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To find wide host range compatible potential inoculant strains with high efficiency in N fixation, we isolated rhizobia from soybean root nodules in Sichuan. Most of the isolated rhizobia were no- or low-effective strains. Eight out of 75 isolates promoted significantly the growth of soybean cultivar Gongxuan No. 1. When tested with three more soybean cultivars, the isolates assigned as Bradyrhizobium japonicum SCAUs36, Bradyrhizobium diazoefficiens SCAUs46, and Ensifer fredii SCAUs65 promoted significantly (P

Competitiveness for nodulation is one of the major restrictive factors in symbiotic nitrogen fixation between rhizobia and their host legumes. Soybean root exudates that include a variety of compounds are thought to act as signals to trigger the early symbiotic events between Bradyrhizobium diazoefficiens and soybeans, and thus they act as a key determinant of the competitiveness for nodulation. To gain a better understanding of the molecular mechanism of competitiveness at the level of protein expression, we compared the proteomic responses of two B. diazoefficiens strains that demonstrated completely different nodulation abilities, strain 4534 being the most competitive and strain 4222 being the least competitive in nodulation. In the proteomic analysis, 40 of the 65 and 22 of the 29 differential proteins were identified in response to soybean root exudates in strain 4534 and strain 4222, respectively. Compared to strain 4222, a higher amount and a number of differential proteins were detected in strain 4534, including S-adenosylmethionine synthetase (SAMS), PhyR-σEcfG regulon, ABC-type transporters, flagellar proteins, molecular chaperones, and proteins involved in redox state maintenance as well as several unknown proteins. Noteworthy was the induction of the PhyR-σEcfG regulon and flagellar proteins, recently demonstrated to be involved in the competitiveness for nodulation in Bradyrhizobium japonicum. Our results indicate that the role of root exudates can go far beyond inducing the expression of nodulation genes in B. diazoefficiens. Many other proteins/enzymes involved in the metabolism and environmental fitness were also upregulated when exposed to root exudates. More proteins were upregulated by the high nodulation competitive strain than that by the low, and the reasons for this need further investigation. The outcome of such study may contribute to our understanding of molecular mechanisms of different competitiveness in B. diazoefficiens as well as specific adaptation in the legume host.

Plant–microorganism associations have long been studied, but their exploitation in agriculture partially or fully replacing chemical fertilizers is still modest. In this study, we evaluated the combined action of rhizobial and plant growth-promoting rhizobacteria inoculants on the yields of soybean and common bean. Seed inoculation with rhizobia (1.2 × 106 cells seed−1) was compared to co-inoculation with Azospirillum brasilense in-furrow (different doses) or on seeds (1.2 × 105 cells seed−1) in nine field experiments. The best in-furrow inoculant dose was 2.5 × 105 cells of A. brasilense seed−1 for both crops. Inoculation with Bradyrhizobium japonicum increased soybean yield by an average 222 kg ha−1 (8.4 %), and co-inoculation with A. brasilense in-furrow by an average 427 kg ha−1 (16.1 %); inoculation always improved nodulation. Seed co-inoculation with both microorganisms resulted in a mean yield increase of 420 kg ha−1 (14.1 %) in soybean relative to the non-inoculated control. For common bean, seed inoculation with Rhizobium tropici increased yield by 98 kg ha−1 (8.3 %), while co-inoculation with A. brasilense in-furrow resulted in the impressive increase of 285 kg ha−1 (19.6 %). The cheaper, more sustainable inoculated treatment produced yields equivalent to the more expensive non-inoculated + N-fertilizer treatment. The results confirm the feasibility of using rhizobia and azospirilla as inoculants in a broad range of agricultural systems, replacing expensive and environmentally unfriendly N-fertilizers.

Elucidating the biodiversity of CO2-assimilating bacterial communities under different land uses is critical for establishing an integrated view of the carbon sequestration in agricultural systems. We therefore determined the abundance and diversity of CO2 assimilating bacteria using terminal restriction fragment length polymorphism and quantitative PCR of the cbbL gene (which encodes ribulose-1,5-biphosphate carboxylase/oxygenase). These analyses used agricultural soils collected from a long-term experiment (Pantang Agroecosystem) in subtropical China. Soils under three typical land uses, i.e., rice–rice (RR), upland crop (UC), and paddy rice–upland crop rotation (PU), were selected. The abundance of bacterial cbbL (0.04 to 1.25 × 108 copies g−1 soil) and 16S rDNA genes (0.05–3.00 × 1010 copies g−1 soil) were determined in these soils. They generally followed the trend RR > PU > UC. The cbbL-containing bacterial communities were dominated by facultative autotrophic bacteria such as Mycobacterium sp., Rhodopseudomonas palustris, Bradyrhizobium japonicum, Ralstonia eutropha, and Alcaligenes eutrophus. Additionally, the cbbL-containing bacterial community composition in RR soil differed from that in upland crop and paddy rice–upland crop rotations soils. Soil organic matter was the most highly statistically significant factor which positively influenced the size of the cbbL-containing population. The RR management produced the greatest abundance and diversity of cbbL-containing bacteria. These results offer new insights into the importance of microbial autotrophic CO2 fixation in soil C cycling.

Carrier-based (soil/FYM, 1:1) plant growth-promoting rhizobacteria (PGPR) isolates (Bacillus subtilis, Klebsiella planticola and Proteus vulgaris) were tested individually and in combination with Bradyrhizobium japonicum and Rhizobium leguminosarum biovar viciae under field conditions on soybean and lentil crops, respectively, under field conditions. Inoculation of soybean (Glycine max) cv. Pusa 22 with B. subtilis produced maximum nodule number, mass and nitrogenase activity (acetylene reduction activity, ARA) followed by B. japonicum (SB 271). Maximum soybean yield was registered with the coinoculation of B. japonicum and B. subtilis over an uninoculated control. Maximum nodulation in the lentil (Lens culinaris) cv. L 4147 was obtained with a combination of R. leguminosarum (L-12-87) and P. vulgaris inoculation followed by a single inoculation with Rhizobium and B. subtilis. None of the PGPR isolates either singly or in coinoculation with R. leguminosarum could significantly influence the yield of the lentil crop.

Due to their ecologic and economic importance, bradyrhizobia have been extensively studied in recent years. Since 1992, Bradyrhizobium elkanii SEMIA 587 and SEMIA 5019 and Bradyrhizobium japonicum SEMIA 5079 and SEMIA 5080 have been widely used in most Brazilian soybean fields. The objective of this work was to estimate the genetic variability of bradyrhizobial isolates recovered from soils under rhizobial inoculation and different soil managements. Only 25% of the isolates demonstrated high similarities to the original strains, and a strong correlation was obtained between the bradyrhizobial genetic variability and soil management. A high level of genetic diversity was observed both within isolates (H = 5.46) as well as among the different soil practices. Soil under no-tillage presented a higher bradyrhizobia diversity compared with bradyrhizobia isolated from soil under conventional tillage. Serological characterization also indicated that B. elkanii strains SEMIA 587 and SEMIA 5019 were more competitive and presented a higher nodular occupancy capacity than strains belonging to B. japonicum species in Southern Brazilian soils.

A study on the diversity, phylogeny, and host specificity of soybean (Glycine max L.) and peanut (Arachis hypogaea L.) bradyrhizobia was conducted based on the 16S ribosomal RNA (rRNA) restriction fragment length polymorphisms (RFLPs), 16S rRNA sequencing, and 16S–23S rRNA intergenetic spacer (IGS) RFLP assays. Based on 16S rRNA RFLP assay, tested bradyrhizobia were divided into five genotypes, which could be further clustered into five groups by IGS RFLP assays. According to the 16S rRNA sequencing, strains of IGS-II, IV, and V were phylogenetically related to Bradyrhizobium liaoningense, Bradyrhizobium japonicum, and Bradyrhizobium elkanii, while strains of IGS-Ic and IGS-III related to Bradyrhizobium yuanmingense and Bradyrhizobium canariense, respectively. All isolates could crossly nodulate Phaseolus vulgaris, forming small white nodules. Strains of IGS-II originally isolated from peanut could efficiently nodulate Glycine soja, and two strains isolated from soybean could also nodulate peanut.

The purpose of this study was to evaluate, during the phenological stages of inoculated soybean crop [Glycine max (L.) Merrill], the effect of different N fertilization levels and inoculation with Bradyrhizobium japonicum on N 2O emissions from the soil. Gas emissions were evaluated at field conditions by the static-chamber method. Nitrogen fertilization increased N 2O emissions significantly (P

Understanding the interaction mechanisms between plant growth-promoting rhizobacteria (PGPR), leguminous crops, and rhizobia is necessary to effectively use PGPR in increasing the biological nitrogen fixation of legumes. We determined the coinoculation effects of Bradyrhizobium japonicum A1017 and a gusA-marked strain of Pseudomonas fluorescens 2137, P. fluorescens WCS365, Azomonas agilis 125, and Azospirillum lipoferum 137 on soybean [Glycine max (L.) Merr] cv. Enrei grown under axenic conditions. The gusA-marked rhizobacteria effectively colonized the root tips and surfaces near the roots tips with a colonization rate ranging from 7.50 to 8.62 log colony forming units (cfu) gfw–1. P. fluorescens 2137 had the highest colonization activity on soybean roots whether inoculated alone or coinoculated with B. japonicum A1017. Coinoculation of P. fluorescens 2137 and B. japonicum A1017 increased the colonization of B. japonicum A1017 on soybean roots, nodule number, and acetylene reduction activity (ARA) at 10 and 20 days after inoculation. Moreover, the addition of sterile spent medium of P. fluorescens 2137 increased the growth of B. japonicum A1017 in yeast mannitol broth (YMB), indicating that P. fluorescens 2137 may have released substances that increased the rhizobial population. The results of this study suggest that the enhanced nodulation and ARA of soybean due to the high colonization of P. fluorescens on soybean roots could depend on the production of growth-promoting substances that stimulate the growth of B. japonicum. However, coinoculation with P. fluorescens WCS365 decreased the nodule number and ARA, despite its slight stimulation of the growth of B. japonicum on the roots, indicating that coinoculation effects are strain dependent.

The symbiotic effectiveness of 30 fast-growing rhizobial strains (doubling times of 85–225 min and acid reaction in yeast mannitol medium) isolated from soybean nodules in Brazil and of Sinorhizobium fredii reference strains was evaluated under greenhouse and field conditions. Most Brazilian fast-growing strains were genetically related to the Rhizobium tropici-Rhizobium genomic species Q-Agrobacterium spp. branch and five to the Bradyrhizobium japonicum and B. elkanii species. Under axenic conditions, some of the fast-growing strains fixed as much N2 as the B. japonicum/B. elkanii strains carried in Brazilian commercial inocula. However, in a co-inoculation experiment, very few strains were able to compete against B. elkanii strain SEMIA 5019. Although isolated from acid soils (pH 3.0–5.1), the competitiveness of Brazilian fast growers and of S. fredii reference strains against B. japonicum/B. elkanii was low under acid conditions (pH 5.1 and pH 5.4), but increased when the pH was raised to 6.8 and 7.9. Therefore, as the great majority of Brazilian soils are acidic and show a very high population of naturalized B. japonicum/B. elkanii, the low competitiveness of S. fredii and of the Brazilian rhizobial strains investigated in this study, under the given conditions, limits, at this time, their recommendation for use in commercial inocula.

Bradyrhizobium japonicum strain CB 1809 was recently chosen to replace strain WB 1 in commercial soybean [Glycine max (L.) Merr.] inoculants in South Africa, the selection criterion being N2-fixing effectiveness. Nodulation competitiveness is an additional characteristic required of inoculants and was determined for CB 1809 and WB 1 as well as two other strains, USDA 110 and a Brazilian strain 965, using the gusA marker gene to identify strains. Initial experiments with plants grown in sterile sand showed that the competitive index of strain WB 1 was less than that of the other strains. Further comparisons used plants grown in five soils containing established populations of B. japonicum. When strains were applied in peat inoculum to seed at a rate of 1,000 cells per seed in a soil containing 300 rhizobia g–1, significant differences in nodule occupancy were detected and strains ranked in the order 965>CB 1809>USDA 110>WB 1. The remaining four soils each contained about 106 rhizobia g–1 and 5×106 cells were applied per seed. Nodule occupancy by inoculant strains ranged from 22% to 81% between soils. In this experiment, WB 1 was consistently the poorest performer and its competitiveness was significantly less than CB 1809. The competition results supported the recent decision to replace WB 1 with CB 1809 in commercial inoculants. Although WB 1 had been used in inoculants over a period of 19 years, this strain was detected in only one soil, where it comprised 8% of isolates. In contrast, a substantial proportion (32–78%) of isolates from the soils corresponded serologically to a former inoculant strain WB 66, which had been discontinued in 1966. This illustrates the difficulty of replacing a resident population with an introduced strain. The effect of naturalized populations on the establishment of CB 1809 in South African soils will need monitoring

Soybean cultivars capable of nodulating with indigenous Bradyrhizobium spp. have been developed by the International Institute of Tropical Agriculture (IITA) and national programs in Africa in order to avoid artificial inoculation by resource-poor farmers in Africa. The current selection procedure for enhanced N2 fixation is based on an assessment of nodule formation which does not directly quantify the proportions of crop N derived from the atmosphere. We have monitored N accumulation patterns and N2 fixation in nine promiscuous soybean cultivars with different maturity periods, using the 15N dilution technique. Nodule development generally peaked at the early podfill stage for all cultivars except Tgx 1519-1D and Tgx 1447-2D in which it continued to increase. The proportion of crop N derived from fixation (%NDFA) ranged between 51% and 67%, 77% and 84%, and 66% and 73% at full bloom, early podfill, and physiological maturity stages, respectively. Total N accumulation increased in all soybean genotypes with increasing plant age. Significant correlations (P

In a previous study soybean Bradyrhizobium strains, used in Brazilian studies and inoculants over the last 30 years, and strains adapted to the Brazilian Cerrados, a region frequently submitted to environmental and nutritional stresses, were analyzed for 32 morphological and physiological parameters in vivo and in vitro. A cluster analysis allowed the subdivision of these strains into species Bradyrhizobium japonicum, Bradyrhizobium elkanii and a mixed genotype. In this study, the bacteria were analyzed for nodulation, N2 fixation capacity, nodule occupancy and the ability to increase yield. The goal was to find a relationship between the strain groups and the symbiotic performance. Two strains of Brazilian B. japonicum showed higher rates of N2 fixation and nodule efficiency (mg of N mg–1 of nodules) under axenic conditions. These strains also showed greater yield increases in field experiments when compared to B. elkanii strains. However, no differences were detected between B. japonicum and B. elkanii strains when comparing nodule occupancy capacity. The adapted strains belonging to the serogroup B. elkanii SEMIA 566, most clustered in a mixed genotype, were more competitive than the parental strain, and some showed a higher capacity of N2 fixation. Some of the adapted strains, such as S-370 and S-372, have shown similar N2 fixation rates and nodulation competitiveness to two Brazilian strains of B. japonicum. This similarity demonstrates the possibility of enhancing N2 fixing ability, after local adaptation, even within B. elkanii species. Differences in the DNA profiles were also detected between the parental SEMIA 566 and the adapted strains by analyses with the ERIC and REP-PCR techniques. Consequently, genetic, morphological and physiological changes can be a result of adaptation of rhizobia to the soil. This variability can be used to select strains capable of increasing the contribution of N2 fixation to soybean nutrition.

The fungicide thiram, widely used as a chemical seed protectant, induces a strong inhibition of primary nodulation in the crown zone of soybean roots. The present work reports on the isolation of Bradyrhizobium japonicum strains resistant to thiram, some of which (T3B, A86 and A2) maintained their capacity for nodulation and were still efficient symbionts, but some (A1, C1 and C6) lost the ability to stimulate nodulation. Characterization tests such as growth at different pH, denitrifying ability, salt tolerance, production of siderophores and phosphate solubilization were performed on the resistant strains. Inoculants produced from these strains could be appropriate for use with thiram-treated seeds, without causing a loss of bacteria viability.

Several years of research have shown that there is a high genetic and physiological variability among Bradyrhizobium japonicum strains, culminating in a subdivision into two bacterial genotypes, and the description of the new species B. elkanii. In Brazil, large-scale soybean inoculation started in 1960 and today 15 million doses of inoculants are sold per year for an estimated area of 12 million ha. Efforts have been made to find strains able to fix high amounts of N2 under Brazilian soil conditions, but few laboratories cover basic studies on N2 fixation, such as strain classification into the two Bradyrhizobium species. In this study several characteristics of 40 soybean Bradyrhizobium strains, including 4 reference strains of B. japonicum (genotype I) species, 3 of B. elkanii (genotype II) and 1 of a mixed genotype were evaluated. The parameters analysed in vitro were: colony morphology, serological grouping, intrinsic resistance to antibiotics, synthesis of indole acetic acid, expression of hydrogenase activity and growth in a medium enriched with asparagine. In vivo, analyses performed included the nodulation of Rj 4 soybean cultivar Hill and the detection of symptoms caused by rhizobitoxine. These evaluations allowed a phenotypic grouping which positioned most of the strains utilized in Brazilian inoculants and studies, as well as some new strains isolated from the Cerrado region, within the species B. elkanii. However, environmental stresses and adaptation of Bradyrhizobium strains to the soil caused a large physiological and genetic variability in some isolates from the Cerrado soils in relation to the putative parental strain introduced 15 years ago, placing these isolates in an intermediate position between the two Bradyrhizobium species.

Interstrain competitiveness is a key factor affecting the performance of rhizobium inoculant. In the present study five native strains of Bradyrhizobium japonicum, namely SSF 4, SSF 5, SSF 6, SSF 7 and SSF 8, were assessed for their competitiveness in nodulating soybean using serological methods. The strains were inoculated individually or with the type strain USDA 110 at a 1:1 ratio. Nodule occupancy determined by immunofluorescence and dot immunoblot assay revealed that under in vitro conditions SSF 8 is more competitive than USDA 110 whereas the others were less competitive. The competitive ability of these strains was also estimated in pot culture in the field. In red soil both SSF 8 and USDA 110 were equally competitive whereas in black soil SSF 8 competed better than USDA 110 and produced more nodules. In a black soil field trial using a randomized block design, USDA 110 or SSF 8, when inoculated alone, occupied the majority of the nodules and enhanced nodule dry weight and shoot biomass. SSF 8 was more competitive when the strains were co-inoculated.

In 1993 and 1994, 12 bacterial isolates were isolated from root nodules of cicer milkvetch (Astragalus cicer). In the tests for nodulation of A. cicer by these bacterial isolates, five were found to form hypertrophic structures, while only two formed true nodules. These true nodules were formed in a sterilized soil system. This system might be able to act as a DNA donor to provide residual DNA to other microbes in the soil. The rhizobial isolates were thought to have lost genetic material crucial to nodulation during the isolation process. This hypothesis was supported by an experiment in which isolate B2 was able to nodulate A. cicer in vermiculite culture after being mixed with heat-killed rhizobia, Rhizobium leguminosarum bv. trifolii and R. loti. The nodulation would not occur in vermiculite culture system without the heat-killed rhizobia. Based on the biochemical data, the B2 and 9462L, which formed true nodules with A. cicer, were closely related. The rhizobia type cultures that nodulate A. cicer include Bradyrhizobium japonicum, Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viceae, and R. loti. All of these rhizobia were from different cross-inoculation groups. The B2 and 9462L isolates could only nodulate Medicago sativa, Phaseolus vulgaris, and Melilotus officinalis, but not these species within the genus from which they were isolated: Astragalus. The traditional cross-inoculation group concept obviously does not fit well in the classification of rhizobia associated with Astragalus. The rhizobia isolated from A. cicer can be quite different, and the rhizobia able to renodulate A. cicer also quite diverse.

The effects of inoculation with Bradyrhizobium japonicum and Azospirillum brasilense strains on the growth of soybean were evaluated with regard to the estimation of N2 fixation using the 15N isotope dilution technique. Inoculation, in general, increased the dry mass of soybean as well as nitrogen content. Dual inoculation with a mixture of B. japonicum and A. brasilense strains was superior over single inoculation with B. japonicum. Nitrogen fixed (Ndfa) varied according to inoculant and soil conditions. Percentages of nitrogen derived from air (% Ndfa) using a non-nodulating isoline were 72% and 76% for B. japonicum and B. japonicum plus A. brasilense, respectively, in non-sterile soil. A similar but higher trend was recorded in sterilized soil, in which the percentages of N2 fixed were 81% and 86% for single and dual inoculation, respectively. The correlation coefficient between N2 fixed and N uptake (r=0.94) and dry mass (r=0.89) was significant. Application of special bacterial inoculants in agricultural systems of Egypt seems to be a promising technology and could be used for improving soybean growth as well as soil fertility, thus minimizing environmental pollution.

N2 fixation, photosynthesis of whole plants and yield increases in soybeans inoculated with mixed cultures of Bradyrhizobium japonicum 110 and Pseudomonas fluorescens 20 or P. fluorescens 21 as well as Glomus mosseae were found in pot experiments in gray forest soil carried out in a growth chamber. The effects of pseudomonads and vesicular-arbuscular (VA) mycorrhizal fungus on these parameters were found to be the same. Dual inoculation of soybeans with mixed cultures of microorganisms stimulated nodulation, nitrogenase activity of nodules and enhanced the amount of “biological” nitrogen in plants as determined by the 15N dilution method in comparison to soybeans inoculated with nodule bacteria alone. An increased leaf area in dually infected soybeans was estimated to be the major factor increasing photosynthesis. P. fluorescens and G. mosseae stimulated plant growth, photosynthesis and nodulation probably due to the production of plant growth-promoting substances. Increasing phosphorus fertilizer rates within the range of 5–40 mg P 100 g-1 1:1 (v/v) soil: sand in a greenhouse experiment led to a subsequent improvement in nodulation, and an enhancement of N2 fixation and yield in soybeans dually inoculated with B. japonicum 110 and P. fluorescens 21. These indexes were considerably higher in P-treated plants inoculated with mixed bacterial culture than in plants inoculated with nodule bacteria alone.

The survival of an antibiotic-resistant mutant of a commercial inoculant Bradyrhizobium japonicum strain, A1017ks, was studied in a volcanic ash soil (Andosol) in comparison with a non-volcanic ash soil (Fluvisol) over a period of 84 days. In a non-sterile soil system, the population decline in the Andosol (15% or 1.2 log units) was larger than in the Fluvisol (6% or 0.54 log units). In both soils, however, the inoculant bradyrhizobium survived at fairly high population levels after the period of incubation [106 and 107 colony-forming units (CFU) g-1 dry soil in the Andosol and Fluvisol, respectively]. In sterile control soil, viable bradyrhizobium cells could not be detected after 1 week of incubation in the Andosol, whereas in the Fluvisol population of introduced bradyrhizobium was maintained throughout the period of incubation. Overall changes in the population of indigenous bacteria and fungi were also monitored. However, no clear pattern of interaction between the inoculant Bradyrhizobium japonicum and the indigenous microbes could be identified. The antibiotic-resistant mutant maintained its resistance in the Fluvisol throughout the 3-month period of incubation, making it a useful model for conducting ecological studies in the soil.

The effects of low temperature and reduced light on a Glycine-Bradyrhizobium-Glomus spp. symbiosis were examined in pot experiments. Soybean plants, Glycine max L. Merr. cv. Tachiyutaka, were grown with N fertilization or inoculation with Bradyrhizobium japonicum plus P fertilization or inoculation with Glomus mosseae in the glasshouse. After the flowering stage, half the pots with soybean plants were subjected to low temperature (15°C 14h/13°C 10 h) with light reduced by shading. At 0, 7, 16, and 28 days after the application of the treatments, the growth, nodulation, vesicular-arbuscular mycorrhizal (VAM) infection and the N and P contents of the soybean plants were measured. In all symbiont-fertilization combinations, the low-temperature treatment reduced the production of dry matter by the soybeans. Nodulation (weight and number) was slightly reduced by this treatment but the proportion of larger nodules was increased. The root length infected by the VAM fungus was little affected by the low-temperature treatment. Both the nodule weight and the infected root length were linearly related to shoot dry weight regardless of treatment and of the symbiont-fertilization combination used. These results suggest that the growth of the symbionts on the root was in balance with the shoot growth of the host, irrespective of climatic conditions, and imply a considerable degree of host control. P inflows to root systems were greatly affected by low-temperature treatment regardless of the symbiont-fertilization combination. This suggests that a simple comparison of P inflows between mycorrhizal and non-mycorrhizal plants may give misleading information on the effects of low temperature or reduced light conditions on P uptake by mycorrhizal plants.

Several commercial fungicide seed treatments were evaluated for their possible effect on the survival of Bradyrhizobium japonicum on seeds and on the nodulation and yield of soybeans in a greenhouse and a field experiment. quinolate Pro (carbendazim and oxine copper), Vitavax 200FF (carboxin and thiram), and Monceren (pencycuron) had a small effect or no effect on the survival of B. japonicum and on the nodulation and yield of soybeans. They can thus be considered compatible with soybean seed inoculation. Germipro UFB (carbendazim and iprodione), Apron 35J (metalaxyl), and Tachigaren (hymexazol) decreased B. japonicum survival and the nodulation and yield of soybeans and thus cannot be considered compatible with soybean seed inoculation.

In Japan some paddy fields are used for upland crops for several years and then returned to paddy fields (paddy-upland rotation). Soybeans (Glycine max L.) are an important summer crop. The ability to denitrify and some characteristics of denitrification by isolated strains of Bradyrhizobium japonicum were investigated to clarify the frequency of denitrifiers in indigenous populations of B. japonicum in fileds under paddyupland rotation. Eight field plots with different cropping systems at two sites were used. The fields consisted of a Gray Lowland Soil, and either soybeans or paddy rice (Oryza sativa L.) was grown as a summer crop, with barley (Hordeum distichum L.) or wheat (Triticum aestivum L.) as a winter crop. All B. japonicum strains present in the plots were able to denitrify. Isolated strains fell into two main groups (groups I and II) according to the rate of denitrification. Strains of group I evolved N2O with C2H2 at a rate comparable to that of Alcaligenes denitrificans IAM 12370, whereas the denitrification activity of group II strains was 100 times lower than that of group I strains. Both group I and II strains occurred in each plot. Amounts of N2O produced by indigenous strains with and without C2H2 suggested that strains of group I and II evolved N2 or N2O, respectively, as the end product of denitrification. One strain (S 107) that was isolated had the highest denitrifying ability with an end product of N2O. These results indicate that indigenous bradyrhizobia may partly contribute to denitrification of field soil under a paddy-upland rotation.

A method was developed to improve the colonizing ability of inoculated strains of root-nodule bacteria using aliette (aluminum tris-O-ethyl phosphonate), a basipetally translocated fungicide. Aliette applied to seeds of alfalfa inoculated with an aliette-resistant strain of Rhizobium meliloti increased the numbers of R. meliloti in the rhizosphere after 3 but not 37 days, increased the number of nodules, and with some seed treatments, increased the growth of alfalfa. The enhanced colonization by R. meliloti as a result of seed treatment with aliette lasted for at least 31 days for alfalfa, although plant weights did not increase, Colonization by R. meliloti was further enhanced if seeds and foliage were treated with the fungicide. Coating seeds or sparaying the foliage with aliette also increased the number and weight of nodules and nitrogenase activity in soybeans inoculated with an aliette-resistant strain of Bradyrhizobium japonicum. The stimulation of B. japonicum in the rhizosphere and of nodulation was evident with successive plantings of soybeans if the seeds for each planting were treated with the chemical, but aliette did not increase the yield of inoculated soybeans in the subsequent plantings. With only the seeds of the first planting of inoculated soybeans treated with aliette, the numbers of B. japonicum in the rhizosphere of subsequent plantings were only occasionally greater and the numbers of nodules on the later plantings were not increased. We suggest that root colonization, nodulation, and N2 fixation by Rhizobium and Bradyrhizobium may be enhanced by the use of basipetally translocated antimicrobial compounds together with root-nodule bacteria that are resistant to those compounds.

β-(Isoxazolin-5-on-2-yl)-alanine (βIA), a heterocyclic non-protein amino acid from root extracts and root exudates of pea seedlings, acts as a potent growth inhibitor of several eukaryotic organisms, including yeasts, phytopathogenic fungi, unicellular green algae, and higher plants. The antibiotic effect on baker's yeast was reversed by l-methionine, l-cysteine, and l-homocysteine. Phytopathogenic fungi such as Botrytis cinerea, Pythium ultimum, and Rhizoctonia solani grown on agar containing βIA were inhibited in the growth of mycelia or in the production of sclerotia. In contrast, no significant inhibition of either Gram-positive or Gram-negative bacteria was observed. Rhizobium leguminosarum, the compatible microsymbiont of Pisum spp., and Rhizobium meliloti were able to tolerate up to 2.9 mM βIA (500 ppm) without any effect on the growth rate. Bradyrhizobium japonicum even gave a positive chemotactic response to βIA. The ecological significance of βIA as a preformed plant protectant during the seedling stage of Pisum spp. and other βIA-containing legumes is discussed.

The diversity of Bradyrhizobium japonicum in agricultural fields has not been well characterized. Therefore a study was conducted to determine the serotypic diversity of B. japonicum both within and among six fields in the Coastal Plain and Piedmont of North Carolina where soybeans [Glycine max (L.) Merr.] are grown. Nodule samples were collected from non-inoculated standing soybean crops. Both nodules and isolates were typed by the enzyme-linked immunosorbent assay (ELISA) technique. Serotypes and their proportions varied both within and among locations. Common serotypes in order of abundance across all sites were 76, M1 (multiple reaction beyween 31 and 94), 94, 24, and 122, and together accounted for over 66% of the typable reactions. No cultivar effect on serotype distribution was observed. Unknown types ranged from 4 to 24%. Based on the total number of serotypes identified and the Shannon diversity index (H′), the mean population diversity was 0.76 for the Piedmont sites and 0.91 for the Coastal Plain sites.

Previous laboratory and greenhouse studies have shown that phages significantly reduce soil populations of homologous rhizobia. Reductions in nodulation and N2 fixation have also been observed. The purpose of the current study was to examine the effect of a phage specific ofBradyrhizobium japonicum USDA 117 on nodulation, nodule occupancy, N2 fixation and soybean growth and yield under field conditions. The phage was inoculated in combination withB. japonicum USDA 117 and/orB. japonicum USDA 110 (resistant strain) into a rhizobia-free sandy loam soil and planted toGlycine max (L.) Merr. “Williams”. When the phage was applied to soil inoculated withB. japonicum USDA 117 alone, significant reductions in nodule weight and number, shoot weight, foliar N, nitrogenase activity, and seed index were observed. When, however, the soil also contained the non-homologous strain,B. japonicum USDA 110, no significant effects on any of these parameters were found. Nodule occupancy by competing strains ofB. japonicum USDA 110 and USDA 117 was also affected by the phage. In soil which did not contain the phage, 46% and 44% of the identified nodules were occupied by USDA 110 and 117, respectively. When the phage was present in the soil, nodule occupancy byB. japonicum USDA 117 was reduced to 23%, while occupancy byB. japonicum USDA 110 was increased to 71%. These results suggest that nodulation by selected strains of rhizobia can be restricted and nodulation by more effective, inoculated strains can be increased through the introduction of a homologous phage to soils.

The genetic relationships among six strains of rhizobia, including three strains of Rhizobium fredii and three strains of Bradyrhizobium japonicum, was determined using random amplified polymorphic DNA (RAPD) technique. In this study, 46 arbitrary 10mer primers were employed for RAPD, generating a total of 251 informative fragments. A dendrogram of phylogenetic relationships among the six strains was constructed. The results indicated that geographical distribution may affect phylogeny, as there were closer relationships among the four Taiwanese strains, SB138, SB562, SB368 and SB651, than between these strains and USDA192, which originated from mainland China. The strain USDA110, obtained from the United States, was used in the parsimony analysis. The greatest similarity (55.6%), existed between two strains of B. japonicum, SB562 and SB138, which both, and the lowest R. fredii (44.4%) between two strains of R. fredii, SB368 and USDA192. We also found a RAPD marker specific to the four Taiwanese SB strains used in the study. The RAPD technique is a potential tool for the identification of the genetics and systematics of different populations.

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.

Hydrogenase activities and N2-fixing capacities of soybean nodules (Glycine max. cv. Hodgson), inoculated with strains ofBradyrhizobium japonicum andRhizobium fredii from different geographical regions, were measured after 35 days of culture under controlled conditions. Of the strains tested, 47% induced nodules with bacteroids which recycled H2. The data obtained suggest that H2-recycling ability is not a major factor influencing early N2-fixation which depends essentially on the precocity and intensity of the initial nodulation.

The existence of a balance survival rate of different B. japonicum strains in soils has been well established. Balance levels (M) were shown to be independent of inoculum size but differences between strains in a soil could not be discussed partly because of the instability of the recovery's efficiency in the area of the plateau. In this paper, a dynamic model, the Gompertz equation, is fitted to the data, to estimate the rate (A) at which equilibrium is reached. For the three strains in the three soils previously studied, this rate is also independent of inoculum size and the soil-strain interaction is highly significant. Some data from the literature are also well fitted by the model. We propose to characterize the saprophytic potential of a strain with the pair of parameters A-M. An optimal experimental procedure is proposed to obtain the most precise estimates for these parameters. Their biological significance is discussed, but the fact that they are not dependent upon inoculation level is of basic importance for agronomic practice.

Experiments to assess the ability of free-living cells of six strains of soybean rhizobia (Bradyrhizobium japonicum USDA 76, 94, 110, 122, 123, and 135) to denitrify nitrate in five soils showed that although some strains ofB. japonicum have the capacity to rapidly denitrify nitrate in soils under anaerobic conditions, it is unlikely that the numbers of soybean rhizobia commonly found under field conditions are sufficient to significantly influence either the extent or the products of denitrification in soil. It is our general conclusion that the advantages, if any, that the ability to denitrify conveys to rhizobia or to the rhizobia-legume symbiosis are not offset by increased losses of plant-available N when denitrifying strains of rhizobia are present as free-living cells in soil.

Mixed infections of Bradyrhizobium japonicum strains in early and late nodules of four soybean cultivars were studied in a field soil. Nodule occupants were identified by immunofluorescence using serogroup specific antibodies prepared against B. japonicum strains USDA 110, USDA 123, and USDA 138. Double infection was determined directly by combined examination of the same microscopic field by fluorescence and phase contrast microscopy. Double strain occupancy was observed consistently, and its occurrence did not differ substantially in pouch, soil pot, and field experiments, ranging in incidence from 12% to 32%. No significant differences in the incidence or nature of double infection could be attributed to cultivar, seed inoculation, or plant maturity. Strains reactive to strain USDA 123-fluorescent antibody were dominant in both singly and doubly infected nodules irrespective of cultivar, plant age, or seed inoculation with strain USDA 110.