Legumes select symbiosis island sequence variants in Bradyrhizobium

Bradyrhizobium strains sampled from 14 legume genera native to eastern North America showed substantial host‐related phylogenetic clustering at three loci in the symbiotic island (SI) region (nodC, nifD, nifH), indicating selection of distinct suites of SI lineages by different legumes. Bacteria assorted consistently with particular legumes across two regions separated by 800 km, implying recurrent assembly of the same symbiotic combinations. High genetic polymorphism of all three SI loci relative to four nonsymbiotic loci supported the inference that a form of multiple‐niche balancing selection has acted on the SI region, arising from differential symbiont utilization by different legume taxa. Extensive discordance between the tree for SI variants and a phylogenetic tree inferred for four housekeeping loci implied that lateral transfer of the symbiosis island region has been common (at least 26 transfer events among 85 Bradyrhizobium strains analysed). Patterns of linkage disequilibrium also supported the conclusion that recombination has impacted symbiotic and nonsymbiotic regions unequally. The high prevalence of lateral transfer suggests that acquisition of a novel SI variant may often confer a strong selective advantage for recipient cells.     

Symbiovars in rhizobia reflect bacterial adaptation to legumes

Legume specificity is encoded in rhizobial genetic elements that may be transferred among species and genera. Dissemination (by lateral transfer) of gene assemblies dictating host range accounts for the existence of the same biological variant (biovar) in distinct microbiological species. Different alternative biovars may exist in a single species expanding their adaptation to different niches (legume nodules). A review of all reported biovars is presented. Instead of the term biovar, symbiotic variant (symbiovar) is proposed as a parallel term to pathovar in pathogenic bacteria. Symbiovars should be determined based on the symbiotic capabilities in plant hosts, distinguished by the differences in host range and supported by symbiotic gene sequence information.     

Diverse rhizobia associated with Sophora alopecuroides grown in different regions of Loess Plateau in China

A total of seventy-five symbiotic bacterial strains isolated from root nodules of wild Sophora alopecuroides grown in different regions of China's Loess Plateau were characterized. Based on the combined RFLP patterns, thirty-five genotypes were defined among the rhizobia and they were classified into nine genomic species, including Mesorhizobium alhagi and M. gobiense as the main groups, as well as Agrobacterium tumefaciens, M. amorphae, Phyllobacterium trifolii, Rhizobium giardinii, R. indigoferae, Sinorhizobium fredii and S. meliloti as the minor groups according to the 16S rRNA and recA gene analyses. Five and three lineages of nodA and nifH were found, respectively, in these strains, implying that the symbiotic genes of the S. alopecuroides rhizobia had different origins or had divergently evolved. Results of correspondence analysis showed that there was a correlation between rhizobial genotypes and the geographic origins. Possible lateral transfer of the recA and 16S rRNA genes between the P. trifolii and A. tumefaciens strains, and that of symbiotic genes (nodA, nifH) between different genera, was shown by discrepancies of the phylogenetic relationships of the four gene loci. These results revealed diverse rhizobia associated with wild S. alopecuroides grown in different regions of China's Loess Plateau, and demonstrated for the first time the existence of symbiotic A. tumefaciens strains in root nodules of S. alopecuroides.     

Diversity of rhizobia and interactions among the host legumes and rhizobial genotypes in an agricultural-forestry ecosystem

To investigate the diversity of rhizobia and interactions among the host legumes and rhizobial genotypes in the same habitat, a total of 97 rhizobial strains isolated from nine legume species grown in an agricultural-forestry ecosystem were identified into seven genomic species and 12 symbiotic genotypes within the genera Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium based upon analyses of genomic DNA regions and symbiotic genes. The results evidenced that the symbiotic genotypes of rhizobia were consistent with their hosts of origin; revealed that vertical transfer was the main mechanism in rhizobia to maintain the symbiotic genes but lateral transfer of symbiotic genes might have happened between the closely related rhizobial species; suggested the existence of co-distribution and co-evolution among the legume hosts and compatible rhizobia. All of these data demonstrated that the biogeography of rhizobia was a result of interactions among the host legumes, bacterial genomic backgrounds and environments.     

Identification and structure of the Rhizobium galegae common nodulation genes: evidence for horizontal gene transfer

Rhizobia are soil bacteria able to fix atmospheric nitrogen in symbiosis with leguminous plants. In response to a signal cascade coded by genes of both symbiotic partners, a specific plant organ, the nodule, is formed. Rhizobial nodulation (nod) genes trigger nodule formation through the synthesis of Nod factors, a family of chitolipooligosaccharides that are specifically recognized by the host plant at the first stages of the nodulation process. Here, we present the organization and sequence of the common nod genes from Rhizobium galegae, a symbiotic member of the RHIZOBIACEAE: This species has an intriguing phylogenetic position, being symbiotic among pathogenic agrobacteria, which induce tumors instead of nodules in plant shoots or roots. This apparent incongruence raises special interest in the origin of the symbiotic apparatus of R. galegae. Our analysis of DNA sequence data indicated that the organization of the common nod gene region of R. galegae was similar to that of Sinorhizobium meliloti and Rhizobium leguminosarum, with nodIJ downstream of nodABC and the regulatory nodD gene closely linked to the common nod operon. Moreover, phylogenetic analyses of the nod gene sequences showed a close relationship especially between the common nodA sequences of R. galegae, S. meliloti, and R. leguminosarum biovars viciae and trifolii. This relationship in structure and sequence contrasts with the phylogeny based on 16S rRNA, which groups R. galegae close to agrobacteria and separate from most other rhizobia. The topology of the nodA tree was similar to that of the corresponding host plant tree. Taken together, these observations indicate that lateral nod gene transfer occurred from fast-growing rhizobia toward agrobacteria, after which the symbiotic apparatus evolved under host plant constraint.     

Population genetics and phylogenetic inference in bacterial molecular systematics: the roles of migration and recombination in Bradyrhizobium species cohesion and delineation

A combination of population genetics and phylogenetic inference methods was used to delineate Bradyrhizobium species and to uncover the evolutionary forces acting at the population-species interface of this bacterial genus. Maximum-likelihood gene trees for atpD, glnII, recA, and nifH loci were estimated for diverse strains from all but one of the named Bradyrhizobium species, and three unnamed "genospecies," including photosynthetic isolates. Topological congruence and split decomposition analyses of the three housekeeping loci are consistent with a model of frequent homologous recombination within but not across lineages, whereas strong evidence was found for the consistent lateral gene transfer across lineages of the symbiotic (auxiliary) nifH locus, which grouped strains according to their hosts and not by their species assignation. A well resolved Bayesian species phylogeny was estimated from partially congruent glnII+recA sequences, which is highly consistent with the actual taxonomic scheme of the genus. Population-level analyses of isolates from endemic Canarian genistoid legumes based on REP-PCR genomic fingerprints, allozyme and DNA polymorphism analyses revealed a non-clonal and slightly epidemic population structure for B. canariense isolates of Canarian and Moroccan origin, uncovered recombination and migration as significant evolutionary forces providing the species with internal cohesiveness, and demonstrated its significant genetic differentiation from B. japonicum, its sister species, despite their sympatry and partially overlapped ecological niches. This finding provides strong evidence for the existence of well delineated species in the bacterial world. The results and approaches used herein are discussed in the context of bacterial species concepts and the evolutionary ecology of (brady)rhizobia.     

斜茎黄芪根瘤菌结瘤基因nodA PCR扩增及PCR-RFLP分析

对采自我国北方地区的16株斜茎黄芪根瘤菌代表菌株的共同结瘤基因nodA进行了PCR扩增及PCR-RFLP分析研究。来自Mesorhizobium和Rhizobium系统发育分支的代表菌株都得到了nodA PCR扩增产物;而来自Agrobacterium系统发育分支的代表菌株都没有得到nodA PCR扩增产物。进一步的nodAPCR-RFLP分析结果表明斜茎黄芪根瘤菌具有很大的nodA基因遗传多样性,具有4种不同的16S rDNAPCR-RFLP遗传图谱类型的12株斜茎黄芪根瘤菌具有8种不同的nodA PCR-RFLP遗传图谱类型。但是斜茎黄芪根瘤菌nodA基因遗传多样性随种群而变化,来自M.septentrionale的具有相同的16S rDNA PCR-RFLP遗传图谱类型的4个代表菌株具有4种不同的nodA PCR-RFLP遗传图谱类型;而来自M.tempera-tum的具有相同的16S rDNA PCR-RFLP遗传图谱类型3个代表菌株则具有相同的nodA PCR-RFLP遗传图谱类型。此外,来自不同种的具有不同16S rDNA PCR-RFLP遗传图谱类型的菌株却具有相同的nodA PCR-RFLP遗传图谱类型,说明nodA基因可能在根瘤菌的不同种间发生了水平转移。     

rep-PCR fingerprinting and taxonomy based on the sequencing of the 16S rRNA gene of 54 elite commercial rhizobial strains

In tropical soils, diversity and biotechnological potential of symbiotic diazotrophic bacteria are high. However, the phylogenetic relationships of prominent strains are still poorly understood. In addition, in countries such as Brazil, despite the broad use of rhizobial inoculants, molecular methods are rarely used in the analysis of strains or determination of inoculant performance. In this study, both rep-PCR (BOX) fingerprintings and the DNA sequences of the 16S rRNA gene were obtained for 54 rhizobial strains officially authorized for the production of commercial inoculants in Brazil. BOX-PCR has proven to be a reliable fingerprinting tool, reinforcing the suggestion of its applicability to track rhizobial strains in culture collections and for quality control of commercial inoculants. On the other hand, the method is not adequate for grouping or defining species or even genera. Nine strains differed in more than 1.03% (15) nucleotides of the 16S rRNA gene in relation to the closest type strain, strongly indicative of new species. Those strains were distributed across the genera Burkholderia, Rhizobium, and Bradyrhizobium.     

Symbiotic conditions induce structural modifications of Sinorhizobium sp. NGR234 surface polysaccharides

When the rhizosphere is starved of nitrogen, the soil bacteria Rhizobium are able to infect legume roots and invade root nodules, where they can fix atmospheric nitrogen. Nod boxes, the nod gene promoters located on the rhizobial symbiotic plasmid, are activated by means of flavonoids present in the legume root exudates, leading to the synthesis of lipochitooligomers: the Nod factors. Several recent works pointed out the importance of rhizobial surface polysaccharides in establishing the highly specific symbiosis between rhizobia and legumes. Lipopolysaccharides (LPSs) exhibit specific active roles in the later stages of the nodulation processes, such as the penetration of the infection thread into the cortical cells or the setting up of the nitrogen-fixing phenotype. The study reported here concerns the structural modifications affecting surface (lipo)polysaccharides when Sinorhizobium sp. NGR234 strains are grown with nod gene induction under nitrogen starvation. In the absence of induction, NGR234 only produces fast-migrating LPSs. When cultured in the presence of flavonoids, the same strain produces large quantities of a high-molecular-weight rhamnose-rich lipopolysaccharide (RLPS). Because the synthesis of this compound seems to be coded by the symbiotic plasmid under direct or indirect gene induction by flavonoids, this RLPS is thought to be biologically relevant.     

Evolution of glutamine synthetase in heterokonts: evidence for endosymbiotic gene transfer and the early evolution of photosynthesis

Although the endosymbiotic evolution of chloroplasts through primary and secondary associations is well established, the evolutionary timing and stability of the secondary endosymbiotic events is less well resolved. Heterokonts include both photosynthetic and nonphotosynthetic members and the nonphotosynthetic lineages branch basally in phylogenetic reconstructions. Molecular and morphological data indicate that heterokont chloroplasts evolved via a secondary endosymbiosis, involving a heterotrophic host cell and a photosynthetic ancestor of the red algae and this endosymbiotic event may have preceded the divergence of heterokonts and alveolates. If photosynthesis evolved early in this lineage, nuclear genomes of the nonphotosynthetic groups may contain genes that are not essential to photosynthesis but were derived from the endosymbiont genome through gene transfer. These genes offer the potential to trace the evolutionary history of chloroplast gains and losses within these lineages. Glutamine synthetase (GS) is essential for ammonium assimilation and glutamine biosynthesis in all organisms. Three paralogous gene families (GSI, GSII, and GSIII) have been identified and are broadly distributed among prokaryotic and eukaryotic lineages. In diatoms (Heterokonta), the nuclear-encoded chloroplast and cytosolic-localized GS isoforms are encoded by members of the GSII and GSIII family, respectively. Here, we explore the evolutionary history of GSII in both photosynthetic and nonphotosynthetic heterokonts, red algae, and other eukaryotes. GSII cDNA sequences were obtained from two species of oomycetes by polymerase chain reaction amplification. Additional GSII sequences from eukaryotes and bacteria were obtained from publicly available databases and genome projects. Bayesian inference and maximum likelihood phylogenetic analyses of GSII provided strong support for the monophyly of heterokonts, rhodophytes, chlorophytes, and plants and strong to moderate support for the Opisthokonts. Although the phylogeny is reflective of the unikont/bikont division of eukaryotes, we propose based on the robustness of the phylogenetic analyses that the heterokont GSII gene evolved via endosymbiotic gene transfer from the nucleus of the red-algal endosymbiont to the nucleus of the host. The lack of GSIII sequences in the oomycetes examined here further suggests that the GSIII gene that functions in the cytosol of photosynthetic heterokonts was replaced by the endosymbiont-derived GSII gene.     

Associations Among Rhizobial Chromosomal Background, nod Genes, and Host Plants Based on the Analysis of Symbiosis of Indigenous Rhizobia and Wild Legumes Native to Xinjiang

The associations among rhizobia chromosomal background, nodulation genes, legume plants, and geographical regions are very attractive but still unclear. To address this question, we analyzed the interactions among rhizobia rDNA genotypes, nodC genotypes, legume genera, as well as geographical regions in the present study. Complex relationships were observed among them, which may be the genuine nature of their associations. The statistical analyses indicate that legume plant is the key factor shaping both rhizobia genetic and symbiotic diversity. In the most cases of our results, the nodC lineages are clearly associated with rhizobial genomic species, demonstrating that nodulation genes have co-evolved with chromosomal background, though the lateral transfer of nodulation genes occurred in some cases in a minority. Our results also support the hypothesis that the endemic rhizobial populations to a certain geographical area prefer to have a wide spectrum of hosts, which might be an important event for the success of both legumes and rhizobia in an isolated region.     

Three Phylogenetic Groups of nodA and nifH Genes in Sinorhizobium and Mesorhizobium Isolates from Leguminous Trees Growing in Africa and Latin America

The diversity and phylogeny of nodA and nifH genes were studied by using 52 rhizobial isolates from Acacia senegal, Prosopis chilensis, and related leguminous trees growing in Africa and Latin America. All of the strains had similar host ranges and belonged to the genera Sinorhizobium and Mesorhizobium, as previously determined by 16S rRNA gene sequence analysis. The restriction patterns and a sequence analysis of the nodA and nifH genes divided the strains into the following three distinct groups: sinorhizobia from Africa, sinorhizobia from Latin America, and mesorhizobia from both regions. In a phylogenetic tree also containing previously published sequences, the nodA genes of our rhizobia formed a branch of their own, but within the branch no correlation between symbiotic genes and host trees was apparent. Within the large group of African sinorhizobia, similar symbiotic gene types were found in different chromosomal backgrounds, suggesting that transfer of symbiotic genes has occurred across species boundaries. Most strains had plasmids, and the presence of plasmid-borne nifH was demonstrated by hybridization for some examples. The nodA and nifH genes of Sinorhizobium teranga ORS1009^T grouped with the nodA and nifH genes of the other African sinorhizobia, but Sinorhizobium saheli ORS609^T had a totally different nodA sequence, although it was closely related based on the 16S rRNA gene and nifH data. This might be because this S. saheli strain was originally isolated from Sesbania sp., which belongs to a different cross-nodulation group than Acacia and Prosopis spp. The factors that appear to have influenced the evolution of rhizobial symbiotic genes vary in importance at different taxonomic levels.     

Molecular diversity and phylogeny of indigenous <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> strains associated with <Emphasis Type="Italic">Trifolium repens</Emphasis> plants in Romania

The symbiotic nitrogen fixing legumes play an essential role in sustainable agriculture. White clover (Trifolium repens L.) is one of the most valuable perennial legumes in pastures and meadows of temperate regions. Despite its great agriculture and economic importance, there is no detailed available information on phylogenetic assignation and characterization of rhizobia associated with native white clover plants in South-Eastern Europe. In the present work, the diversity of indigenous white clover rhizobia originating in 11 different natural ecosystems in North-Eastern Romania were assessed by a polyphasic approach. Initial grouping showed that, 73 rhizobial isolates, representing seven distinct phenons were distributed into 12 genotypes, indicating a wide phenotypic and genotypic diversity among the isolates. To clarify their phylogeny, 44 representative strains were used in sequence analysis of 16S rRNA gene and IGS fragments, three housekeeping genes (atpD, glnII and recA) and two symbiosis-related genes (nodA and nifH). Multilocus sequence analysis (MLSA) phylogeny based on concatenated housekeeping genes delineated the clover isolates into five putative genospecies. Despite their diverse chromosomal backgrounds, test strains shared highly similar symbiotic genes closely related to Rhizobium leguminosarum biovar trifolii. Phylogenies inferred from housekeeping genes were incongruent with those of symbiotic genes, probably due to occurrence of lateral transfer events among native strains. This is the first polyphasic taxonomic study to report on the MLSA-based phylogenetic diversity of indigenous rhizobia nodulating white clover plants grown in various soil types in South-Eastern Europe. Our results provide valuable taxonomic data on native clover rhizobia and may increase the pool of genetic material to be used as biofertilizers.     

Molecular diversity and phylogeny of rhizobia associated with wild legumes native to Xinjiang, China

A total of 111 rhizobial strains were isolated from wild legumes in Xinjiang, an isolated region of northwest China. Nine genomic species belonging to four genera of Rhizobium, Mesorhizobium, Ensifer, and Bradyrhizobium were defined among these strains based on the characterization of amplified 16S ribosomal DNA restriction analysis (ARDRA), restriction fragment length polymorphism (RFLP) analysis of 16S-23S rDNA intergenic spacers (IGS), 16S rRNA gene sequencing and multilocus sequence analysis (MLSA). Twenty-five nodC types corresponding to eight phylogenetic clades were divided by RFLP and sequence analysis of the PCR-amplified nodC gene. The acid-producing Rhizobium and Mesorhizobium species were predominant, which may be related to both the local environments and the hosts sampled. The present study also showed the limitation of using nod genes to estimate the host specificity of rhizobia.     

Symbiotic effectiveness of rhizobial mutualists varies in interactions with native Australian legume genera

Background and Objectives

Interactions between plants and beneficial soil organisms (e.g. rhizobial bacteria, mycorrhizal fungi) are models for investigating the ecological impacts of such associations in plant communities, and the evolution and maintenance of variation in mutualisms (e.g. host specificity and the level of benefits provided). With relatively few exceptions, variation in symbiotic effectiveness across wild host species is largely unexplored.

Methods

We evaluated these associations using representatives of several legume genera which commonly co-occur in natural ecosystems in south-eastern Australia and an extensive set of rhizobial strains isolated from these hosts. These strains had been previously assigned to specific phylotypes on the basis of molecular analyses. In the first of two inoculation experiments, the growth responses of each host species was evaluated with rhizobial strains isolated from that species. The second experiment assessed performance across genera and the extent of host specificity using a subset of these strains.

Results

While host growth responses to their own (sympatric) isolates varied considerably, rhizobial phylotype was a significant predictor of symbiotic performance, indicating that bacterial species designations on the basis of molecular markers have ecological importance. Hosts responded in qualitatively different ways to sympatric and allopatric strains of rhizobia, ranging from species with a clear preference for their own strains, to those that were broad generalists, through to species that grew significantly better with allopatric strains.

Conclusion

Theory has focused on trade-offs between the provision of benefits and symbiont competitive ability that might explain the persistence of less beneficial strains. However, differences in performance among co-occurring host species could also drive such patterns. Our results thus highlight the likely importance of plant community structure in maintaining variation in symbiotic effectiveness.     

American origin of Cupriavidus bacteria associated with invasive Mimosa legumes in the Philippines

To identify the origins of Cupriavidus nodule symbionts associated with two invasive Mimosa species in the Philippines, 22 isolates were sequenced for portions of three chromosomal genes and two symbiotic plasmid loci. Eleven isolates were identical at all gene loci (2713?bp) to a lineage found in Central America. Four other Philippine isolates were identical to a second Cupriavidus lineage distributed both in Central America and in the Caribbean. None of the remaining Philippine strains had more than 0.6% sequence divergence from American Cupriavidus lineages. These results imply that the Philippine population was founded by multiple introductions from the native range of their Mimosa hosts.     

Genetic diversity and symbiotic evolution of rhizobia from root nodules of Coronilla varia

Ninety symbiotic rhizobial isolates from root nodules of Coronilla varia growing in the Shaanxi province of China were characterized. Combined with the results of RFLP patterns, six genotypes were defined among the rhizobial strains and they were divided into three genomic genera. These included Mesorhizobium sp., M. alhagi, M. amorphae, M. metallidurans/M. gobiense as the dominant group (86.7%), and Rhizobium yanglingense and Agrobacterium tumefaciens as the minor groups, according to analysis of the corresponding 16S rRNA, nodC and nifH genes. Five nodC types, which mainly grouped into the Mesorhizobium genus, were obtained from all the isolates examined, implying that nodC genes probably occurred from the native habitat through lateral transfer and long-term adaptation, finally evolving toward M. alhagi. Four different nifH types, displaying obvious differences compared to those of 16S rRNA and nodC, implied that possible lateral transfer of the symbiotic genes occurred between different genera. The association between soil components and the genetic diversity of the rhizobial population demonstrated that combined genotypes were positively correlated with the pH of soil samples.     

Glutamine synthetase II inRhizobium: Reexamination of the proposed horizontal transfer of DNA from eukaryotes to prokaryotes

Summary We have determined the DNA sequence of aRhizobium meliloti gene that encodes glutamine synthetase II (GSII). The deduced amino acid sequence was compared to that ofBradyrhizobium japonicum GSII and those of various plant and mammalian glutamine synthetases (GS) in order to evaluate a proposal that the gene for this enzyme was recently transferred from plants to their symbiotic bacteria. There is 83.6% identity between theR. meliloti andB. japonicum proteins. The bacterial GSII proteins average 42.5% identity with the plant GS proteins and 41.8% identity with their mammalian counterparts. The plant proteins average 53.7% identity with the mammalian proteins. Thus, the GS proteins are highly conserved and the divergence of these proteins is proportional to the phylogenetic divergence of the organisms from which the sequences were determined. No transfer of genes across large taxonomic gaps is needed to explain the presence of GSII in these bacteria.     

Evolutionary genetics and biogeographic structure of Rhizobium gallicum sensu lato, a widely distributed bacterial symbiont of diverse legumes

We used phylogenetic and population genetics approaches to evaluate the importance of the evolutionary forces on shaping the genetic structure of Rhizobium gallicum and related species. We analysed 54 strains from several populations distributed in the Northern Hemisphere, using nucleotide sequences of three 'core' chromosomal genes (rrs, glnII and atpD) and two 'auxiliary' symbiotic genes (nifH and nodB) to elucidate the biogeographic history of the species and symbiotic ecotypes (biovarieties) within species. The analyses revealed that strains classified as Rhizobium mongolense and Rhizobium yanglingense belong to the chromosomal evolutionary lineage of R. gallicum and harbour symbiotic genes corresponding to a new biovar; we propose their reclassification as R. gallicum bv. orientale. The comparison of the chromosomal and symbiotic genes revealed evidence of lateral transfer of symbiotic information within and across species. Genetic differentiation analyses based on the chromosomal protein-coding genes revealed a biogeographic pattern with three main populations, whereas the 16S rDNA sequences did not resolve that biogeographic pattern. Both the phylogenetic and population genetic analyses showed evidence of recombination at the rrs locus. We discuss our results in the light of the contrasting views of bacterial species expressed by microbial taxonomist and evolutionary biologists.     

Competitive nodulation blocking of cv. Afghanistan pea is related to high levels of nodulation factors made by some strains of Rhizobium leguminosarum bv. viciae

Cultivar Afghanistan peas are resistant to nodulation by many strains of Rhizobium leguminosarum bv. viciae but are nodulated by strain TOM, which carries the host specificity gene nodX. Some strains that lack nodX can inhibit nodulation of cv. Afghanistan by strain TOM. We present evidence that this "competitive nodulation-blocking" (Cnb) phenotype may result from high levels of Nod factors inhibiting nodulation of cv. Afghanistan peas. The TOM nod gene region (including nodX) is cloned on pIJ1095, and strains (including TOM itself) carrying pIJ1095 nodulate cv. Afghanistan peas very poorly but can nodulate other varieties normally. The presence of pIJ1095, which causes increased levels of Nod factor production, correlates with Cnb. Nodulation of cv. Afghanistan by TOM is also inhibited by a cloned nodD gene that increases nod gene expression and Nod factor production. Nodulation of cv. Afghanistan can be stimulated if nodD on pIJ1095 is mutated, thus severely reducing the level of Nod factor produced. Repression of nod gene expression by nolR eliminates the Cnb phenotype and can stimulate nodulation of cv. Afghanistan. Addition of Nod factors to cv. Afghanistan roots strongly inhibits nodulation. The Cnb+ strains and added Nod factors inhibit infection thread initiation by strain TOM. The sym2A allele determines resistance of cv. Afghanistan to nodulation by strains of R. leguminosarum bv. viciae lacking nodX. We tested whether sym2A is involved in Cnb by using a pea line carrying the sym2A region introgressed from cv. Afghanistan; nodulation in the introgressed line was inhibited by Cnb+ strains. Therefore, the sym2A region has an effect on Cnb, although another locus (or loci) may contribute to the stronger Cnb seen in cv. Afghanistan.