Interaction of Bradyrhizobium sp. with an antagonistic actinomycete in culture medium and in soil

Summary Experiments were conducted to determine the extent to which populations of Bradyrhizobium sp. strain Tal 209SR were suppressed by the antagonistic activity of a Streptomyces sp. isolated from a highly weathered tropical soil. The activity of the actinomycete was evaluated in culture medium in the presence or absence of clay minerals and in the soil from which the actinomycete was isolated after sterilization by gamma-irradiation. The presence of the actinomycete in culture medium was associated with a significant reduction in the density of Bradyrhizobium sp. (Tal 209SR). Nevertheless, the density of the Bradyrhizobium sp. surviving in the medium in the presence of the actinomycete was much higher than that normally observed when comparable densities of rhizobia are introduced into non-sterile soils. The presence of the antagonistic actinomycete in culture medium was associated with a drastic decrease in the optical density of the medium. This decrease was accompanied by a significant decrease in the insoluble exopolysaccharide content of the medium in addition to the significant decrease in bradyrhizobial cells. The actinomycete did not appear to significantly influence the growth and survival of the test Bradyrhizobium sp. in sterile soil. The inability of the actinomycete to significantly antagonize the test bacterium in sterile soil was not adequately explained by the presence of clay minerals.Hawaii Institute of Tropical Agriculture and Human Resources Journal Series No. 3129     

Effect of high temperature on plasmid curing of Rhizobium spp. in relation to nodulation of pigeonpea [Cajanus cajan (L.) Millsp.]

Summary Fifty-six isolates of Rhizobium and Bradyrhizobium spp. (Cajanus) were studied for their plasmid profile and N₂-fixation efficacy. One to three plasmids were reproducibly detected in all the Rhizobium spp. strains but no plasmid was detected in the Bradyrhizobium spp. strains. Rhizobium sp. strain P-1 was mutagenized by Tn5 and three nod^– and six nod⁺fix^– were screened for symbiotic parameters. Neomycin-sensitive mutants were isolated by elevated temperatrue (40°C) from tranconjugants carrying Tn5 insertions. The high temperature cured these mutants from the single large plasmid present in the parent strain P-1. All these cured mutants were nod^–, indicating that the genes for nodulation were present on this plasmid, which is readily cured at a high temperature (40°C). The high temperature in the semi-arid zones of Haryana could be responsible for the low nodulation of pigeonpea because the plasmid carrying the nodulation genes is cured at 40°–45°C giving rise to non-nodulating mutants.     

Rhizobium sp. exopolysaccharide production,nodulation, and acetylene reduction activity (N2-fixation) in pigeon pea (Cajanus cajan L.)

Summary There was no correlation between the quantity of exopolysaccharide produced and acetylene reduction activity by Rhizobium spp. or by Bradyrhizobium spp. (Cajanus). The exopolysaccharide-defective mutants of Rhizobium sp. strain P 116 either failed to nodulate or showed a decrease in effectiveness. The deficiency in exopolysaccharide production was corrected by the addition of purified exopolysaccharide from the parent strain, or from Bradyrhizobium sp. strain P 149 or S24 isolated from pigeonpea (Cajanus cajan) and mungbean (Vigna radiata), respectively. However, the nodules so formed were not fully effective compared to those formed by the parent strain.     

Competitive interaction among strains of Rhizobium leguminosarum bv. phaseoli in the nodulation of kidney beans (Phaseolus vulgaris L.)

We examined the competitiveness of five effective Rhizobium leguminosarum biovar phaseoli strains in the nodulation of kidney beans (Phaseolus vulgaris L.), either alone or in pairwise combination, against the indigenous strains. The results showed that the introduced Rhizobium sp. strains (B2, B17, B36, T2, or CIAT 652) occupying 64–79% of the total nodules (as single inocula) were more competitive in nodulation than the native rhizobia. However, the competitiveness of the individual Rhizobium sp. strain either increased or decreased when used in a pairwise combination of double-strain inocula. For example, strain B17, although quite competitive against the indigenous population (68% nodule occupancy), became poorly competitive in the presence of strain B2 (reduced from 68 to 2.5%). A similar reduction in nodule occupancy by strain B17 was observed in the presence of B36 or CIAT 652, indicating that two competitive strains may not always be compatible. These results suggest that it is important to co-select competitive as well as compatible rhizobia for multistrain inoculant formulation.     

Competition between Rhizobium strain NGR234 and Bradyrhizobium strain CP283 for nodulation in Siratro investigated with the GUS reporter gene

The use of the GUS reporter gene was found to be very suitable for studying the competitiveness of the Rhizobium strain NGR234 and Bradyrhizobium strain CP283 for nodulation in siratro. However, the expression from the transposon mTn5SSgusA20 declined in the nodules of old plants, particularly the nodules at 35 d after inoculation and onward. Siratro inoculated with both gusA-marked NGR234 and CP283 strains showed a similar nodulation and acetylene reduction activity (ARA) to those of their parental strains. No major changes in nodulation and symbiotic properties in these marked strains were observed. When the Rhizobium strain NGR234 and Bradyrhizobium strain CP283 were inoculated separately to siratro, both of them were found equally effective for nodulation in the plant. But when the Rhizobium strain NGR234, and gusAmarked Bradyrhizobium strain CP283 were co-inoculated to the plants in a ratio of 1:1, the strain NGR234 occupied 75% of the nodules, while, the strain CP283 occupied only 25%, irrespective of the growth stages and inoculum concentrations. Similar results were also observed in the plants in which the Bradyrhizobium strain CP283 and gusA-marked Rhizobium strain NGR234 had been co-inoculated. Thus, the Rhizobium strain NGR234 was more competitive for nodulation in siratro.     

Influence of a vesicular-arbuscular mycorrhizal fungus on the competitive ability of Bradyrhizobium spp. for nodulation of cowpea Vigna unguiculata (L.) Walp in non-sterilized soil

Summary We examined the influence of a vesicular-arbuscular (VAM) fungus (Glomus pallidum Hall) on the competitive ability of introduced and native Bradyrhizobium strains to nodulate cowpeas [Vigna unguiculata (L) Walp]. Our experiments in non-sterilized soil revealed that in the presence of VAM fungus, introduced Bradyrhizobium spp. strains become more competitive than native rhizobia. For example, strain JRC29 occupied 59.2% of the total nodules when inoculated alone, but this figure increased to 71.2% when JRC29 was used in dual inoculations with VAM fungus. A similar pattern of enhanced competitiveness for nodule formation was observed with the two other strains in the presence of the VAM fungus. Our results suggest that the competitiveness of rhizobia can be enhanced by co-inoculating with a selected strain of a VAM fungus.     

Phosphate-solubilization activity of bacterial strains in soil and their effect on soybean growth under greenhouse conditions

The efficiency of 13 phosphate-solubilizing bacteria (PSB; four Burkholderia sp., five Enterobacter sp., and four Bradyrhizobium sp.) was assessed in a soil plate assay by evaluating soil phosphorus (P) availability. A commercial argentine strain, Pseudomonas fluorescens, was used for comparing solubilizing activity. Burkholderia sp. PER2F, Enterobacter sp. PER3G, and Bradyrhizobium sp. PER2H strains solubilized the largest quantities of P in the soil plate assay after 60 days as compared with the other strains, including the commercial one. The effect of PSB inoculation on growth and nutrient uptake of soybean plants was also studied under greenhouse conditions. Plants inoculated with Burkholderia sp. PER2F had the highest aerial height and showed an appropriate N/P ratio. However, none of the PSB increased P uptake by plants. This suggests that PSB inoculation does not necessarily improve P nutrition in soybean, nor was there any relationship between P availability in the soil plate assay and P content in the soybean shoot in the greenhouse. We concluded that the selection of efficient PSB strains as possible inoculation tools for P-deficient soils should focus on the integral interpretation of soil assays, greenhouse experiments, and field trials.     

Use of poultry manure and plant cultivation for the reclamation of burnt soils

Annual (Pisum sativum L. and Vicia sativa L.) and perennial (Trifolium repens L. and Lotus corniculatus L.) leguminous species were grown in pots containing samples from the ash layers of two Cambisols under Pinus sylvestris L., which has been affected by high-intensity wildfires 3 and 15 days before the sampling. The gramineous Lolium perenne L. was cultivated as a second plant after Trifolium and Lotus harvesting. Three treatments were compared: soils without fertilization and soils fertilized with two doses of poultry manure (1 and 2 g total N kg^-1 dry soil). The aim of the work was to study the capacity of the ash layer to sustain vegetation and the influence of plants and organic manure on the recovery of vegetation cover, ash layer fixation and soil structure formation to avoid erosion. The ash samples were able to sustain vegetation without fertilization. The organic manure increased the yields of all the plants tested, the lower dose being the optimal for the first crop whereas the higher dose was beneficial for the second crop. The annual legumes grew very quickly. The mixture of Trifolium and Lotus seemed very suitable for reclamation of soil degraded by wildfires because Trifolium produced more phytomass than Lotus in the first growing stages whereas the development of Lotus was higher in the later growing stages. Ash layer conditions did not inhibit nodulation, which was, however, stimulated by the organic manure, particularly in the case of Lotus. Lolium after perennial legumes was the best plant combination because it produced the highest phytomass, particularly root phytomass, and thus improved vegetation cover and ash layer fixation. All the plants tested improved the formation of soil aggregates, particularly the combination of perennial legumes and Lolium. However, wet aggregate stability was higher when plants were grown on soils fertilized with poultry manure than when plants were cropped on unmanured soils, which points to the favourable influence of the organic manure on soil aggregation.     

Differential effect of various endomycorrhizal fungi on nodulating ability of green gram by Bradyrhizobium sp. (Vigna) strain S24

Bradyrhizobium sp. (Vigna) strain S24 interacted differentially with eight vesicular-arbuscular mycorrhizal (VAM) fungi and caused significant variations in nodulation and growth parameters of green gram. Coinoculation with Scutellospora calospora resulted in the highest nitrogenase activity and dry biomass. The nodulation competitiveness of strain S24 was significantly higher (60–63%) in the presence of Glomus mosseae, G. fasciculatum and Scutellospora calospora when compared to treatment with single inoculation of S24 (51%). Percentage VAM colonization was higher in treatments having higher nocule occupancy of introduced strain (S24). Received: 26 June 1995     

Competition among strains of Bradyrhizobium and vesicular-arbuscular mycorrhizae for groundnut (Arachis hypogaea L.) root infection and their effect on plant growth and yield

Summary Strains of Bradyrhizobium influenced root colonization by a species of vesicular-arbuscular mycorrhizae (VAM), and species of VAM influenced root nodulation by strains of Bradyrhizobium in pot experiments. In a field experiment, the effects of VAM on competition amongst inoculated bradyrhizobia were less evident, but inoculation with Bradyrhizobium strains increased root colonization by VAM. Certain VAM/Bradyrhizobium inoculum strain combinations produced higher nodule numbers. Plants grown without Bradyrhizobium and VAM, but supplied with ammonium nitrate (300 g ml^–1) and potassium phosphate (16 g ml^–1), produced higher dry-matter yields than those inoculated with both symbionts in the pot experiment. Inoculation with either symbiont in the field did not result in higher pod and haulm yields at harvest.ICRISAT Journal Article No. 886     

高效亲和的花生寄主植物-根瘤菌株组合

本文针对栽培花生，从大、小共生体双方研究了寄主植物－根瘤菌株组合的亲和性。结果指出：（１）在温室水培盆栽条件下，用菌株１４７－３接种的寄主植物结瘤、固氮能力育成品种〉普通型；（２）血清学鉴定出５个不同血清型菌株，其竞争力或回收率与寄主品种、根瘤菌株和土著菌数密切相关；（３）在温室与田间条件下，不同花生寄主一根瘤菌株组合存在着广谱或特异共生亲和性与非亲和性的差异，并鉴定出高效、广谱亲和的品种徐州６－     

Nodulation competitiveness of Rhizobium leguminosarum bv. phaseoli and Rhizobium tropici strains measured by glucuronidase (gus) gene fusion

Summary The nodulation competitiveness of 17 Rhizobium leguminosarum bv. phaseoli and 3 R. tropici strains was analysed in growth pouches, at pH 5.2 and 6.4. All 20 strains were coinoculated with a gus ⁺ strain of R. leguminosarum bv. phaseoli strain KIM5s. The gus⁺ phenotype, carrying the glucuronidase gene, was used to type nodules directly in the growth pouches. Nodule occupancy ranged from 4% for the least competitive to 96% for the most competitive R. leguminosarum bv. phaseoli strain. The R. tropici strains showed low rates of nodule occupancy at pH 6.4 but their competitiveness improved significantly under acid conditions. CIAT 895 was the only R. leguminosarum bv. phaseoli strain that was less competitive (P<0.05) at the lower pH. The competitiveness of all the other R. leguminosarum bv. phaseoli strains was unaffected by pH. Various physiological and genetic properties of the strains were analysed in search of correlations with nodulation competitiveness. Hybridisation patterns with three different DNA probes (nif KDH, common nod genes, and hup genes) and the metabolism of 53 different C sources were compared. No general correlations were found between hybridisation or growth pattern and competitiveness. The less competitive R. tropici strains had a unique DNA hybridisation pattern and were not able to use shikimate, ferulate, coumarate, or asparagine as C sources. Most of the less competitive R. leguminosarum bv. phaseoli strains could not metabolize either ferulate or coumarate. This might indicate a relationship between nodulation competitiveness and the ability to degrade aromatic compounds.     

Nodulation status of native woody legumes and phenotypic characteristics of associated rhizobia in soils of southern Ethiopia

The nodulation of provenances of Acacia seyal, Acacia tortilis and Faidherbia albida, and other indigenous multipurpose tree species were tested in 14 different soil samples collected from diverse agro-ecological zones in southern Ethiopia. Associated rhizobia were isolated from these and from excavated nodules of field standing mature trees, and phenotypically characterized. Indigenous rhizobia capable of eliciting nodules on at least one or more of the woody legume species tested were present in most of the soils. Tree species were markedly different in nodulation in the different site soils. Sesbania sesban and Acacia abyssinica showed higher nodulation ability across the different sites indicating widespread occurrence of compatible rhizobia in the soils. The nodulation patterns of the different provenances of Acacia spp. suggested the existence of intraspecific provenance variations in rhizobial affinity which can be exploited to improve N fixation through tree selection. Altogether, 241 isolates were recovered from the root nodules of trap host species and from excavated nodules. Isolates were differentiated by growth rate and colony morphology and there were very fast-, fast-, slow-, and very slow-growing rhizobia. The bulk of them (68.5%) were fast-growing acid-producing rhizobia while 25.3% were slow-growing alkali-producing types. Fast-growing alkali-producing (2.9%) and slow-growing acid-producing strains (3.3%) were isolated from trap host species and excavated nodules, respectively. All isolates fell into four colony types: watery translucent, white translucent, dull glistering and milky (curdled) type. The diversity of indigenous rhizobia in growth rate and colony morphology suggested that the collection probably includes several rhizobial genera.     

Population size and N2-fixing activity of native peanut rhizobia in soils of Thailand

Summary The objective of this study was to assess the number and effectiveness of peanut rhizobia in soils of the major peanut-growing areas of Thailand. Three cropping areas, (1) continuously cropped with peanuts, (2) continuously cropped with non-legumes, and (3) non-cultivated fields, were chosen in each region. Peanut rhizobia were found in the soil at 38 to 55 sites sampled. Cultivated fields with a peanut cultivation history contained (as estimated by most probable numbers) an average of 1.6×10³ cells g^-1 of soil. The numbers of peanut rhizobia in most of the fallow fields and some of the noncultivated shrub or forest locations were much the same as at the sites where Arachis hypogaea was cultivated. In contrast, there were no or few (28–46 cells g^-1 soil) peanut rhizobia in the majority of fields continuously cultivated with sugarcane, cassava, corn, and pineapple. It appears that in these areas the indigenous peanut rhizobial populations are not adequate in number for a maximal nodulation of peanuts. A total of 343 Bradyrhizobium isolates were tested for effectiveness and were found to vary widely in their ability to fix N₂. In some areas the majority of rhizobia were quite effective while in others they were less effective than the inoculum strain THA 205 recommended in Thailand.     

Survival and competitiveness of Bradyrhizobium sp. in the rhizosphere of pigeonpea (Cajanus cajan)

Summary The survival of Bradyrhizobium Cajanus strain CC1021 in the soil and rhizosphere of pigeonpea [Cajanus cajan (L.) Millsp] was determined by most probable number and fluorescent antibody techniques. The survival of strain CC1021 was poor under field compared with pot-culture conditions. Although the rhizosphere of pigeonpea promoted the growth of native pigeonpea rhizobia, although no increase in the number of rhizobia was observed with the inoculant strain. Under similar conditions of rhizosphere colonization the competitiveness of strain CC1021 with cv UPAS 120 and ICPL 312 was 10% and 66016 during the first year and 8.4% and 33.3%, respectively, during the second year.     

Iron requirement for soybean [Glycine max (L.) Merrill] inoculated with selected exotic and native isolates of Bradyrhizobium sp. under irrigated conditions

A field and greenhouse experiments were conducted to determine the requirement of Fe nutrient supplied through foliar and soil application in soybean inoculated with different selected isolates of exotic and native Bradyrhizobium spp. in saline soils. Six soybean genotypes and three Bradyrhizobium spp. were used for the greenhouse experiments, whereas only two soybean genotypes, namely TGx-1336424 and GIZA, were selected for further study under field conditions. Two levels of FeSO₄ (0 and 4 mg Fe kg^?1 soil) directly supplied to the soil and three levels of Fe-ethylenediaminetetraacetic acid (0–2% of Fe) through foliar application were used for greenhouse and field experiments, respectively. The results of the greenhouse experiment indicated a non-significant effect of Fe application on nodulation and shoot biomass in soybean. Fe application did not improve the grain yield and total biomass yield in soybean inoculated with UK isolate and local isolate but showed remarkable improvement with TAL-379. High soil native N might be the cause for insignificant effect of Fe applied at 2% in highly effective inoculated plants. Therefore, it can be concluded that the symbiotic effectiveness of Bradyrhizobium sp. and the native soil N would affect the soybean Fe requirement supplied through foliar application.     

Growth and yield of soybean varieties inoculated with <Emphasis Type="Italic">Bradyrhizobium</Emphasis> spp in N-deficient calcareous soils

This research reports the effect of Bradyrhizobium spp strains on dry matter yield, nodulation and seed yield of soybean varieties grown in N-deficient soil in Uzbekistan in pot and field experiments, in 2001 and 2002. The results of experiments revealed that significant positive effects on growth, nodule number and yield of soybean were obtained after inoculation with Bradyrhizobium spp strains; and, generally, strains S62 and S63 were more effective than strain S61. The protein content of seeds also increased after inoculation. Both Orzu and Uzb2 soybean varieties gave a higher response than the Uzb6 variety.     

Displacement of native rhizobia nodulating chickpea (Cicer arietinum L.) by an inoculant strain through soil solarization

Summary Soil solarization greatly reduced the native chickpea Rhizobium population. With inoculation, it was possible to increase the population of the Rhizobium in solarized plots. In the 1st year, 47% nodulation was obtained with chickpea inoculant strain IC 59 when introduced with a cereal crop 2 weeks after the soil solarization and having a native Rhizobium count of <10 g^-1 soil, and only 13% when introduced 16 weeks after solarization at the time the chickpeas were sown, with 2.0×10² native rhizobia g^-1 soil. In the non-solarized plots inoculated with 5.6×10³ native rhizobia g^-1 soil, only 6% nodulation was obtained with the inoculant. In the succeeding year, non-inoculated chickpea was grown on the same plots without any solarization or Rhizobium inoculation. The treatment that showed good establishment of the inoculant strain in year 1 formed 68% inoculant nodules. Other treatments indicated a further reduction in inoculant success, from 1%–13% to 1%–9%. Soil solarization thus allowed an inoculant strain to successfully displace the high native population in the field and can serve as a research tool to compare strains in the field, irrespective of competitive ability. In year 1, Rhizobium inoculation of chickpea gave increased nodulation and increased plant growth 20 and 51 days after sowing, and increased dry matter, grain yield, and grain protein yield at maturity. These beneficial effects of inoculation on plant growth and yield were not measured in the 2nd year.Submitted as Journal Article No. JA 945 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, Andhra Pradesh 502 324, India     

Competition and persistence of Rhizobium tropici and Rhizobium etli in tropical soil during successive bean (Phaseolus vulgaris L.) cultures

Strains of Rhizobium tropici IIB, CIAT899 and F98.5, both showing good N₂ fixation, and a R. etli strain W16.3SB were introduced into a field which had no history of bean culture. Plant dilution estimates showed that in the presence of its host (Phaseolus vulgaris cv. Carioca) during the cropping seasons and the subsequent fallow summer periods, the bean rhizobial populations increased from less than 30 to 10³ g^–1 dry soil after 1 year and to 10⁴ g^–1 dry soil after 2 years. In the 1st year crop, the inoculated strains occupied most of the nodules, which resulted in a higher nodulation and C₂H₂ reduction activity. Without reinoculation for the second and third crops, however, little R. tropici IIB was recovered from the nodules and the bean population consisted mainly of R. etli, R. leguminosarum bv. phaseoli, and R. tropici IIA. Reinoculation with our superior R. tropici IIB strains before the second crop resulted in R. tropici IIB occupying the main part of the nodules and a positive effect on nodulation and C₂H₂ reduction activity, but reintroduction of the inoculant strain in the third season did not have any effect.