production of B vitamins was dependent on the C source and pH of the growth medium. By random Tn5 mutagenesis, thiamine and niacin auxotrophs were isolated from P. fluorescens strain 267 and mutants were used to evaluate the vitamin production on colonization of clover roots under controlled conditions. Red clover root colonization decreased by about 1 order of magnitude in the case of the niacin auxotroph. The vitamin auxotrophs of P. fluorescens in a mixed inoculation of clover with R. leguminosarum bv. trifolii strain 24.1 showed no plant growth-promotion activity. Received: 23 May 2000     

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.     

Effect of calcium carbonate, sand, and organic matter levels on mortality of five species of Azospirillum in natural and artificial bulk soils

 Five bacterial strains, one from each of the five known species of the plant growth-promoting bacteria (PGPB) Azospirillum (A. brasilense, A. lipoferum, A. amazonense, A. halopraeference, and A. irakense) were inoculated into two natural, semiarid soils (terra rosa and loessial sandy) from Israel, and two artificial soils constructed to simulate the native soils. Within 60 days, the populations of all five Azospirillum species declined significantly in a linear fashion, in both the native soils and in the homologous artificial soils. Increased levels of CaCO₃, and fine and rough sand, had significant detrimental effects on the survival of the five Azospirillum species, whereas increased organic matter content improved survival. In contrast, when the bacterial strains were incubated in the rhizosphere of tomato seedlings grown in the artificial soils, manipulation of these soil variables had only a marginal effect on bacterial survival; all Azospirillum species survived well in the tomato rhizosphere under conditions that are otherwise detrimental. This study indicates that most cells of the strains of five known species of Azospirillum died out linearly over time in two semiarid soils, and that only the major soil components affected Azospirillum survival in soil. Because mortality was similar in native soils and in artificial homologous soils, artificial soils can be used to study the soil behavior of Azospirillum. Received: 9 April 1999     

Identification and quantification of auxins in culture media of Azospirillum and Klebsiella and their effect on rice roots

 The production of auxins [indoleacetic acid (IAA) and related compounds] by Azospirillum brasilense and Klebsiella pneumoniae in culture media supplemented with tryptophan was investigated. Extracts from the supernatants of cultures incubated for different periods of time were partitioned into two fractions and each fraction was analysed separately by using HPLC. The chromatography was performed by using different optimized mobile phases and corresponding standards. Both N-fixing strains produced high quantities of extracellular IAA and tryptophol. The production of both compounds increased during the stationary phase of the culture. However, very low levels of indoleacetaldehyde and indolepyruvate produced by K. pneumoniae in the exponential phase of the culture, were only recorded when the samples were analysed immediately after the extraction. In addition, the effect of different concentrations of filter-sterilized culture supernatants of both strains on the development of rice roots grown in hydroponic culture medium was studied. Addition of the optimum concentrations (6–8%) of bacterial supernatants to such hydroponic cultures increased root elongation, root surface area, root dry matter and development of lateral roots and root hairs compared to untreated roots. On the other hand, the addition of high concentrations of the supernatant (more than 10%) strongly inhibited root elongation, lateral root development, and caused root outgrowths, i.e. round nodule-like tumours. Received: 7 January 1998     

Field survival of the phytostimulator Azospirillum lipoferum CRT1 and functional impact on maize crop, biodegradation of crop residues, and soil faunal indicators in a context of decreasing nitrogen fertilisation

Heavy nitrogen fertilisation is often implemented in maize cropping systems, but it can have negative environmental effects. Nitrogen-fixing, phytohormone-producing Azospirillum plant growth-promoting rhizobacteria (PGPR) have been proposed as crop inoculants to maintain high yield when decreasing nitrogen fertilisation. In this context, agronomic and ecological effects of the inoculation of maize seeds with the PGPR Azospirillum lipoferum CRT1 were studied in two consecutive years. The inoculant was recovered from maize at 10⁵ CFU g⁻¹ root or higher. Inoculation enhanced root growth and development based on results of root biomass, rooting depth and/or parameters describing root system architecture, and a transient positive effect on shoot height was observed in the first year. Inoculation did not increase yield, but reducing mineral nitrogen fertilisation had only a minor effect on yield. This suggests that the lack of positive effect of the PGPR on yield was due to the fact that the whole field was heavily fertilised in years prior to the start of the experiment. Soil nitrogen levels decreased during the 2 years of the study, and the inoculant had no effect on residual soil nitrogen levels at harvest. Inoculation had no impact on Fusarium symptoms and concentration of the mycotoxin deoxynivalenol in maize kernels, but both were influenced by the interaction between inoculation and nitrogen fertilisation level. Inoculation did not influence meso/macrofaunal soil populations, but had a small but significant effect (smaller than the effect of added nitrogen) on decomposition, nitrogen mineralisation and mesofaunal colonisation of maize leaves (in litter bags). Overall, the ecological impact of seed inoculation with the PGPR A. lipoferum CRT1 was small, and its magnitude was smaller than that of chemical nitrogen fertilisation.     

Genetics and molecular biology of Azospirillum

 Genetic manipulation of Azospirillum spp. has facilitated a better understanding of the mode of action of this plant-growth promoting bacterium and should help to improve its ability to stimulate plant growth and development. This review considers and discusses Agospirillum plasmids, promoter sequences, the isolation of Azospirillum mutants, the genetic transformation of Azospirillum, the transfer of foreign genes into Azospirillum by conjugation and the Azospirillum genes that have been isolated and characterized. The Azospirillum genes that are discussed include genes involved in nitrogen fixation, plant root attachment, phytohormone biosynthesis, tryptophan biosynthesis, carbon metabolism and a few other less well characterized processes. Received: 11 December 1997     

Nodule distribution on legume roots: Specificity and response to the presence of soil

Roots of siratro, cowpea, and two cultivars of soybean were inoculated with either Rhizobium sp. 3G4b16 or R. fredii 191. The numbers and distribution of nodules were determined after hydroponic growth of the plants for 10 days in plastic growth pouches. The uppermost nodules on the primary roots of both siratro and cowpea were clustered near the position occupied by the root tip at the time of inoculation. This is the region thought to be maximally susceptible to nodulation. Appreciable numbers of nodules also were scattered down the length of the primary root. Nodulation of McCall and Vicoja soybean (in growth pouches) was very different to that in an autoclaved greenhouse soil mixture. Strain × cultivar-specific differences in the numbers of nodules on primary roots were obliterated by soil. Soil reduced the extent of scattering of nodules along primary roots and shifted the ratios of primary root nodules to lateral root nodules. Soil also caused a 95% reduction in the number of McCall root hairs infected by strain 3G4b16. The soil environment appears to obliterate strain × cultivar-specific variation that is expressed when inoculated plants are grown hydroponically in growth pouches.     

Diurnal and seasonal patterns of nitrogenase activity of red alder in comparison with white clover in silvopastoral agroforestry systems

 Simultaneous measurements were made to assess the diurnal and seasonal patterns of nitrogenase activity of red alder (Alnus rubra Bong.) and white clover (Trifolium repens L.) growing together in a silvopastoral agroforestry system using the acetylene reduction assay. Diurnal measurements were made in the summer and autumn at 3-h intervals whereas seasonal nitrogenase activity was assessed based on observations made at midday in July, September and January to represent the summer, autumn and winter seasons, respectively. No obvious diurnal patterns of nitrogenase activity were found in either red alder or white clover in summer and no significant variations in nitrogenase activity were observed between day and night. However, in autumn, pronounced diurnal patterns were observed in both species. Significantly higher rates of nitrogenase activity per unit dry weigh (dwt) of nodules were detected at 1500 hours in red alder, whereas, in white clover, significantly higher rates were obtained at 2100 hours. There was no significant correlation between diurnal nitrogenase activity and air temperature, photosynthetically active radiation and soil temperature at 10 cm depth in either red alder or white clover. Seasonal rates of nitrogenase activity showed significantly higher activity in summer, which subsequently decreased in autumn, to reach very low levels in the winter. The rates of nitrogenase activity of white clover were consistently higher than those of red alder both diurnally and seasonally. In the three seasons sampled, the average nitrogenase activity for white clover was 66.42 μmol C₂H₄ g dwt^–1 h^–1, which was 3.5 times higher than the 18.67 μmol C₂H₄ g dwt^–1 h^–1 obtained for red alder. Received: 11 November 1997