Evaluation of strain competitiveness in Rhizobium leguminosarum bv phaseoli using a nod + fix− natural mutant

Competition from native soil rhizobia is likely to be an important factor limiting Phaseolus vulgaris L. inoculant response in Latin America. We used UMR 1116, a nod ⁺ fix^– natural mutant of Rhizobium leguminosarum bv phaseoli strain CC511, as a reference strain to study competition for nodulation sites in this species. When P. vulgaris cv Carioca was planted in soils containing different proportions of UMR 1116 and the effective and competitive strain UMR 1899, UMR 1116 occupied more than 50% of the nodules at all inoculant ratios tested, though increasing the proportion of UMR 1899 in the inoculant did enhance the number and percentage of effective nodules and plant dry weight. Sixty two strains of bean rhizobia were tested in competition with UMR 1116. An inoculant ratio of 1:1 was used, with all strains applied to the soil rather than to seeds. Strains varied in the number and percentage of effective nodules produced in competition with UMR 1116, and in plant dry weight, and there was a strong correlation between variation in each of these traits and plant N accumulation. Seven of the strains (UMR 1073, 1084, 1102, 1125, 1165, 1378 and 1384) were identified as both superior in competitive ability and active in N₂ fixation. Site of placement of the inoculant and ambient temperature influenced strain response.Journal paper 16736, Agricultural Experiment Station, University of Minnesota, St. Paul, MN 55108, USA     

Semienclosed Tube Cultures of Bean Plants (Phaseolus vulgaris L.) for Enumeration of Rhizobium phaseoli by the Most-Probable-Number Technique

The semienclosed tube culture technique of Gibson was modified to permit growth of common bean (Phaseolus vulgaris L.) roots in humid air, enabling enumeration of the homologous (nodule forming) symbiont, Rhizobium phaseoli, by the most-probable-number plant infection method. A bean genotype with improved nodulation characteristics was used as the plant host. This method of enumeration was accurate when tubes were scored 3 weeks after inoculation with several R. phaseoli strains diluted from aqueous suspensions, peat-based inoculants, or soil. A comparison of population sizes obtained by most-probable-number tube cultures and plate counts indicated that 1 to 3 viable cells of R. phaseoli were a sufficient inoculant to induce nodule formation.     

Host-Controlled Restriction of Nodulation by Bradyrhizobium japonicum Strains in Serogroup 110

We previously reported the identification of a soybean plant introduction (PI) genotype, PI 417566, which restricts nodulation by Bradyrhizobium japonicum MN1-1c (USDA 430), strains in serogroup 129, and USDA 110 (P. B. Cregan, H. H. Keyser, and M. J. Sadowsky, Appl. Environ. Microbiol. 55:2532-2536, 1989, and Crop Sci. 29:307-312, 1989). In this study, we further characterized nodulation restriction by PI 417566. Twenty-four serogroup 110 isolates were tested for restricted nodulation on PI 417566. Of the 24 strains examined, 62.5% were restricted in nodulation by the PI genotype. The remainder of the serogroup 110 strains tested (37.5%), however, formed significant numbers of nodules on PI 417566, suggesting that host-controlled restriction of nodulation by members of serogroup 110 is strain dependent. Analysis of allelic variation at seven enzyme-encoding loci by multilocus enzyme electrophoresis indicated that the serogroup 110 isolates can be divided into two major groups. The majority of serogroup 110 isolates which nodulated PI 417566 belonged to the same multilocus enzyme electrophoresis group. B. japonicum USDA 110 and USDA 123 were used as coinoculants in competition-for-nodulation studies using PI 417566. Over 98% of the nodules formed on PI 417566 contained USDA 123, whereas less than 2% contained USDA 110. We also report the isolation of a Tn5 mutant of USDA 110 which has overcome nodulation restriction conditioned by PI 417566. This mutant, D4.2-5, contained a single Tn5 insertion and nodulated PI 417566 to an extent equal to that seen with the unrestricted strain USDA 123. The host range of D4.2-5 on soybean plants and other legumes was unchanged relative to that of USDA 110, except that the mutant nodulated Glycine max cv. Hill more efficiently. While strain USDA 110 has the ability to block nodulation by D4.2-5 on PI 417566, the nodulation-blocking phenomenon was not seen unless strain USDA 110 was inoculated at a 100-fold greater concentration than the mutant strain.     

Plant phenology and genetic variability in root and nodule development strongly influence genetic structuring of Rhizobium leguminosarum biovar viciae populations nodulating pea

The symbiotic relationships between legumes and their nitrogen (N(2))-fixing bacterial partners (rhizobia) vary in effectiveness to promote plant growth according to both bacterial and legume genotype. To assess the selective effect of host plant on its microsymbionts, the influence of the pea (Pisum sativum) genotype on the relative nodulation success of Rhizobium leguminosarum biovar viciae (Rlv) genotypes from the soil populations during plant development has been investigated. Five pea lines were chosen for their genetic variability in root and nodule development. Genetic structure and diversity of Rlv populations sampled from nodules were estimated by molecular typing with a marker of the genomic background (rDNA intergenic spacer) and a nodulation gene marker (nodD region). Differences were found among Rlv populations related to pea genetic background but also to modification of plant development caused by single gene mutation. The growth stage of the host plant also influenced structuring of populations. A particular nodulation genotype formed the majority of nodules during the reproductive stage. Overall, modification in root and nodule development appears to strongly influence the capacity of particular rhizobial genotypes to form nodules.     

Root hairs and phosphorus acquisition of wheat and barley cultivars

Several genes that restrict nodulation with specific Bradyrhizobiumstrains are known in Glycine max (soybean), and a similar system of nodulation restriction has recently been discovered in the related North American legume Amphicarpaea bracteata. We analyzed how nodulation-restrictive genotypes of each plant interacted with Bradyrhizobium strains sampled from the other host species. Ten bacterial isolates from A. bracteata that nodulated differentially with genotypes of their homologous host legume showed uniform responses to two soybean isogenic lines that differed at the Rj4 locus controlling nodulation restriction: all isolates formed nodules of normal size and morphology on both isolines. However, little or no nitrogen fixation occurred in any of these symbioses. A. bracteata genotypes that displayed broad vs. restricted symbiotic phenotypes toward naturally-associated bradyrhizobia were also tested with two bacterial isolates from soybean (USDA 76 and USDA 123). Both isolates formed nodules and fixed nitrogen in association with both A. bracteata genotypes. However, symbiotic effectiveness (as measured by acetylene reduction assays) was normal only for the combination of USDA 76 with the restrictive A. bracteata genotype. Overall, these results indicate that plant genes that restrict nodulation by certain naturally-associated bradyrhizobia do not confer comparable specificity when plants interact with bacteria from another related legume species.     

A Legume Genetic Framework Controls Infection of Nodules by Symbiotic and Endophytic Bacteria

Legumes have an intrinsic capacity to accommodate both symbiotic and endophytic bacteria within root nodules. For the symbionts, a complex genetic mechanism that allows mutual recognition and plant infection has emerged from genetic studies under axenic conditions. In contrast, little is known about the mechanisms controlling the endophytic infection. Here we investigate the contribution of both the host and the symbiotic microbe to endophyte infection and development of mixed colonised nodules in Lotus japonicus. We found that infection threads initiated by Mesorhizobium loti, the natural symbiont of Lotus, can selectively guide endophytic bacteria towards nodule primordia, where competent strains multiply and colonise the nodule together with the nitrogen-fixing symbiotic partner. Further co-inoculation studies with the competent coloniser, Rhizobium mesosinicum strain KAW12, show that endophytic nodule infection depends on functional and efficient M. loti-driven Nod factor signalling. KAW12 exopolysaccharide (EPS) enabled endophyte nodule infection whilst compatible M. loti EPS restricted it. Analysis of plant mutants that control different stages of the symbiotic infection showed that both symbiont and endophyte accommodation within nodules is under host genetic control. This demonstrates that when legume plants are exposed to complex communities they selectively regulate access and accommodation of bacteria occupying this specialized environmental niche, the root nodule.     

Single-Strain versus Multistrain Inoculation: Effect of Soil Mineral N Availability on Rhizobial Strain Effectiveness and Competition for Nodulation on Chick-Pea, Soybean, and Dry Bean

The nitrogen-fixing effectiveness of multistrain inoculants was found to be determined by both the effectiveness of the component strains and the percentage of the nodules occupied by them. Multistrain formulations were always either as good as the most effective single-strain inoculant or intermediate between the most and the least effective. The percentage of nodules occupied and the amount of nitrogen fixed by the component strains of a multistrain inoculant showed highly significant linear correlation. The availability of soil N had a significant influence on the nitrogen fixation potential of each strain. The mineral N status of the soil was clearly a significant factor in affecting the competition pattern of Rhizobium loti (chick-pea) and Bradyrhizobium japonicum strains. Differences between the effectiveness of strains were masked under conditions of soil N availability. However, when soil N was immobilized with sugarcane bagasse, the differences became significant. In the chick-pea system, R. loti TAL 1148 (Nit 27A8) was the most effective but not the most competitive of the three strains used. In the soybean and dry bean systems, B. japonicum TAL 102 (USDA 110) and R. leguminosarum bv. phaseoli TAL 182, respectively, were consistently the most effective and, more often than not, the most competitive of the strains used for each species.     

Infection density of Wolbachia endosymbiont affected by co-infection and host genotype

Infection density is among the most important factors for understanding the biological effects of Wolbachia and other endosymbionts on their hosts. To gain insight into the mechanisms of infection density regulation, we investigated the adzuki bean beetles Callosobruchus chinensis and their Wolbachia endosymbionts. Double-infected, single-infected and uninfected host strains with controlled nuclear genetic backgrounds were generated by introgression, and infection densities in these strains were evaluated by a quantitative polymerase chain reaction technique. Our study revealed previously unknown aspects of Wolbachia density regulation: (i) the identification of intra-specific host genotypes that affect Wolbachia density differently and (ii) the suppression of Wolbachia density by co-infecting Wolbachia strains. These findings shed new light on symbiont-symbiont and host-symbiont interactions in the Wolbachia-insect endosymbiosis and strongly suggest that Wolbachia density is determined through a complex interaction between host genotype, symbiont genotype and other factors.     

NONRANDOM GENOTYPIC ASSOCIATIONS IN A LEGUME—BRADYRHIZOBIUM MUTUALISM

Genetically divergent lineages often coexist within populations of the annual legume Amphicarpaea bracteata. At one site dominated by two such lineages (termed biotypes “C” and “S”), isolates of root-nodule bacteria (Bradyrhizobium sp.) were sampled from both hosts and analyzed by enzyme electrophoresis. Symbiont populations on the two plant biotypes were highly distinct. Out of 15 bacterial multilocus genotypes detected (among 51 isolates analyzed), only one was shared in common by the two plant biotypes. Cluster analysis revealed three bacterial lineages (designated I, II, and III), with lineage I found exclusively on biotype C plants, and the two other lineages almost completely restricted to biotype S hosts. Laboratory inoculation tests indicated that lineage I bacteria were strictly specialized on biotype C hosts, forming few or no nodules on plants of the other host biotype. Bacterial lineages II and III were capable of forming nodules on both kinds of plants, but nodule numbers were often significantly higher on biotype S hosts. The nonrandom association between plant and bacterial lineages at this site implies that genetic diversity of hosts is an important factor in the maintenance of polymorphism within the symbiont population.     

Widespread fitness alignment in the legume-rhizobium symbiosis

Although 'cheaters' potentially destabilize the legume-rhizobium mutualism, we lack a comprehensive review of host-symbiont fitness correlations. Studies measuring rhizobium relative or absolute fitness and host benefit are surveyed. Mutant studies are tallied for evidence of pleiotropy; studies of natural strains are analyzed with meta-analysis. Of 80 rhizobium mutations, 19 decrease both partners' fitness, four increase both, two increase host fitness but decrease symbiont fitness and none increase symbiont fitness at the host's expense. The pooled correlation between rhizobium nodulation competitiveness and plant aboveground biomass is 0.65 across five experiments that compete natural strains against a reference, whereas, across 14 experiments that compete rhizobia against soil populations or each other, the pooled correlation is 0.24. Pooled correlations between aboveground biomass and nodule number and nodule biomass are 0.76 and 0.83. Positive correlations between legume and rhizobium fitness imply that most ineffective rhizobia are 'defective' rather than 'defectors'; this extends to natural variants, with only one significant fitness conflict. Most studies involve non-coevolved associations, indicating that fitness alignment is the default state. Rhizobium mutations that increase both host and symbiont fitness suggest that some plants maladaptively restrict symbiosis with novel strains.     

Some USDA studies on the soybean-Rhizobium symbiosis

Summary The ecology, strain evaluation, genetics of host strain interactions and physiology of nitrogen fixation ofRhizobium japonicum in association with the soybean,Glycine max, were studied. Results of inoculation experiments with selected strains ofRhizobium japonicum indicated that indigenous strains occupied most of the nodules of soybeans grown in highRhizobium japonicum populated soils. Nodule sampling indicated that inoculation did not result in quicker nodulation or a higher incidence of root nodules (primary or secondary) than uninoculated checks. Rhizosphere studies indicated that colonization by introduced strains did occur but did not compete successfully with field strains for nodule sites. Recovery of specific serological types from nodules was influenced by planting intervals. The distribution of the serotypes varied with the time of planting and the age of the plant. Temperature studies indicated that the distribution of serotypes recovered from the nodules was influenced by temperature. Field studies showed the selectivity of soybean genotypes on strains ofRhizobium japonicum. Some strains were more common in the nodules of some varieties than in others. Closely related varieties had similar populations in their nodules. Three genes which control nodule response in soybeans are reported. Nitrogen fixation profiles were determined for some variety-strain interactions. Combinations previously classified as inefficient showed some nitrogenase activity as measured by the acetylene reduction technique. Research Microbiologist; Research Agronomist; Research Plant Physiologist, Soybean Investigations, Crops Research Division, Beltsville, Md. (USDA, ARS); and Plant Pathologist currently located at Michigan State University, East Lansing, Michigan.     

Symbiotic Characteristics of Rhizobium leguminosarum bv. trifolii Isolates Which Represent Major and Minor Nodule-Occupying Chromosomal Types of Field-Grown Subclover (Trifolium subterraneum L.)

The symbiotic effectiveness and nodulation competitiveness of Rhizobium leguminosarum bv. trifolii soil isolates were evaluated under nonsoil greenhouse conditions. The isolates which we used represented both major and minor nodule-occupying chromosomal types (electrophoretic types [ETs]) recovered from field-grown subclover (Trifolium subterraneum L.). Isolates representing four ETs (ETs 2, 3, 7, and 8) that were highly successful field nodule occupants fixed between 2- and 10-fold less nitrogen and produced lower herbage dry weights and first-harvest herbage protein concentrations than isolates that were minor nodule occupants of field-grown plants. Despite their equivalent levels of abundance in nodules on field-grown subclover plants, ET 2 and 3 isolates exhibited different competitive nodulation potentials under nonsoil greenhouse conditions. ET 3 isolates generally occupied more subclover nodules than isolates belonging to other ETs when the isolates were mixed in 1:1 inoculant ratios and inoculated onto seedlings. In contrast, ET 2 isolates were less successful at nodulating under these conditions. In many cases, ET 2 isolates required a numerical advantage of at least 6:1 to 11:1 to occupy significantly more nodules than their competitors. We identified highly effective isolates that were as competitive as the ET 3 isolates despite representing serotypes that were rarely recovered from nodules of field-grown plants. When one of the suboptimally effective isolates (ET2-1) competed with an effective and competitive isolate (ET31-5) at several different inoculant ratios, the percentages of nodules occupied by the former increased as its numerical advantage increased. Although subclover yields declined as nodule occupancy by ET2-1 increased, surprisingly, this occurred at inoculant ratios at which large percentages of nodules were still occupied by ET31-5.     

Relative genetic structure of a population of Rhizobium meliloti isolated directly from soil and from nodules of alfalfa (Medicago sativa) and sweet clover (Melilotus alba)

Insertion sequence (IS) hybridization was used to define the structure of a population of Rhizobium meliloti isolated directly from soil and from nodules of Medicago sativa (alfalfa) and Melilotus alba (sweet clover) grown under controlled conditions and inoculated with a suspension of the same soil. The detection of R. meliloti isolated from soil on agar plates was facilitated by use of a highly species specific DNA probe derived from ISRm5. All R. meliloti obtained directly from soil proved to be symbiotic (i.e. nodulated and fixed nitrogen with alfalfa). Analysis of 293 R. meliloti isolates revealed a total of 17 distinct IS genotypes of which 9, 9 and 15 were from soil, M. alba and M. sativa, respectively; 8 genotypes were common to soil and both plant species. The frequency of R. meliloti genotypes from soil differed markedly from that sampled from nodules of both legume species: 5 genotypes represented about 90% of the isolates from soil whereas a single genotype predominated among isolates from nodules accounting for more than 55% of the total. The distribution of genotypes differed between M. sativa and M. alba indicating species variation in nodulation preferences for indigenous R. meliloti. The data are discussed in the context of competition for nodulation of the host plant and the selection of Rhizobium strains for use in legume inoculants. This study has ecological implications and suggests that the composition of R. meliloti populations sampled by the traditionally used host legume may not be representative of that actually present in soil.     

Performance of phaseolus bean rhizobia in soils from the major production sites in the Nile Delta

The symbiotic and competitive performances of two highly effective rhizobia nodulating French bean P. vulgaris were studied in silty loam and clayey soils. The experiments were carried out to address the performance of two rhizobia strains (CE3 and Ph. 163] and the mixture thereof with the two major cultivated bean cultivars in two soil types from major growing French bean areas in Egypt. Clay and silty loam soils from Menoufia and Ismailia respectively were planted with Bronco and Giza 6 phaseolus bean cultivars. The data obtained from this study indicated that rhizobial inoculation of Giza 6 cultivar in clayey soil showed a positive response to inoculation in terms of nodule numbers and dry weight. This response was also positive in dry matter and biomass accumulation by the plants. The inoculant of strain CE3 enhanced plant growth and N-uptake relative to Ph. 163. However, the mixed inoculant strains were not always as good as single strain inoculants. The competition for nodulation was assessed using two techniques namely fluorescent antibody testing (FA) and REP-PCR fingerprinting. The nodule occupancy by inoculant strain Ph. 163 in both soils occupied 30-40% and 38-50 of nodules of cultivar Bronco. The mixed inocula resulted in higher proportions of nodules containing CE3 in silty loam soil and Ph. 163 in clayey soil. The native rhizobia occupied at least 50% of the nodules on the Bronco cultivar. For cultivar Giza 6, the native rhizobia were more competitive with the inoculant strains. Therefore, we suggest using the studied strains as commercial inocula for phaseolus bean.     

Phage sensitivity and host range of Rhizobium strains isolated from root nodules of temperate legumes

Twenty-five Rhizobium strains were isolated from root nodules of Astragalus spp. (10), Hedysarum alpinum (7), Glycyrrhiza pallidiflora (3) and Ononis arvensis (5). The sensitivity of these strains to bacteriophages of Rhizobium loti, R. meliloti, R. galegae and R. leguminosarum was studied. Phages specific to R. loti strains were shown to induce the phage lysis of several Astragalus, Hedysarum and Ononis rhizobia. Ten R. loti strains tested for nodulation abilities on the plant hosts under investigation were able to develop nitrogen-fixing nodules on the Ononis arvensis roots. On the other hand, rhizobia from Ononis and Glycyrrhiza could form an effective symbiosis with Lotus corniculatus plants, so these bacteria are considered to belong to the Rhizobium loti taxon. Bacterial strains isolated from Astragalus and Hedysarum were observed to cross-nodulate their plant hosts as well as Oxytropis campestris, Glycyrrhiza uralensis and Ononis arvensis plants, whereas they could not nodulate Lotus plants. It is concluded that these Rhizobium strains comprise a cross-inoculation group related to Rhizobium loti. ei]{gnR O D}{fnDixon}     

Bradyrhizobium strains and the nodulation,nodule efficiency and growth of soybean (Glycine max L.) in Egyptian soils

Six strains and a commercial inoculant ofBradyrhizobium japonicum were evaluated in association withGlycine max (L.) cultivar Clark. Inoculated and uninoculated plants were grown in pot and field experiments. Nodules were counted and weighed and roots and shoots were separated and analysed for total nitrogen. In pot experiments, two of six bacterial strains were superior to the other four, and to the commercial inoculant (Nitragin) in promoting greater root and top growth and plant nitrogen accumulation. In the field experiment, there were indications that environmental conditions may have affected nodulation by the bacteria. The strains could be divided into three groups according to nodule efficiencies, accumulation of plant dry matter, and total nitrogen content. The greater variations in nodule efficiencies of the tested strains could be attributed to the quantities of bacteroid, cytosol protein and leghaemoglobin in the nodules.     

Diversity and compatibility of peanut (Arachis hypogaea L.) bradyrhizobia and their host plants

A high degree of genetic diversity among 125 peanut bradyrhizobial strains and among 32 peanut cultivars collected from different regions of China was revealed by using the amplified fragment length polymorphism (AFLP) technique. Eighteen different peanut bradyrhizobial genotypes and six peanut cultivars were selected for symbiotic cross-inoculation experiments. The genomic diversity was reflected in the symbiotic diversity. The peanut cultivars varied in their ability to nodulate with the strains used. Some cultivars had a more restricted host range than the others. Also the strains displayed a range of nodulation patterns. In yield formation there were clear differences between the plant cultivar/bradyrhizobium combinations. There was good compatibility between some peanut bradyrhizobial strains and selected cultivars, with inoculation resulting in well-nodulated, high-yielding symbiotic combinations, but no plant cultivar was compatible with all strains used. The strains displayed a varying degree of effectiveness, with some strains being fairly effective with all cultivars and others with selected ones. The AFLP genotypes of the strains did not explain the symbiotic behavior, whereas the yield formation of the plant cultivars was more related to the genotype. It is concluded that to obtain optimal nitrogen fixation efficiency of peanut in the field, compatible plant cultivar-bradyrhizobium combinations should be selected either by finding inoculant strains compatible with the plant cultivars used, or plant cultivars compatible with the indigenous bradyrhizobia.     

Response of introduced Bradyrhizobium strains infectinga promiscuous soybean cultivar

Two field experiments were established to assess the competitiveness of foreign bradyrhizobia in infecting the promiscuous soybean cultivar TGX 536-02D. Seeds were inoculated with antibiotic mutants of the bradyrhizobia strains before planting after land preparation. Soybean plants were harvested at pre-determined days after planting for estimating nodule number, nodule dry weight, nodule occupancy, shoot dry weight and seed yield. Results show that nodule number and dry weight significantly increased and showed great variability at 84 days after planting (DAP), probably due to differences in the ability of inoculant bradyrhizobia to form nodules with the soybean cultivar TGX 536-02D. Increased shoot dry weight, %N, total N and seed yield were a result of increased nodulation by the effective and competitive inoculant Bradyrhizobium strains. Strain USDA 110 occupied the highest percentage of nodule sites because it was more competitive than the other Bradyrhizobium strains. These results show that there was high potential for increasing growth and seed yield of the promiscuous soybean cultivar TGX 536-02D by inoculation with foreign Bradyrhizobium strains.     

Nodulation of annual medicago by strains ofR. Meliloti in a commercial inoculant as influenced by soil phosphorus and pH

Summary Four species of annual medics showed poor growth and little or no nodulation in an inoculated, acid soil low in available phosphorus. Nodulation was significantly increased by adding phosphorus to the soil. The number of nodules found on the roots of each species was approximately the same for soil at pH 5.5 or 6.8. A commercial inoculant ofRhizobium meliloti was adequate for nodulation of all four species and two strains of rhizobia predominated in the nodules which were assayed by immunodiffusion.     

Exclusion of inefficient Bradyrhizobium japonicum serogroups by soybean genotypes

Soybean [Glycine max (L.) Merr.] forms a symbiosis with serogroups of Bradyrhizobium japonicum that differ in their dinitrogen fixing abilities. The objectives of this study were to identify soybean genotypes that would restrict nodulation by relatively inefficient serogroups indigenous to a large portion of the southeastern USA, and then characterize the nodulation responses of selected genotypes with specific bradyrhizobial strains under controlled conditions. From field screening trials followed by controlled single and competitive inoculations of serogroups USDA 31, 76 and 110, twelve soybean genotypes out of 382 tested were identified with varying levels of exclusion abilities. Soybean nodule occupancies and nodulation characteristics were influenced by plant genotype, environment (i.e. field or greenhouse), bradyrhizobial serogroup, and location of nodules (i.e. tap or lateral root). The cultivar Centennial sustains high seed yields even though it nodulates to a high degree with the inefficient serogroup USDA 31. In contrast, data from the released cultivars Braxton, Centennial and Coker 368 indicate that they may have been selected to exclude the inefficient serogroup USDA 76 from their tap root nodules, possibly contributing to high seed yield.