Rhizobitoxine production and symbiotic compatibility of Bradyrhizobium from Asian and North American lineages of amphicarpaea.

Reciprocal inoculations with Bradyrhizobium sp. isolates from the North American legume Amphicarpaea bracteata (L.) Fern. (Phaseoleae-Glycininae) and from a Japanese population of its close relative Amphicarpaea edgeworthii (Benth.) var. japonica were performed to analyze relative symbiotic compatibility. Amphicarpaea edgeworthii plants formed few or no nodules with any North American bradyrhizobial strains isolated from A. bracteata, but all A. bracteata lineages formed effective nitrogen-fixing nodules with Japanese Bradyrhizobium isolates from A. edgeworthii. However, one group of A. bracteata plants (lineage Ia) when inoculated with Japanese bradyrhizobia developed a striking leaf chlorosis similar to that known to be caused by rhizobitoxine. The beta-cystathionase inhibition assay demonstrated that significant amounts of rhizobitoxine were present in nodules formed by these Japanese bradyrhizobia. No North American bradyrhizobial isolate from A. bracteata induced chlorosis on any plants, and the beta-cystathionase assay failed to detect rhizobitoxine in nodules formed by these isolates. The role of rhizobitoxine in A. edgeworthii nodulation development was tested by inoculating plants with a Bradyrhizobium elkanii rhizobitoxine-producing strain, USDA 61, and two mutant derivatives, RX17E and RX18E, which are unable to synthesize rhizobitoxine. Amphicarpaea edgeworthii inoculated with wild-type USDA 61 developed >150 nodules per plant, while plants inoculated with RX17E and RX18E developed fewer than 10 nodules per plant. Thus, efficient nodule development in A. edgeworthii appears to be highly dependent on rhizobitoxine production by Bradyrhizobium strains.     

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.     

Nodulation and Competition for Nodulation of Selected Soybean Genotypes among Bradyrhizobium japonicum Serogroup 123 Isolates

Twenty recently obtained field isolates of Bradyrhizobium japonicum serogroup 123 were tested for their nodule mass production on the standard commercial soybean (Glycine max (L.) Merr. cv. Williams) and on two soybean plant introduction (PI) genotypes previously determined to restrict nodulation by strain USDA 123. Four of the field isolates showed similar restricted nodulation on the two genotypes, while all 20 isolates produced a normal amount of nodules on G. max cv. Williams. Serological analyses with adsorbed fluorescent antibodies showed that members of the 123 serotype ranked low in nodulation of the two PIs, in contrast to members of serotypes 127 and 129. Competition studies on the PIs indicated that isolates which were restricted were not competitive for nodule occupancy against strain USDA 110. However, unrestricted isolates of serogroup 123 were very competitive against USDA 110. On G. max cv. Williams, all serogroup 123 isolates tested were very competitive against USDA 110.     

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.     

Genotypic Characterization of Phage-Typed Indigenous Soybean Bradyrhizobia and Their Host Range Symbiotic Effectiveness

Analysis of genetic diversity among indigenous rhizobia and its symbiotic effectiveness with soybean cultivar is important for development of knowledge about rhizobial ecology. In India, little is known about the genetic resources and diversity of rhizobia nodulating soybean. Indigenous bradyrhizobia isolated from root nodules of soybean plants, collected from traditional cultivating regions of two states (Madhya Pradesh and Uttar Pradesh) of India, were screened for bacteriophage sensitivity to identify successful broad host range symbiotic effectivity. Of 172 rhizobial isolates, 91 showed sensitivities to eight lytic phages and form ten groups on the basis of sensitivity patterns. The genetic diversity of 23 isolates belonging to different phage groups was assessed along with that of strains USDA123 and USDA94 by the restriction fragment length polymorphism (RFLP) analysis of 16S rDNA, intergenic spacer (IGS) (16S–23S rDNA), and DnaK regions. RFLP analysis of 16S rDNA formed 5 groups, whereas 19 and 9 groups were revealed by IGS and the DnaK genes, respectively. The IGS regions showed many amplified polymorphic bands. Nine isolates which revealed high RFLP polymorphism in the abovementioned regions (16S rRNA, IGS, DnaK) were used for 16S rRNA sequence analyses. The results indicate that taxonomically, all isolates were related to Rhizobium etli, Bradyrhizobium spp., and Bradyrhizobium yuanmingense. The doubling time of isolates varied from 9 h (MPSR155) to 16.2 h (MPSR068) in YM broth. Five isolates which did not show cross infectivity with isolated phage strains were studied for symbiotic efficiency. All isolates showed broad host range symbiotic effectiveness forming effective nodules on Vigna mungo, Vigna radiata, Vigna unguiculata, and Cajanus cajan. The present study provides information on genetic diversity and host range symbiosis of indigenous soybean rhizobia typed by different phages.     

Restriction of Nodulation by Bradyrhizobium japonicum Is Mediated by Factors Present in the Roots of Glycine max

Reciprocal grafting experiments done using soybean plant introduction genotypes indicated that restriction of nodulation by Bradyrhizobium japonicum is determined by the genotype of the root and is dependent on plant growth temperature. Microscopic analyses indicated that the soybean plant introduction genotypes restrict nodulation of B. japonicum at symbiotic stages which occur both before and after the formation of nodule primordia.     

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.     

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.     

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.     

Relationships of bradyrhizobia from the legumes Apios americana and Desmodium glutinosum.

Multilocus enzyme electrophoresis, partial 23S rRNA sequences, and nearly full-length 16S rRNA sequences all indicated high genetic similarity among root-nodule bacteria associated with Apios americana, Desmodium glutinosum, and Amphicarpaea bracteata, three common herbaceous legumes whose native geographic ranges in eastern North America overlap extensively. A total of 19 distinct multilocus genotypes (electrophoretic types [ETs]) were found among the 35 A. americana and 33 D. glutinosum isolates analyzed. Twelve of these ETs (representing 78% of all isolates) were either identical to ETs previously observed in A. bracteata populations, or differed at only one locus. Within both 23S and 16S rRNA genes, several isolates from A. americana and D. glutinosum were either identical to A. bracteata isolates or showed only single nucleotide differences. Growth rates and nitrogenase activities of A. bracteata plants inoculated with isolates from D. glutinosum were equivalent to levels found with native A. bracteata bacterial isolates, but none of the three A. americana isolates tested had high symbiotic effectiveness on A. bracteata. Phylogenetic analysis of both 23S and 16S rRNA sequences indicated that both A. americana and D. glutinosum harbored rare bacterial genotypes similar to Bradyrhizobium japonicum USDA 110. However, the predominant root nodule bacteria on both legumes were closely related to Bradyrhizobium elkanii.     

Microbial influence on gene-for-gene interactions in legume-Rhizobium symbioses

Recent advances in our understanding of the molecular genetics of legume-Rhizobium symbioses have indicated that relatively few bacterial genes are required for nodulation. While some of these genes are functionally similar and shared among microsymbionts nodulating genetically diverse legumes, others appear to encode host-specific nodulation (hsn) functions which allow for nodulation of plants within a given legume genus. More recently, genotype-specific nodulation (GSN) determinants have been identified in R. leguminosarum bv. viceae strain TOM and in B. japonicum strain USDA 110. GSN determinants refer to those bacterial sequences that allow for nodulation of specific plant genotypes within a given legume species. In contrast to the avr loci of several plant pathogens, rhizobia host-range determinants (hsn and GSN) have been shown to positively affect nodulation. That is, the introduction of exogenous hsn and GSN loci extends host-range. Since GSN loci have been reported to interact with single host plant alleles, it suggests that gene-for-gene interactions occur in rhizobial-legume symbioses and contribute to nodulation specificity at the host genotype level.     

New betaproteobacterial Rhizobium strains able to efficiently nodulate Parapiptadenia rigida (Benth.) Brenan

Among the leguminous trees native to Uruguay, Parapiptadenia rigida (Angico), a Mimosoideae legume, is one of the most promising species for agroforestry. Like many other legumes, it is able to establish symbiotic associations with rhizobia and belongs to the group known as nitrogen-fixing trees, which are major components of agroforestry systems. Information about rhizobial symbionts for this genus is scarce, and thus, the aim of this work was to identify and characterize rhizobia associated with P. rigida. A collection of Angico-nodulating isolates was obtained, and 47 isolates were selected for genetic studies. According to enterobacterial repetitive intergenic consensus PCR patterns and restriction fragment length polymorphism analysis of their nifH and 16S rRNA genes, the isolates could be grouped into seven genotypes, including the genera Burkholderia, Cupriavidus, and Rhizobium, among which the Burkholderia genotypes were the predominant group. Phylogenetic studies of nifH, nodA, and nodC sequences from the Burkholderia and the Cupriavidus isolates indicated a close relationship of these genes with those from betaproteobacterial rhizobia (beta-rhizobia) rather than from alphaproteobacterial rhizobia (alpha-rhizobia). In addition, nodulation assays with representative isolates showed that while the Cupriavidus isolates were able to effectively nodulate Mimosa pudica, the Burkholderia isolates produced white and ineffective nodules on this host.     

Diversity among Field Populations of Bradyrhizobium japonicum in Poland

Genetic structure in field populations of Bradyrhizobium japonicum isolated in Poland was determined by using several complementary techniques. Of the 10 field sites examined, only 4 contained populations of indigenous B. japonicum strains. The Polish bradyrhizobia were divided into at least two major groups on the basis of protein profiles on polyacrylamide gels, serological reaction with polyclonal antisera, repetitive extragenic palindromic PCR fingerprints of genomic DNA, and Southern hybridization analyses with nif and nod gene probes. Serological analyses indicated that 87.5% of the Polish B. japonicum isolates tested were in serogroups 123 and 129, while seven (12.5%) of the isolates tested belonged to their own unique serogroup. These seven strains also could be grouped together on the basis of repetitive extragenic palindromic PCR fingerprints, protein profiles, and Southern hybridization analyses. Cluster analyses indicated that the seven serologically undefined isolates were genetically dissimilar from the majority of the Polish B. japonicum strains. Moreover, immuno-cross-adsorption studies indicated that although the Polish B. japonicum strains reacted with polyclonal antisera prepared against strain USDA123, the majority failed to react with serogroup 123- and 129-specific antisera, suggesting that Polish bradyrhizobia comprise a unique group of root nodule bacteria which have only a few antigens in common with strains USDA123 and USDA129. Nodulation studies indicated that members of the serologically distinct group were very competitive for nodulation of Glycine max cv. Nawiko. None of the Polish serogroup 123 or 129 isolates were restricted for nodulation by USDA123- and USDA129-restricting soybean plant introduction genotypes. Taken together, our results indicate that while genetically diverse B. japonicum strains were isolated from some Polish soils, the majority of field sites contained no soybean-nodulating bacteria. In addition, despite the lack of long-term soybean production in Poland, field populations of unique B. japonicum strains are present in some Polish soils and these strains are very competitive for nodulation of currently used Polish soybean varieties.     

Vigna mungo, V. radiata and V. unguiculata plants sampled in different agronomical–ecological–climatic regions of India are nodulated by Bradyrhizobium yuanmingense

Vigna mungo, Vigna radiata and Vigna unguiculata are important legume crops cultivated in India, but little is known about the genetic resources in native rhizobia that nodulate these species. To identify these bacteria, a core collection of 76 slow-growing isolates was built from root nodules of V. mungo, V. radiata and V. unguiculata plants grown at different sites within three agro-ecological-climatic regions of India. The genetic diversity of the bacterial collection was assessed by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified DNA fragments of the 16S–23S rDNA intergenic spacer (IGS) region, and the symbiotic genes nifH and nodC. One rDNA IGS type grouped 91% of isolates, but more diversity was found at the symbiotic loci (17 symbiotic genotypes). Overall, no host plant specificity was shown, the three host plant species sharing common bradyrhizobial genotypes that represented 62% of the collection. Similarly, the predominant genotypes were found at most sampling sites and in all agro-ecological-climatic regions. Phylogenies inferred from IGS sequencing and multi-locus sequence analysis of the dnaK, glnII and recA genes indicated that all isolates but one were clustered with the Bradyrhizobium yuanmingense species. The nifH phylogeny also grouped the different nif haplotypes within a cluster including B. yuanmingense, except for one infrequent nif haplotype which formed a new lineage within the Bradyrhizobium genus. These results may reflect a long history of co-evolution between B. yuanmingense and Vigna spp. in India, while intra-species polymorphism detected in the symbiotic loci may be linked with the long history of diversification of B. yuanmingense coinciding with that of its host legumes.     

Host range,RFLP, and antigenic relationships betweenRhizobium fredii strains andRhizobium sp. NGR234

Rhizobium fredii is a nitrogen-fixing symbiont from China that combines broad host range for nodulation of legume species with cultivar specificity for nodulation of soybean. We have compared 10R. fredii strains withRhizobium sp. NGR234, a well known broad host range strain from Papua New Guinea. NGR234 nodulated 16 of 18 tested lugume species, and nodules on 14 of the 16 fixed nitrogen. TheR. fredii strains were not distinguishable from one another. They nodulated 13 of the legumes, and in only nine cases were nodules effective. All legumes nodulated byR. fredii were included within the host range of NGR234. Restriction fragment length polymorphisms (RFLPs) were detected with four DNA hybridization probes: the regulatory and commonnod genes,nodDABC; the soybean cultivar specificity gene,nolC; the nitrogenase structural genes, nifKDH; and RFRS1, a repetitive sequence fromR. fredii USDA257. A fifth locus, corresponding to a second set of soybean cultivar specificity genes,nolBTUVWX, was monomorphic. Using antisera against whole cells of threeR. fredii strains and NGR234, we separated the 11 strains into four serogroups. The anti-NGR234 sera reacted with a singleR. fredii strain, USDA191. Only one serogroup, which included USDA192, USDA201, USDA217, and USDA257, lacked cross reactivity with any of the others. Although genetic and phenotypic differences amongR. fredii strains were as great as those between NGR234 andR. fredii, our results confirm that NGR234 has a distinctly wider host range thanR. fredii.     

Molecular aspects of soybean cultivar-specific nodulation by Sinorhizobium fredii USDA257

Sinorhizobium fredii USDA257 forms nitrogen-fixing nodules in association with the primitive soybean cultivar 'Peking' but fails to initiate nodules on many advanced soybean cultivars, including 'McCall'. This distinction is controlled by a set of nodulation genes termed nolXWBTUV. Inactivation of any of these genes enables USDA257 to nodulate McCall and many other improved soybean cultivars. Mutation in the nolXWBTUV locus also alters the Nod factor structure resulting in the production of a novel molecule with glucose incorporated into the chitin backbone. Some of the genes located in the nolXWBTUV locus reveal sequence homologies to known components of the type III secretion system (TTSS) of plant and animal pathogenic bacteria. Recent studies have demonstrated the presence of a complete TTSS in USDA257 and few other symbiotic bacteria. The TTSS cluster of USDA257 contains 27 open reading frames out of which 10 code for the structural components of the TTSS. USDA257, when grown in presence of flavonoids, secrete several proteins called Nops (Nodulation Outer Proteins) into the extracellular environment. Genes located in the TTSS of USDA257 encode some of the extracellular proteins, such as NopX, NopB, and NopL. These type III secreted proteins appear to play an important role in regulating nodulation in a host-dependent manner. Failure to elaborate the Nops results in a drastic phenotypic effect on soybean nodulation, indicating that these proteins may play a pivotal role in soybean cultivar specificity. The secretion of Nops appears to be facilitated by novel filamentous appendages (pili) that are produced by USDA257 upon induction by flavonoids. Biochemical studies have demonstrated the close association of several Nops with the purified pili. However, it remains to be seen if the filamentous appendages can function as conduits for delivery of Nops into the host cell. This review examines the current state of our knowledge on the molecular aspects of soybean cultivar-specific nodulation by USDA257.     

Nodulation gene regulation and quorum sensing control density-dependent suppression and restriction of nodulation in the Bradyrhizobium japonicum-soybean symbiosis

The nodulation of Glycine max cv. Lambert and the nodulation-restricting plant introduction (PI) genotype PI 417566 by wild-type Bradyrhizobium japonicum USDA110 is regulated in a population-density-dependent manner. Nodulation on both plant genotypes was suppressed (inhibited) when plants received a high-density inoculum (10(9) cells/ml) of strain USDA110 grown in complex medium, and more nodules were produced on plants receiving a low-cell-density inoculum (10(5) cells/ml). Since cell-free supernatants from strain USDA110 grown to high cell density in complex medium decreased the expression of an nodY-lacZ fusion, this phenomenon was attributed to bradyoxetin-induced repression of nod gene expression. Inoculation of either the permissive soybean genotype (cv. Lambert) or PI 417566 with 10(9) cells/ml of the nodD2, nolA, nodW, and nwsB mutants of USDA110 enhanced nodulation (up to 24%) relative to that seen with inoculations done with 10(5) cells/ml of the mutants or the wild-type strain, indicating that these genes are involved in population-density-dependent nodulation of soybeans. In contrast, the number of nodules produced by an nodD1 mutant on either soybean genotype was less than those seen with the wild-type strain inoculated at a low inoculum density. The nodD2 mutant outcompeted B. japonicum strain USDA123 for nodulation of G. max cv. Lambert at a high or low inoculum density, and the results of root-tip-marking and time-to-nodulate studies indicated that the nolA and nodD2 mutants nodulated this soybean genotype faster than wild-type USDA110. Taken together, the results from these studies indicate that the nodD2 mutant of B. japonicum may be useful to enhance soybean nodulation at high inoculum densities and that NodD2 is a key repressor influencing host-controlled restriction of nodulation, density-dependent suppression of nodulation, perception of bradyoxetin, and competitiveness in the soybean-B. japonicum symbiosis.     

DNA Hybridization Probe for Use in Determining Restricted Nodulation among Bradyrhizobium japonicum Serocluster 123 Field Isolates

Several soybean plant introduction (PI) genotypes have recently been described which restrict nodulation of Bradyrhizobium japonicum serocluster 123 in an apparently serogroup-specific manner. While PI 371607 restricts nodulation of strains in serogroup 123 and some in serogroup 127, those in serogroup 129 are not restricted. When DNA regions within and around the B. japonicum I-110 common nodulation genes were used as probes to genomic DNA from the serogroup strains USDA 123, USDA 127, and USDA 129, several of the probes differentially hybridized to the nodulation-restricted and -unrestricted strains. One of the gene regions, cloned in plasmid pMJS12, was subsequently shown to hybridize to 4.6-kilobase EcoRI fragments from DNAs from nodulation-restricted strains and to larger fragments in nodulation-unrestricted strains. To determine if the different hybridization patterns could be used to predict nodulation restriction, we hybridized pMJS12 to EcoRI-digested genomic DNAs from uncharacterized serocluster 123 field isolates. Of the 36 strains examined, 15 were found to have single, major, 4.6-kilobase hybridizing EcoRI fragments. When tested for nodulation, 80% (12 of 15) of the strains were correctly predicted to be restricted for nodulation of the PI genotypes. In addition, hybridization patterns obtained with pMJS12 and nodulation phenotypes on PI 371607 indicated that there are at least three types of serogroup 127 strains. Our results suggest that the pMJS12 gene probe may be useful in selecting compatible host-strain combinations and in determining the suitability of field sites for the placement of soybean genotypes containing restrictive nodulation alleles.     

New nodulation mutants responsible for infection thread development in Lotus japonicus

Legume plants develop specialized root organs, the nodules, through a symbiotic interaction with rhizobia. The developmental process of nodulation is triggered by the bacterial microsymbiont but regulated systemically by the host legume plants. Using ethylmethane sulfonate mutagenesis as a tool to identify plant genes involved in symbiotic nodule development, we have isolated and analyzed five nodulation mutants, Ljsym74-3, Ljsym79-2, Ljsym79-3, Ljsym80, and Ljsym82, from the model legume Lotus japonicus. These mutants are defective in developing functional nodules and exhibit nitrogen starvation symptoms after inoculation with Mesorhizobium loti. Detailed observation revealed that infection thread development was aborted in these mutants and the nodules formed were devoid of infected cells. Mapping and complementation tests showed that Ljsym74-3, and Ljsym79-2 and Ljsym79-3, were allelic with reported mutants of L. japonicus, alb1 and crinkle, respectively. The Ljsym82 mutant is unique among the mutants because the infection thread was aborted early in its development. Ljsym74-3 and Ljsym80 were characterized as mutants with thick infection threads in short root hairs. Map-based cloning and molecular characterization of these genes will help us understand the genetic mechanism of infection thread development in L. japonicus.     

Host Plant Effects on Nodulation and Competitiveness of the Bradyrhizobium japonicum Serotype Strains Constituting Serocluster 123

Strains in Bradyrhizobium japonicum serocluster 123 are the major indigenous competitors for nodulation in a large portion of the soybean production area of the United States. Serocluster 123 is defined by the serotype strains USDA 123, USDA 127, and USDA 129. The objective of the work reported here was to evaluate the ability of two soybean genotypes, PI 377578 and PI 417566, to restrict the nodulation and reduce the competitiveness of serotype strains USDA 123, USDA 127, and USDA 129 in favor of the highly effective strain CB1809 and to determine how these soybean genotypes alter the competitive relationships among the three serotype strains in the serocluster. The soybean genotypes PI 377578 and PI 417566 along with the commonly grown cultivar Williams were planted in soil essentially free of soybean rhizobia and inoculated with single-strain treatments of USDA 123, USDA 127, USDA 129, or CB1809 and six dual-strain competition treatments of USDA 123, USDA 127, or USDA 129 versus CB1809, USDA 123 versus USDA 127, USDA 123 versus USDA 129, and USDA 127 versus USDA 129. PI 377578 severely reduced the nodulation and competitiveness of USDA 123 and USDA 127, while PI 417566 similarly affected the nodulation and competitiveness of USDA 129. Thus, the two soybean genotypes can reduce the nodulation and competitiveness of each of the three serocluster 123 serotype strains. Our results indicate that host control of restricted nodulation and reduced competitiveness is quite specific and effectively discriminates between B. japonicum strains which are serologically related.