A spontaneous chlorate-resistant mutant of Azospirillum brasilense Sp245 displays defects in nitrate reduction and plant root colonization

Azospirillum, a soil bacterium capable of colonizing plant roots, can reduce NO₃^-. In this work, a spontaneous chlorate-resistant mutant of Azospirillum brasilense Sp245, named Sp245chl1, was phenotypically characterized. The mutant is defective in both assimilatory and periplasmic dissimilatory nitrate reductase activity. Using the gusA reporter gene methodology, Sp245chl1 was found to be significantly affected in its ability to colonize roots of wheat and rice seedlings.     

Can Azospirillum strains capable of growing at a sub-optimal temperature perform better in field-grown-wheat rhizosphere

Two Azospirillum brasilense strains, CDJA and A40, capable of growing and producing plant growth-promoting (PGP) substances at the sub-optimal temperature (SOT) of 22°C, were tested for their ability to survive, colonize and enhance wheat growth and yield under field conditions upon inoculation. The response was compared with that of A. brasilense strain, A9, impaired in growth and PGP activities at SOT (22°C) but otherwise comparable to CDJA and A40 at 37°C. A field experiment was carried out in a split-plot design with four levels of N as main plots and three strains and an uninoculated control as subplots. A differential response in the establishment of the strains and in plant growth and yield was obtained, due to the categories of strains, particularly at lower levels of N (0 kg and 40 kg N ha^-1). The results clearly demonstrated that strains capable of growing and producing PGP substances at SOT are better inocula for wheat.     

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.     

Time course of N<Subscript>2</Subscript> fixation and growth of chickpea

The ¹⁵N isotopic dilution technique was used to assess N₂ fixation in desi chickpea (Cicer arientinum L.) cv. Myles at different growth stages as influenced by inoculation method. In this growth chamber study, no significant differences in nodule dry weight, amount of N₂ fixed and plant dry matter were observed between seed inoculation with seed-applied peat inoculant and soil-applied granular inoculant placed 2.5 cm below the seed. However, seed inoculation with liquid inoculant was inferior to the seed-applied peat or the granular inoculant for all parameters measured at all sampling dates. The seed-applied peat and granular inoculant treatments fixed 4.8 and 4.1 mg N plant^-1, respectively, by the late vegetative stage, and reached a maximum of 20.6 and 25.6 mg N plant^-1, respectively, by the late pod-filling stage. These values accounted for 30.5% and 34.9% of the total plant N for the peat and granular inoculant, respectively, by late pod-filling. For the liquid inoculant treatment, the amount of N₂ fixed increased from 2.3 mg N plant^-1 by the late vegetative stage to a maximum of 9.6 mg N plant^-1 which was 22.2% of total plant N by the mid pod-filling stage. The highest daily N₂ fixation rate for the peat and granular inoculant was 0.9 mg N plant^-1 and occurred between flowering and early pod-filling, whereas that for the liquid occurred between early and mid pod-filling stages (0.23 mg N plant^-1). After the mid pod-filling stage, little or no N₂ was fixed in all treatments. Plant dry matter increased from the late vegetative stage to physiological maturity but the greatest dry matter accumulation occurred between the late vegetative and early pod-filling stages in all treatments.     

Yield response of wheat and barley to inoculation of plant growth promoting rhizobacteria at various levels of nitrogen fertilization

The yield response of a wheat (Kirik) and a barley (Tokak 157/37) cultivar to inoculation with Azospirillum brasilense Sp246 and Bacillus sp. OSU‐142 was studied in relation to three levels of N fertilization (0, 40, and 80 kg ha^–1) under field conditions in Erzurum, Turkey, in 1999 and 2000. Seed inoculation with A. brasilense Sp246 significantly affected yield and yield components, both in wheat and barley. On average of years and N doses, inoculation with A. brasilense Sp246 increased spike number per m², grain number per spike, grain yield, and crude protein content by 7.2, 5.9, 14.7, and 4.1 % in wheat and by 6.6, 8.1, 17.5, and 5.1 % in barley, respectively, as compared to control. Inoculation with Bacillus sp. OSU‐142 significantly increased kernel number per spike in wheat, but no significant effect was determined in the other characteristics. Grain yields and yield components were also higher at all levels of nitrogen fertilizer in the inoculated plots as compared to the control. However, these increases diminished at high fertilizer levels. These results suggest that application of the growth promoting bacteria A. brasilense Sp246 may have the potential to be used as a biofertilizer for spring wheat and barley cultivation in organic and low‐N input agriculture.     

Yield promotion and phosphorus solubilization by plant growth–promoting rhizobacteria in extensive wheat production in Turkey

Plant growth–promoting rhizobacteria (PGPR) have been reported to stimulate the growth and yield of grain crops, particularly when nutrient supply is poor. However, the mechanisms underlying stimulation of plant growth may vary depending not only on growth conditions and crop management but also on plant and bacterial species. The present study assessed the effect of an inoculation with single or multiple PGPR strains on phosphorus (P)‐solubilization processes in the soil and on grain yield in wheat. Single inoculation with Bacillus subtilis OSU‐142, Bacillus megaterium M3, or Azospirillum brasilense Sp245 increased grain yield by 24%, 19%, and 19%, respectively, while a mixed inoculation with OSU‐142, M3, and Sp245 increased grain yield by 33% relative to noninoculated plants. Single inoculations with Paenibacillus polymyxa RC05 or Bacillus megaterium RC07 were less effective. Single or mixed treatments with OSU‐142, M3, and Sp245 increased the concentrations of the labile and moderately labile P fractions in rhizosphere soil. The growth‐stimulating effect of OSU‐142, M3, and Sp245 was also reflected by higher P concentrations in most plant organs. Among all inocula tested, the highest plant P acquisition was obtained in the presence of M3 and accompanied by the highest microbial P levels and the highest phosphatase activities in the rhizosphere soil. In conclusion, seed inoculation with mixed PGPR strains may effectively substitute for a part of P‐fertilizer application in extensive wheat production, and in particular M3 appears to improve the solubilization of inorganic soil P.     

Response of neem (Azadirachta indica A. Juss) to indigenous arbuscular mycorrhizal fungi, phosphate-solubilizing and asymbiotic nitrogen-fixing bacteria under tropical nursery conditions

Neem (Azadirachta indica A. Juss) seedlings were inoculated with arbuscular mycorrhizal (AM) fungi, Glomus intraradices Schenck and Smith and G. geosporum (Nicol. and Gerd.) Walker, Azospirillum brasilense, and phosphate-solubilizing bacteria (PSB) individually or in various combinations in unsterile soil under nursery conditions. Seedlings were harvested at 60 and 120 days after transplantation. Microbial inoculation resulted in increased mycorrhizal colonization, greater plant height, leaf area and number, root collar diameter, biomass, phosphorus, nitrogen and potassium content, and seedling quality. Inoculated seedlings also had low root/shoot ratios and low nutrient utilization efficiencies. Populations of PSB declined with seedling growth; contrarily populations of A. brasilense increased. A. brasilense and PSB populations were related to each other and influenced root colonization by AM fungi. Microbial inoculation effects were greatest when seedlings were inoculated with a combination of microbes rather than individually. This clearly indicates that these microorganisms act synergistically when inoculated simultaneously, with maximum response being when both AM fungi were coinoculated with A. brasilense and PSB. The results emphasize the importance of microbial inoculations for the production of robust, rapidly growing seedlings in nurseries and illustrate the advantage of inoculating soils of a low microbial population with indigenous microbes.     

Relationship between in vitro production of auxins by rhizobacteria and their growth-promoting activities in Brassica juncea L.

Rhizobacteria were isolated from the rhizosphere of different Brassica species and assayed for their ability to produce auxins in vitro. The isolates varied greatly in their potential for auxin production (ranging from 0.33 to 11.40 µg ml^-1). L-Tryptophan (an auxin precursor) addition to the media increased the auxin production by several fold. Based upon in vitro auxin production and growth promotion of B. juncea seedlings caused by various isolates under gnotobiotic conditions, promising isolates were selected and tested in pot trial to observe their effects on growth, yield and oil content of the same Brassica species. Results showed that seed inoculation with different isolates of rhizobacteria significantly increased plant height (up to 56.5%), stem diameter (up to 11.0%), number of branches (up to 35.7%), number of pods per plant (up to 26.7%), 1,000-grain weight (up to 33.9%), grain yield (up to 45.4%) and oil content (up to 5.6%) over the uninoculated control. Isolate S54 gave the most promising and consistent results. Highly significant correlations between L-TRP-derived auxin production by plant growth-promoting rhizobacteria (PGPR) in vitro and grain yield (r =0.77^**), number of pods (r =0.78^**) and number of branches per plant (r =0.77^**) of B. juncea were found. It was hypothesized that these PGPR may influence the growth and yield of inoculated plants by production of auxins in the rhizosphere of inoculated plants from the L-TRP present in the root exudates, although other mechanisms of action might have also contributed.     

Cultivar-specific dinitrogen fixation in Vicia faba studied with the nitrogen-15 natural abundance method

N fixation by different faba bean (Vicia faba) cultivars was studied using the natural abundance method. The delta ¹⁵N ('¹⁵N) values of the faba beans and the reference plants differed by 4.6-7.0‰. The non-nodulating V. faba cv. F48 seems to be the best reference plant for nodulated and N₂-fixing V. faba. Significant differences occurred in the quantity of N₂ fixation of six V. faba cultivars. The average fraction of N derived from air (FNdfa) estimated from leaf material ranged between 69 and 80%. Shoot-based estimates of N fixation varied between 200 and 360 kg N ha^-1. N fixation was affected more by differences in FNdfa than by differences in total N accumulation. Fixation data calculated with the non-nodulated reference plant V. faba cv. F48 were lower than those calculated with cabbage (Brassica oleracea) and ryegrass (Lolium perenne) as reference plants. Of all reference plants, non-N₂-fixing V. faba cv. F48 has a root system and temporal pattern of N assimilation that is the one most similar to that of N₂-fixing V. faba plants. Cv. F48 showed senescence as did the other V. faba cultivars after pod-fill was complete, whereas cabbage, ryegrass and camomile had a later senescence period. N fixation during pod-filling appears more important for a good yield than N₂ fixation abilities in the earlier growth period. The best V. faba cultivars left about 100 kg N ha^-1 in residual material on the field as fertilization for the following crops.     

Copper binding capacity of root exudates of cultivated plants and associated weeds

Cu binding to root exudates of two cultivated plants, wheat (Triticum aestivum) and rape (Brassica napus), and two weeds associated with wheat, dog daisy (Matricaria inodora) and cornflower (Centaurea cyanus), was studied in vitro under hydroponic and sterile conditions. Nutrient solutions were prepared with or without P. A MetPLATE microbiological test was used to assess the metal complexing capacity of root exudates. In the P-deficient solutions, no exudation was observed for any of the four plants; consequently, no Cu binding occurred. When P was present in the nutrient solutions, the plant exudates displayed differing abilities to complex Cu. No difference was detected in the binding capacity of the dog daisy or cornflower, and the blank [heavy metal binding capacity (HMBC)=1.07, 1.40 and 1.00, respectively]; however, the rape and wheat exudates were found to complex Cu in rhizospheric solutions (HMBC=1.73 and 3.00, respectively). The concentrations of exuded organic C were 1.2, 10.8, 15.3 and 15.7 mg l^-1 for the dog daisy, cornflower, wheat and rape, respectively. These results suggest that the nature, as well as the amount, of the organic compounds exuded by plant root, is important in determining the extent of Cu complexation.     

Comparison of the decomposition and N and P mineralization of canola, pea and wheat residues

Insight into nutrient cycling is gained by understanding the dynamics and quantifying nutrient mineralization from decomposing crop residues. Since wheat (Triticum aestivum L.), canola (Brassica napus L.) and pulse crops such as pea (Pisum sativum L.) are commonly grown in rotation, our objectives were to: (1) compare, using the mesh bag technique, the dry matter (DM) loss and release of N and P of straw and root residues of those crops in the 10-11 months following harvest, and (2) determine the influence of N fertilizer on residue decomposition and nutrient release. The no-tillage study started in autumn 1997 when straw residues were placed on the soil surface and root residues were buried in the soil, and sampled periodically through the 1998 growing season. Wheat was grown in 1998 and received 0 or 60 kg N ha^-1. The study was repeated in 1998/1999. Wheat straw decomposed more slowly than canola or pea straw (losing an average of 12%, 24% and 25%, respectively, of initial DM in 10-11 months), however, the converse was noted for root residues (42%, 26% and 19% of initial DM). Average net N mineralization from wheat, canola and pea straw was essentially 0, 0.7 and 5.6 kg N ha^-1, respectively. Phosphorus released from straw ranged from 0.5 kg ha^-1 for pea to 0.75 kg ha^-1 for canola. Net N and P mineralization from root varied little between crop species: 0.9-1.6 kg N ha^-1 and 0.1-0.3 kg P ha^-1. Nitrogen fertilization increased DM loss, and N and P release from straw residues.     

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.     

Phenotypic variation in Azospirillum brasilense Sp7 does not influence plant growth promotion effects

The Azospirillum genus comprises free-living, plant growth-promoting, nitrogen-fixing bacteria found in the rhizosphere of plant roots. Azospirilla are able to promote plant growth mainly through improvement of root development. Bacterial surface components, such as extracellular polysaccharides and proteins, are involved in root colonization. Phase variation – or phenotypic variation – is one of the mechanisms by which microorganisms adapt to environmental changes. This phenomenon is characterized by the presence of a sub-population of the bacteria presenting a different phenotype relative to the major population. In this study we characterized phenotypic variation of Azospirillum brasilense Sp7. When plated on solid media, some A. brasilense colonies were shown to possess a much more mucoid morphology, producing 7.5–8 times more exopolysaccharide with different monosaccharide composition than the parental strain Sp7. The rate of appearance of this kind of variant colonies was 1 in 5000, in agreement with the accepted rate for the phase/phenotypic variation phenomenon. The variants were significantly more resistant to heat and UV-exposure than the parental strain and displayed genomic changes as seen by the different band patterns following ERIC-PCR, BOX-PCR and RAPD analyses. In plant inoculation experiments under greenhouse conditions, with maize, wheat, soybean and peanuts, the EPS overproducing variants performed as similar as the parental strain. Therefore, EPS overproduction did not confer an apparent advantage to A. brasilense in terms of induction of plant growth promotion.     

Macronutrient accumulation in wheat crop (Triticum aestivum L.) with Azospirillum brasilense associated with nitrogen doses and sources

Abstract

New studies are needed to optimize the nitrogen (N) amount that can be applied to utilize the Azospirillum brasilense benefits. In addition, information regarding the interaction between the urease inhibitor and biological nitrogen fixation (BNF) and how they affect the macronutrients accumulation are also needed. We evaluate the effect of N sources and doses associated with A. brasilense regarding the macronutrients accumulation in straw and grains and wheat grain yield in tropical conditions. A randomized block experimental design was used with four replications in a 2?×?5?×?2 factorial arrangement as follows: two N sources (urea and urea with urease enzyme inhibitor NBPT; five N doses (0, 50, 100, 150, and 200?kg ha^?1) applied in topdressing; with and without A. brasilense inoculation. We found that an increase in N doses positively influenced the accumulation of macronutrients in straw and grains and the wheat grain yield. N sources have similar effects. Inoculation with A. brasilense increased accumulation of Mg and S in straw and P, Ca, and Mg in grains, regardless of the N dose. The inoculation with A. brasilense associated with 140?kg ha^?1 of N increased wheat grain yield. The inoculation can contribute in a more sustainable way to wheat nutrition and optimizing N fertilization.     

Use of Plant-Growth-Promoting Rhizobacteria (PGPR) Seed Inoculation as Alternative Fertilizer Inputs in Wheat and Barley Production

In this study we aimed to investigate the effects of plant-growth-promoting rhizobacteria (PGPR) on seed incubation of spring wheat and barley. Three bacterial strains were applied singly and in combinations. Seed inoculation with strains significantly affected grain yield (GY), straw (SWY), total yield (TY), and plant nutrient element (PNE) content. In field trials, compared to the control, single inoculations gave GY, SWY, and TY increases by 27.5–31.9%, 1.1–5.3%, and 1.3–11.3% in wheat and 15.1–27.8%, 10.8–15.5%, and 14.5–18.5% in barley, respectively, but mixtures of strains gave increases in GY, SWY, and TY by 54.7%, 2.1%, and 6.7% in wheat and 57.8%, 14.6%, and 17.5% in barley, respectively. According to the results, it was concluded that seed inoculations with PGPR and mixture inoculation might satisfy nitrogen requirements, but Bacillus megaterium M3 and MIX (Bacillus subtilis OSU142, B. megaterium M3, Azospirillum brasilense Sp245) inoculation provided greater PNE concentrations than mineral fertilizer application for wheat and barley under field conditions.     

Effects of dicyandiamide, farmyard manure and irrigation on crop yields and ammonia volatilization from an alluvial soil under a rice (Oryza sativa L.)-wheat (Triticum aestivum L.) cropping system

Volatilization of NH₃ from soil is a major N-loss mechanism that reduces the efficiency of applied N fertilizers, and causes environmental pollution. Strategies are needed to reduce the loss. The influences of dicyandiamide (DCD), farmyard manure (FYM) and irrigation on NH₃ volatilization from an alluvial soil in rice (Oryza sativa L.)-wheat (Triticum aestivum L.) cropping system was studied using the acid trap method. The loss of NH₃ in the rice-wheat system ranged from 38.6 kg N ha^-1 from the unfertilized soil to 69.0 kg N ha^-1 in the treatment with urea+DCD. Substitution of 50% N provided through urea by FYM reduced NH₃-N volatilization by 10% in rice and wheat as compared to the urea treatment. Application of DCD increased NH₃ volatilization in wheat by 7% but in rice it had no effect. The irrigation level had no effect on NH₃ volatilization in rice but fewer irrigations with fewer splits of N in wheat resulted in higher NH₃ volatilization. Application of DCD and FYM with urea had similar effects on grain yield and N uptake by rice and wheat as that of the urea treatment. The study showed that integrated use of organic manure and chemical fertilizer has the potential to reduce the loss of N due to volatilization and thereby minimize environmental pollution. Nitrification inhibitors, which are reported to be useful in increasing the N-use efficiency by reducing the leaching and denitrification losses of N, however, may increase N loss due to volatilization.     

Assessment of soil factors limiting growth and establishment of Mucuna in farmers' fields in the derived savanna of the Benin Republic

Mucuna pruriens was been used to control Imperata cylindrica and improve soil fertility in maize and cassava cropping systems in the derived savanna of the Benin Republic, West Africa. However, field observations showed that Mucuna had poor establishment in some farmers' fields. This could be due in part to the poor symbiotic effectiveness of Mucuna and/or its poor nutrition because of mineral deficiencies in the soil. A short-term survey was carried out in 34 farmers' fields located in four different sites (Zouzouvou, Eglime, Tchi, and Niaouli) in the derived savanna to assess the natural nodulation and mycorrhizal infection of Mucuna. This survey was followed by a nutrient-omission trial conducted in a pot experiment using soil collected from two groups of farmers' fields at Zouzouvou where Mucuna had poor establishment. Mycorrhizal infection ranged from 2 to 31% and correlated positively with nodulation and shoot dry matter production of plants grown only in one site at Zouzouvou. The number of rhizobia ranged between <0.05 (near the detection limit) and 15 cells g^-1 soil depending on the plot history and the fields. Nodulation occurred in 79% of the fields with numbers of nodules ranging from 0 to 135 plant^-1. The nutrient-omission trial showed that when N and P were absent in the complete fertilizer treatment, biomass production decreased significantly, on average by 69% (N) and 33% (P). Mg, S, K and micronutrient deficiencies did not reduce significantly the biomass production in the two groups. However, N fertilizer applied additionally each week to some treatments drastically reduced Mucuna nodulation. Strategies to enhance Mucuna establishment and growth are discussed.     

Implications of Azospirillum brasilense inoculation and nutrient addition on maize in soils of the Brazilian Cerrado under greenhouse and field conditions

The combination of nitrogen and Azospirillum can ensure greater nutrient absorption and crop yield in agricultural areas using high technology. Thus, the objective was to evaluate maize response to Azopirillum brasilense (AZ) inoculation and nutrient (macronutrients and micronutrients) application under greenhouse and field conditions in clay and sandy soils of the Brazilian Cerrado. In the greenhouse assays, the following parameters were measured: shoot dry weight (SDW), root dry weight (RDW), and root volume (RV). In the field experiments, the maize yield was determined after drying the grains at 60 °C for 48 h. In clay soil, there was a significant increase in the SDW, RDW and RV in the treatment with AZ concentrated (10¹¹ cells ml⁻¹ of inoculum) when compared with the control treatment and the treatment with AZ diluted (10⁶ cells ml⁻¹ of inoculum). In this soil, adding micronutrients did not affect the maize response under greenhouse conditions. In sandy soil, there was no difference between the AZ treatment and the control, except for treatments where nutrients and AZ were both added leading to a significant increase in the maize response. In both soils, the RV:RDW ratio was higher in the treatment with AZ concentrated compared to that in the treatment with AZ diluted, but the yield response depended on the addition of nutrients. Inoculation with A. brasilense gave comparable yield to the nitrogen treatment. The grain production was increased by 29% in the treatment with A. brasilense and nitrogen compared to nitrogen fertilization alone. In this study, the yield response was affected significantly when maize was inoculated with A. brasilense, but this response was dependent on the soil type under greenhouse conditions.     

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.     

Evaluation of inoculation procedures for Calliandra calothyrsus Meisn. grown in tree nurseries

A series of inoculation experiments was conducted in glasshouses in Senegal and Kenya to evaluate inoculation procedures designed to optimise nodulation and N₂ fixation of Calliandra calothyrsus Meisn. seedlings. Nodulation and plant growth were used as indices of inoculation success. In an experiment carried out in sterile peat/vermiculite mixture, it was established that inoculation of C. calothyrsus with an effective rhizobial strain at the low rate of 1᎒² rhizobia per seedling was satisfactory for nodulation and growth, but further response occurred at rates of up to 1᎒⁹. A second experiment in (unsterilised) Sangalkam soil (Senegal) containing indigenous rhizobia demonstrated that the most successful form of inoculation was liquid inoculant applied around the root collar immediately after transplanting. This method was more successful than seed inoculation or application of alginate bead inoculant. A third experiment was conducted using filtermud inoculant in Leonard jars and unsterilised Muguga nursery soil from Kenya, containing a large population of indigenous rhizobia. Application of liquid inoculant to seedlings was better than seed inoculation. On the basis of our study, we recommend that C. calothyrsus seedlings raised in the nursery should be inoculated with a liquid inoculant immediately or soon after germination.