Near isogenic lines of soybeans as tools to identify nodulation specificmutants of Bradyrhizobium elkanii

The dominant allele Rj4 in soybean interdicts or restricts the nodulation of plants by certain strains of bacteria, most of which are classified as Bradyrhizobium elkanii, while the recessive allele permits normal nodulation with the same strains. The near isogenic lines BARC-2 (Rj4) and BARC-3 (rj4) are calculated to be 99.95% identical in their nuclear DNA, but differ specifically in the allele present at the Rj4 locus. These lines were used to identify spontaneous mutants of the Rj4-restricted Bradyrhizobium elkanii strain USDA 61 Nal^r that had the ability to effectively nodulate plants of the Rj4 genotype. Of the eight rare nodules found on roots of soybean plants of the Rj4 genotype inoculated with the genetically marked strain USDA 61 Nal^r, four were identified as containing mutants with the ability to overcome the effects of the Rj4 allele.     

Host genetic control of symbiosis in soybean (Glycine max L.)

Genes controlling nitrogen-fixing symbioses of legumes with specialized bacteria known as rhizobia are presumably the products of many millions of years of evolution. Different adaptative solutions evolved in response to the challenge of survival in highly divergent complexes of symbionts. Whereas efficiency of nitrogen fixation appears to be controlled by quantitative inheritance, genes controlling nodulation are qualitatively inherited. Genes controlling nodulation include those for non-nodulation, those that restrict certain microsymbionts, and those conditioning hypernodulation, or supernodulation. Some genes are naturally occurring polymorphisms, while others were induced or were the result of spontaneous mutations. The geographic patterns of particular alleles indicate the role of coevolution in determining symbiont specificites and compatibilities. For example, the Rj4 allele occurs with higher frequency (over 50%) among the soybean (G. max) from Southeast Asia. DNA homology studies of strains of Bradyrhizobium that nodulate soybean indicated two groups so distinct as to warrant classification as two species. Strains producing rhizobitoxine-induced chlorosis occur only in Group II, now classified as B. elkanii. Unlike B. japonicum, B. elkanii strains are characterized by (1) the ability to nodulate the rj1 genotype, (2) the formation of nodule-like structures on peanut, (3) a relatively high degree of ex planta nitrogenase activity, (4) distinct extracellular polysaccharide composition, (5) distinct fatty acid composition, (6) distinct antibiotic resistance profiles, and (7) low DNA homology with B. japonicum. Analysis with soybean lines near isogenic for the Rj4 versus rj4 alleles indicated that the Rj4 allele excludes a high proportion of B. elkanii strains and certain strains of B. japonicum such as strain USDA62 and three serogroup 123 strains. These groups, relatively inefficient in nitrogen fixation with soybean, tend to predominate in soybean nodules from many US soils. The Rj4 allele, the most common allelic form in the wild species, has a positive value for the host plants in protecting them from nodulation by rhizobia poorly adapted for symbiosis.     

Fine mapping of the Rj4 locus,a gene controlling nodulation specificity in soybean

Leguminous plants have the ability to make their own nitrogen fertilizer by forming a root nodule symbiosis with nitrogen-fixing soil bacteria, collectively called rhizobia. This biological process plays a critical role in sustainable agriculture because it reduces the need for external nitrogen input. One remarkable property of legume–rhizobial symbiosis is its high level of specificity, which occurs at both inter- and intra-species levels and takes place at multiple phases of the interaction, ranging from initial bacterial infection and nodulation to late nodule development associated with nitrogen fixation. Knowledge of the molecular mechanisms controlling symbiotic specificity will facilitate the development of new crop varieties with improved agronomic potential for nitrogen-fixing symbiosis. In this report, we describe fine mapping of the Rj4 locus, a gene controlling nodulation specificity in soybean (Glycine max). The Rj4 allele prevents the host plant from nodulation with many strains of Bradyrhizobium elkanii, which are frequently present in soils of the southeastern USA. Since B. elkanii strains are poor symbiotic partners of soybean, cultivars containing an Rj4 allele are considered favorable. We have delimited the Rj4 locus within a 57-kb genomic region on soybean chromosome 1. The data reported here will facilitate positional cloning of the Rj4 gene and the development of genetic markers for marker-assisted selection in soybean.     

The Rj4 allele in soybean represses nodulation by chlorosis-inducing bradyrhizobia classified as DNA homology group II by antibiotic resistance profiles

Summary To determine the relationship between nodulation restriction by the Rj4 allele of soybean, rhizobitoxine-induced chlorosis, and taxonomic grouping of bradyrhizobia, 119 bradyrhizobial isolates were tested in Leonard jar culture for nodulation response and chlorosis induction. In addition to strain USDA 61, the strain originally reported as defining the Rj4 response, eight other isolates (i.e., USDA 62, 83, 94, 238, 252, 259, 260, and 340) were discovered to elicit the nodulation interdiction of the Rj4 allele. Only 16% of all the bradyrhizobial strains tested induced chlorosis, but seven of the nine strains (78%) interdicted by the Rj4 allele were chlorosis-inducing strains. Furthermore, in tests for antibiotic resistance profile, eight of the nine interdicted strains (89%) were classed in DNA homology group II. This evidence suggests that the Rj4 allele has a positive value to the host plant in shielding it from nodulation by certain chlorosis-inducing bradyrhizobia of a DNA homology group with impaired efficiency of nitrogen fixation with soybean.     

Survival of Bradyrhizobium sp. (Arachis) on fungicide-treated peanut seed in relationship to plant growth and yield

Survival and viability of Bradyrhizobium inoculant on fungicide-treated peanut seed and the resulting effects on nitrogen fixation, plant growth and seed yield were determined. Vitavax and Benomyl had the most and least lethal actions against Bradyrhizobium strains grown on YEM medium containing a fungicide, respectively, while Thiram and Captan effects were intermediate. Survival of Bradyrhizobium USDA 3384 and USDA 3456, as single strain peat inoculants, on peanut (Arachis hypogaea L. var. Florunner) seeds treated with Benomyl or Vitavax at the rate of 3g/kg seed was also examined. Both fungicides inhibited the growth and affected the survival of strain USDA 3384 on peanut seed. Vitavax killed the inoculant in 9 h. In contrast, USDA 3456 resisted both fungicides, and survived for up to 72h. Nodule formation on greenhouse-grown plants inoculated with USDA 3384 was inhibited by all fungicides. Shoot dry weight and plant nitrogen content significantly decreased as compared to controls. Fungicides, except Vitavax, had a slight effect on nodulation and plant growth when USDA 3456 was used as inoculant. The agronomic importance of fungicide-inoculant interaction was examined in field experiments conducted in Egypt in soil free of peanut-nodulating Bradyrhizobium, where seeds were treated with a combination of two fungicides and a single strain peat inoculant of either USDA 3384 or USDA 3456. All fungicides decreased nodulation, nitrogen fixation, plant growth and seed yield, especially with USDA 3384 as inoculant. Fungicides inhibited viability and survival of Bradyrhizobium on peanut seeds which decreased nodule formation leading to reduced peanut seed yield.     

A Role of Bradyrhizobium elkanii and Closely Related Strains in the Degradation of Methoxychlor in Soil and Surface Water Environments

We have reported that a leguminous bacterial strain, Bradyrhizobium sp. strain 17-4, isolated from river sediment, phylogenetically very close to Bradyrhizobium elkanii, degraded methoxychlor through O-demethylation and oxidative dechlorination. In the present investigation, we found that B. elkanii (USDA94), a standard species deposited in the Culture Collection, degraded methoxychlor. Furthermore, Bradyrhizobium sp. strain 4-1, also very close to B. elkanii, isolated from Japanese paddy field soil, degraded methoxychlor. These B. elkanii and closely related strains degraded methoxychlor through almost identical metabolic pathways, and cleaved the phenyl ring and mineralized. In contrast, another representative Bradyrhizobium species, B. japonicum (USDA110), did not degrade methoxychlor at all. Based on these findings, B. elkanii and closely related strains are likely to play an important role not only in providing the readily biodegradable substrates but also in completely degrading (mineralizing) methoxychlor by themselves in the soil and surface water environment.     

Soybean cultivars affect nodulation competition of Bradyrhizobium japonicum strains

The influence of five Thai soybean cultivars on nodulation competitiveness of four Bradyrhizobium japonicum strains was investigated. Cultures of B. japonicum strains THA5, THA6, USDA110 and SEMIA5019 were mixed with each other prior to inoculating germinated soybean seeds growing in Leonard jars with nitrogen-free nutrient solution. At harvest, nodule occupancy by each strain was determined by a fluorescent antibody technique. The term ‘general competitive ability’ was introduced to describe the average competitive nodule occupancy of a strain in paired co-inoculation with a number of strains on soybean. The nodule occupancies by an individual strain were directly correlated with the proportions of that strain in the inoculum mixtures. USDA110 showed higher nodulation competitiveness than the other strains on three of the five cultivars. The Thai strain THA6 appeared to be more competitive than USDA110 on cultivar SJ5. Thus, nodulation competitiveness of the B. japonicum strains was affected by the cultivars of soybean used. This revised version was published online in August 2006 with corrections to the Cover Date.     

Detection of Homoserine Lactone-Like Quorum Sensing Molecules in Bradyrhizobium Strains

One hundred and forty-two Bradyrhizobium strains were screened for their ability to produce N-acyl homoserine lactone-like molecules (AHLs) by using an Agrobacterium tumefaciens biosensor strain containing a traI-lacZ fusion. Approximately 22% (31 of 142) of the tested strains produced AHLs that induced moderate to elevated β-galactosidase activity levels in the biosensor strain. Bradyrhizobium japonicum and Bradyrhizobium elkanii strains were both shown to produce AHLs. Age of culture, and media composition were each shown to influence production of AHL(s), with greater production occurring in 2 day-old cultures grown in rich media. Reverse-phase high-performance liquid chromatography and thin-layer chromatography analyses indicated that the B. japonicum strain USDA 290 produced at least two types of AHLs. Our results indicate that the production AHL-like autoinducers is widespread among both B. japonicum and B. elkanii strains.     

Rhizobitoxine Production by Bradyrhizobium elkanii Enhances Nodulation and Competitiveness on Macroptilium atropurpureum

Application of 1-aminoocyclopropane-1-carboxylic acid, an ethylene precursor, decreased nodulation of Macroptilium atropurpureum by Bradyrhizobium elkanii. B. elkanii produces rhizobitoxine, an ethylene synthesis inhibitor. Elimination of rhizobitoxine production in B. elkanii increased ethylene evolution and decreased nodulation and competitiveness on M. atropurpureum. These results suggest that rhizobitoxine enhances nodulation and competitiveness of B. elkanii on M. atropurpureum.     

Effect of lectin on nodulation by wild-type Bradyrhizobium japonicum and a nodulation-defective mutant.

The nodulation characteristics of wild-type Bradyrhizobium japonicum USDA 110 and mutant strain HS111 were examined. Mutant strain HS111 exhibits a delayed-nodulation phenotype, a result of its inability to initiate successful nodulation promptly following inoculation of the soybean root. Previously, we showed that the defect in initiation of infection leading to subsequent nodulation which is found in HS111 can be phenotypically reversed by pretreatment with soybean root exudate or soybean seed lectin. This effect is not seen after pretreatment with root exudates and lectins obtained from other plant species. Treatment of strain HS111 with as little as 10 soybean seed lectin molecules per bacterium (3.3 X 10 (-12) M) resulted in enhancement of nodule formation. Pretreatment of wild-type B. japonicum USDA 110 with soybean root exudate or seed lectin increased nodule numbers twofold on 6-week-old plants. Wild-type strain USDA 110 cells inoculated at 10(4) cells per seedling exhibited a delay in initiation of infection leading to subsequent nodulation. Wild-type cells pretreated in soybean root exudates or seed lectin did not exhibit a delay in nodulation at this cell concentration. Mutant strain HS111 pretreated in seed lectin for 0 or 1 h, followed by washing with the hapten D-galactose to remove the lectin, exhibited a delay in initiation of nodulation. Phenotypic reversal of the delayed-nodulation phenotype exhibited by strain HS111 was seen if incubation was continued for an additional 71 h in plant nutrient solution following 1 h of lectin pretreatment. Reversal of the delayed-nodulation phenotype of HS111 through lectin pretreatment was prevented by chloramphenicol or rifampin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rj4, a Gene Controlling Nodulation Specificity in Soybeans,Encodes a Thaumatin-Like Protein But Not the One Previously Reported

Rj4 is a dominant gene in soybeans (Glycine max) that restricts nodulation by many strains of Bradyrhizobium elkanii. The soybean-B. elkanii symbiosis has a low nitrogen-fixation efficiency, but B. elkanii strains are highly competitive for nodulation; thus, cultivars harboring an Rj4 allele are considered favorable. Cloning the Rj4 gene is the first step in understanding the molecular basis of Rj4-mediated nodulation restriction and facilitates the development of molecular tools for genetic improvement of nitrogen fixation in soybeans. We finely mapped the Rj4 locus within a small genomic region on soybean chromosome 1, and validated one of the candidate genes as Rj4 using both complementation tests and CRISPR/Cas9-based gene knockout experiments. We demonstrated that Rj4 encodes a thaumatin-like protein, for which a corresponding allele is not present in the surveyed rj4 genotypes, including the reference genome Williams 82. Our conclusion disagrees with the previous report that Rj4 is the Glyma.01G165800 gene (previously annotated as Glyma01g37060). Instead, we provide convincing evidence that Rj4 is Glyma.01g165800-D, a duplicated and unique version of Glyma.01g165800, that has evolved the ability to control symbiotic specificity.Legumes are capable of forming a root nodule symbiosis with nitrogen-fixing soil bacteria called rhizobia. Remarkably, this symbiosis shows a high level of specificity (Broughton et al., 2000; Perret et al., 2000; Wang et al., 2012). The specificity occurs at both between- and within-species levels, such that each legume species or genotype can establish an efficient symbiosis with only a specific group of rhizobial species or strains. Genetic control of symbiosis specificity is complex, involving an exchange of multiple molecular signals between the symbiotic partners. Understanding the molecular mechanisms underlying symbiosis specificity would allow for development of tools for genetic improvement of biological nitrogen fixation in legumes.In most but not all legumes, bacterial infection and nodule organogenesis is mediated by specific perception of bacterially derived lipo-chitooligosaccharides (called Nod factors) by the cognate plant receptors (Lerouge et al., 1990; Geurts et al., 1997; Limpens et al., 2003; Radutoiu et al., 2003, 2007). The Nod factors carry various species-specific chemical decorations, and this structural variation is widely thought to play an important role in defining the recognition specificity at the species level (Lerouge et al., 1990; Perret et al., 2000; Radutoiu et al., 2007). In addition to Nod factors, rhizobial surface polysaccharides, such as exopolysaccharides, lipopolysaccharides, capsular polysaccharides, and cyclic glucans, are also important for development of infected root nodules and for modulating host specificity (D’Haeze and Holsters, 2004; Jones et al., 2008; Deakin and Broughton, 2009). Recently, an exopolysaccharide receptor has been identified in Lotus japonicus that controls rhizobial infection and distinguishes between compatible and incompatible exopolysaccharides (Kawaharada et al., 2015).Despite their unique attributes, the legume-rhizobial interactions share many common features with pathogenic plant-bacterial interactions (D’Haeze and Holsters, 2004; Deakin and Broughton, 2009). As such, plant immunity triggered by microbe-associated molecular patterns or bacterial effector proteins also plays a key role in regulation of strain-specific nodulation (D’Haeze and Holsters, 2004; Deakin and Broughton, 2009; Yang et al., 2010; Wang et al., 2012). It has been demonstrated in soybeans (Glycine max) that plants use classical NBS-LRR resistance genes to restrict nodulation with certain rhizobial strains (Yang et al., 2010). In this case, the host range of rhizobial symbionts is determined by the presence of type III effectors in the bacteria and the corresponding resistance genes in the plant.In this report, we describe positional cloning of the soybean Rj4 gene. The Rj4 gene was first identified in 1972 (Vest and Caldwell, 1972) and subject to extensive study in the 1980s and 1990s (e.g. Devine and O’Neill, 1986; Devine et al., 1990; Sadowsky and Cregan, 1992). Soybean genotypes carrying an Rj4 allele restrict nodulation by many strains of Bradyrhizobium japonicum and Bradyrhizobium elkanii (Sadowsky and Cregan, 1992). B. elkanii is a poor symbiotic partner of soybeans because of its low nitrogen-fixation efficiency. In addition, many of the strains also produce rhizobitoxine, a compound that induces chlorosis in the host plant. Thus, cultivars with the Rj4 genotype are favorable in soils where the B. elkanii population is dominant because Rj4 stops those cultivars from forming invaded nodules with it. The Rj4 allele is frequently present in Gly soja, the wild progenitor of soybean, but less frequent in the modern cultivars from North America (Devine and Breithaupt, 1981). We mapped the Rj4 locus within a small genomic region on soybean chromosome 1 (Tang et al., 2014), and validated one of the candidate genes as Rj4 using both complementation tests and CRISPR/Cas9-based gene knockout experiments. We showed that Rj4 encodes a thaumatin-like protein that does not have a corresponding allele in the analyzed rj4 genetic backgrounds. This conclusion disagrees with the previous report that Rj4 is the Glyma.01G165800 gene (Hayashi et al., 2014). Instead, we provide convincing evidence that Rj4 is a duplicate copy of Glyma.01G165800.     

Effect of lectin on nodulation by wild-type Bradyrhizobium japonicum and a nodulation-defective mutant

The nodulation characteristics of wild-type Bradyrhizobium japonicum USDA 110 and mutant strain HS111 were examined. Mutant strain HS111 exhibits a delayed-nodulation phenotype, a result of its inability to initiate successful nodulation promptly following inoculation of the soybean root. Previously, we showed that the defect in initiation of infection leading to subsequent nodulation which is found in HS111 can be phenotypically reversed by pretreatment with soybean root exudate or soybean seed lectin. This effect is not seen after pretreatment with root exudates and lectins obtained from other plant species. Treatment of strain HS111 with as little as 10 soybean seed lectin molecules per bacterium (3.3 X 10 (-12) M) resulted in enhancement of nodule formation. Pretreatment of wild-type B. japonicum USDA 110 with soybean root exudate or seed lectin increased nodule numbers twofold on 6-week-old plants. Wild-type strain USDA 110 cells inoculated at 10(4) cells per seedling exhibited a delay in initiation of infection leading to subsequent nodulation. Wild-type cells pretreated in soybean root exudates or seed lectin did not exhibit a delay in nodulation at this cell concentration. Mutant strain HS111 pretreated in seed lectin for 0 or 1 h, followed by washing with the hapten D-galactose to remove the lectin, exhibited a delay in initiation of nodulation. Phenotypic reversal of the delayed-nodulation phenotype exhibited by strain HS111 was seen if incubation was continued for an additional 71 h in plant nutrient solution following 1 h of lectin pretreatment. Reversal of the delayed-nodulation phenotype of HS111 through lectin pretreatment was prevented by chloramphenicol or rifampin.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

N2 fixation by soybean-Bradyrhizobium combinations under acidity,low P and high Al stresses

Five strains of Bradyrhizobium japonicum (USDA 6, 110, 122, 138, and 143) were screened in cell culture for tolerance to acidity (pH 4.2, 4.4, and 4.6) and Al (0, 3, 4, 5, and 6 mg L^–1) under low P conditions. Each strain was later grown in association with seven soybean [Glycine max. (L) Merr.] cultivars which were also screened for tolerance to the same stresses in nutrient culture to determine which soybean-Bradyrhizobium combinations would establish the most effective symbiotic N₂ fixing relationships. Results indicated that strains USDA 110 and 6 were more tolerant than USDA 122, 138 and 143 with USDA 110 being the most tolerant. Acidity appeared to be the more severe stress; but even when strains showed tolerance to the stresses, cell numbers were significantly reduced. This suggests that colonization of soils and soybean roots can be adversely affected under similar conditions in the field which may result in reduced nodulation. The strains found to be more tolerant to the stresses were more effective N₂ fixers in symbiosis with all soybean cultivars, with USDA 110 being definitely superior. The association between the more tolerant strains and cultivars had the largest nitrogenase activity. Further studies on the inclusion of tolerant Bradyrhizobium strains in inoculum used on tolerant soybean cultivars in the field are warranted.     

Identification of a characteristic antioxidant,anthrasesamone F,in black sesame seeds and its accumulation at different seed developmental stages

Assay-guided fractionation of the methanol extract from black seeds of sesame (Sesamum indicum L.) led to the isolation of an active compound that had a 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. This antioxidant was confirmed to be anthrasesamone F, an anthraquinone derivative previously isolated from different black sesame seeds and biogenetically related to other anthrasesamones in sesame roots. The radical scavenging assay showed that anthrasesamone F had more potent activity than Trolox. The content of anthrasesamone F in different parts and at different developmental stages of black sesame seeds was investigated to clarify the accumulation pattern of this antioxidant in the black seeds. Anthrasesamone F was localized in the seed coat of black seeds and accumulated after the seed coat color changed to black. The content of anthrasesamone F increased gradually with seed maturation and drastically on air-drying, the final stage in sesame cultivation.     

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.     

Bradyrhizobium elkanii Induces Outer Cortical Root Swelling in Soybean

When soybean roots were inoculated with Bradyrhizobium elkaniiUSDA76, USDA94 or USDA31, a localized swelling of the root outercortical cells was observed within 10 days after inoculation.This phenotype was designated outer cortical swelling (OCS).OCS was not observed in roots inoculated with B.japonicum USDA110or USDA122. Therefore, the OCS response appears to be specificfor B. elkanii. To determine the involvement of the nodulationgenes in OCS formation, we constructed a nod^- mutant, strainUSDA94     

The Soybean Rj4 Allele Restricts Nodulation by Bradyrhizobium japonicum Serogroup 123 Strains

Of nine Bradyrhizobium japonicum serogroup 123 strains examined, 44% were found to be restricted for nodulation by cultivar Hill. Nodulation studies with soybean isoline BARC-2 confirmed that the soybean Rj4 allele restricts nodulation by the same serogroup 123 isolates. Immunological analyses indicated that B. japonicum strains in serogroups 123 and 31 share at least one surface somatic antigen.