The Genome of the Acid Soil-Adapted Strain Rhizobium favelukesii OR191 Encodes Determinants for Effective Symbiotic Interaction With Both an Inverted Repeat Lacking Clade and a Phaseoloid Legume Host.

Although Medicago sativa forms highly effective symbioses with the comparatively acid-sensitive genus Ensifer, its introduction into acid soils appears to have selected for symbiotic interactions with acid-tolerant R. favelukesii strains. Rhizobium favelukesii has the unusual ability of being able to nodulate and fix nitrogen, albeit sub-optimally, not only with M. sativa but also with the promiscuous host Phaseolus vulgaris. Here we describe the genome of R. favelukesii OR191 and genomic features important for the symbiotic interaction with both of these hosts. The OR191 draft genome contained acid adaptation loci, including the highly acid-inducible lpiA/acvB operon and olsC, required for production of lysine- and ornithine-containing membrane lipids, respectively. The olsC gene was also present in other acid-tolerant Rhizobium strains but absent from the more acid-sensitive Ensifer microsymbionts. The OR191 symbiotic genes were in general more closely related to those found in Medicago microsymbionts. OR191 contained the nodA, nodEF, nodHPQ, and nodL genes for synthesis of polyunsaturated, sulfated and acetylated Nod factors that are important for symbiosis with Medicago, but contained a truncated nodG, which may decrease nodulation efficiency with M. sativa. OR191 contained an E. meliloti type BacA, which has been shown to specifically protect Ensifer microsymbionts from Medicago nodule-specific cysteine-rich peptides. The nitrogen fixation genes nifQWZS were present in OR191 and P. vulgaris microsymbionts but absent from E. meliloti-Medicago microsymbionts. The ability of OR191 to nodulate and fix nitrogen symbiotically with P. vulgaris indicates that this host has less stringent requirements for nodulation than M. sativa but may need rhizobial strains that possess nifQWZS for N2-fixation to occur. OR191 possessed the exo genes required for the biosynthesis of succinoglycan, which is required for the Ensifer-Medicago symbiosis. However, 1H-NMR spectra revealed that, in the conditions tested, OR191 exopolysaccharide did not contain a succinyl substituent but instead contained a 3-hydroxybutyrate moiety, which may affect its symbiotic performance with Medicago hosts. These findings provide a foundation for the genetic basis of nodulation requirements and symbiotic effectiveness with different hosts.

High-quality draft genome sequence of Ensifer meliloti Mlalz-1, a microsymbiont of Medicago laciniata (L.) miller collected in Lanzarote, Canary Islands, Spain.

10.1601/nm.1335 Mlalz-1 (INSDC = ATZD00000000) is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen-fixing nodule of Medicago laciniata (L.) Miller from a soil sample collected near the town of Guatiza on the island of Lanzarote, the Canary Islands, Spain. This strain nodulates and forms an effective symbiosis with the highly specific host M. laciniata. This rhizobial genome was sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) sequencing project. Here the features of 10.1601/nm.1335 Mlalz-1 are described, together with high-quality permanent draft genome sequence information and annotation. The 6,664,116 bp high-quality draft genome is arranged in 99 scaffolds of 100 contigs, containing 6314 protein-coding genes and 74 RNA-only encoding genes. Strain Mlalz-1 is closely related to 10.1601/nm.1335 10.1601/strainfinder?urlappend=%3Fid%3DIAM+12611 T, 10.1601/nm.1334 A 321T and 10.1601/nm.17831 10.1601/strainfinder?urlappend=%3Fid%3DORS+1407 T, based on 16S rRNA gene sequences. gANI values of ≥98.1% support the classification of strain Mlalz-1 as 10.1601/nm.1335. Nodulation of M. laciniata requires a specific nodC allele, and the nodC gene of strain Mlalz-1 shares ≥98% sequence identity with nodC of M. laciniata-nodulating 10.1601/nm.1328 strains, but ≤93% with nodC of 10.1601/nm.1328 strains that nodulate other Medicago species. Strain Mlalz-1 is unique among sequenced 10.1601/nm.1335 strains in possessing genes encoding components of a T2SS and in having two versions of the adaptive acid tolerance response lpiA-acvB operon. In 10.1601/nm.1334 strain 10.1601/strainfinder?urlappend=%3Fid%3DWSM+419, lpiA is essential for enhancing survival in lethal acid conditions. The second copy of the lpiA-acvB operon of strain Mlalz-1 has highest sequence identity (> 96%) with that of 10.1601/nm.1334 strains, which suggests genetic recombination between strain Mlalz-1 and 10.1601/nm.1334 and the horizontal gene transfer of lpiA-acvB.

High-quality permanent draft genome sequence of the Bradyrhizobium elkanii type strain USDA 76T, isolated from Glycine max (L.) Merr.

Bradyrhizobium elkanii USDA 76T (INSCD = ARAG00000000), the type strain for Bradyrhizobium elkanii, is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen-fixing root nodule of Glycine max (L. Merr) grown in the USA. Because of its significance as a microsymbiont of this economically important legume, B. elkanii USDA 76T was selected as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria sequencing project. Here the symbiotic abilities of B. elkanii USDA 76T are described, together with its genome sequence information and annotation. The 9,484,767 bp high-quality draft genome is arranged in 2 scaffolds of 25 contigs, containing 9060 protein-coding genes and 91 RNA-only encoding genes. The B. elkanii USDA 76T genome contains a low GC content region with symbiotic nod and fix genes, indicating the presence of a symbiotic island integration. A comparison of five B. elkanii genomes that formed a clique revealed that 356 of the 9060 protein coding genes of USDA 76T were unique, including 22 genes of an intact resident prophage. A conserved set of 7556 genes were also identified for this species, including genes encoding a general secretion pathway as well as type II, III, IV and VI secretion system proteins. The type III secretion system has previously been characterized as a host determinant for Rj and/or rj soybean cultivars. Here we show that the USDA 76T genome contains genes encoding all the type III secretion system components, including a translocon complex protein NopX required for the introduction of effector proteins into host cells. While many bradyrhizobial strains are unable to nodulate the soybean cultivar Clark (rj1), USDA 76T was able to elicit nodules on Clark (rj1), although in reduced numbers, when plants were grown in Leonard jars containing sand or vermiculite. In these conditions, we postulate that the presence of NopX allows USDA 76T to introduce various effector molecules into this host to enable nodulation.

Biogeography of a Novel Ensifer meliloti Clade Associated with the Australian Legume Trigonella suavissima.

Here, we describe a novel clade within Ensifer meliloti and consider how geographic and ecological isolation contributed to the limited distribution of this group. Members of the genus Ensifer are best known for their ability to form nitrogen-fixing symbioses with forage legumes of three related genera, Medicago L., Melilotus Mill., and Trigonella L., which are members of the tribe Trifolieae. These legumes have a natural distribution extending from the Mediterranean Basin through western Asia, where there is an unsurpassed number of species belonging to these genera. Trigonella suavissima L. is unusual in that it is the only species in the tribe Trifolieae that is native to Australia. We compared the genetic diversity and taxonomic placement of rhizobia nodulating T. suavissima with those of members of an Ensifer reference collection. Our goal was to determine if the T. suavissima rhizobial strains, like their plant host, are naturally limited to the Australian continent. We used multilocus sequence analysis to estimate the genetic relatedness of 56 T. suavissima symbionts to 28 Ensifer reference strains. Sequence data were partitioned according to the replicons in which the loci are located. The results were used to construct replicon-specific phylogenetic trees. In both the chromosomal and chromid trees, the Australian strains formed a distinct clade within E. meliloti The strains also shared few alleles with Ensifer reference strains from other continents. Carbon source utilization assays revealed that the strains are also unusual in their ability to utilize 2-oxoglutarate as a sole carbon source. A strategy was outlined for locating similar strains elsewhere.IMPORTANCE In this study, we employed a biogeographical approach to investigate the origins of a symbiotic relationship between an Australian legume and its nitrogen-fixing rhizobia. The question of the ancestral origins of these symbionts is based on the observation that the legume host is not closely related to other native Australian legumes. Previous research has shown that the legume host Trigonella suavissima is instead closely related to legumes native to the Mediterranean Basin and western Asia, suggesting that it may have been introduced in Australia from those regions. This led to the question of whether its rhizobia may have been introduced as well. In this study, we were unable to find persuasive evidence supporting this hypothesis. Instead, our results suggest either that the T. suavissima rhizobia are native to Australia or that our methods for locating their close relatives elsewhere are inadequate. A strategy to investigate the latter alternative is proposed.

High-quality permanent draft genome sequence of the Mimosa asperata - nodulating Cupriavidus sp. strain AMP6.

Cupriavidus sp. strain AMP6 is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from a root nodule of Mimosa asperata collected in Santa Ana National Wildlife Refuge, Texas, in 2005. Mimosa asperata is the only legume described so far to exclusively associates with Cupriavidus symbionts. Moreover, strain AMP6 represents an early-diverging lineage within the symbiotic Cupriavidus group and has the capacity to develop an effective nitrogen-fixing symbiosis with three other species of Mimosa. Therefore, the genome of Cupriavidus sp. strain AMP6 enables comparative analyses of symbiotic trait evolution in this genus and here we describe the general features, together with sequence and annotation. The 7,579,563 bp high-quality permanent draft genome is arranged in 260 scaffolds of 262 contigs, contains 7,033 protein-coding genes and 97 RNA-only encoding genes, and is part of the GEBA-RNB project proposal.

High-quality permanent draft genome sequence of the Parapiptadenia rigida-nodulating Cupriavidus sp. strain UYPR2.512.

Cupriavidus sp. strain UYPR2.512 is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from a root nodule of Parapiptadenia rigida grown in soils from a native forest of Uruguay. Here we describe the features of Cupriavidus sp. strain UYPR2.512, together with sequence and annotation. The 7,858,949 bp high-quality permanent draft genome is arranged in 365 scaffolds of 369 contigs, contains 7,411 protein-coding genes and 76 RNA-only encoding genes, and is part of the GEBA-RNB project proposal.

High-quality permanent draft genome sequence of the Parapiptadenia rigida-nodulating Burkholderia sp. strain UYPR1.413.

Burkholderia sp. strain UYPR1.413 is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from a root nodule of Parapiptadenia rigida collected at the Angico plantation, Mandiyu, Uruguay, in December 2006. A survey of symbionts of P. rigida in Uruguay demonstrated that this species is nodulated predominantly by Burkholderia microsymbionts. Moreover, Burkholderia sp. strain UYPR1.413 is a highly efficient nitrogen fixing symbiont with this host. Currently, the only other sequenced isolate to fix with this host is Cupriavidus sp. UYPR2.512. Therefore, Burkholderia sp. strain UYPR1.413 was selected for sequencing on the basis of its environmental and agricultural relevance to issues in global carbon cycling, alternative energy production, and biogeochemical importance, and is part of the GEBA-RNB project. Here we describe the features of Burkholderia sp. strain UYPR1.413, together with sequence and annotation. The 10,373,764 bp high-quality permanent draft genome is arranged in 336 scaffolds of 342 contigs, contains 9759 protein-coding genes and 77 RNA-only encoding genes.

Stemphylium Leaf Spot of Parsley in California Caused by Stemphylium vesicarium.

From 2009 through 2011, a previously undescribed disease occurred on commercial parsley in coastal (Ventura County) California. Symptoms of the disease consisted of circular to oval, tan to brown leaf spots and resulted in loss of crop quality and, hence, reduced yields. A fungus was consistently isolated from symptomatic parsley. Morphological and molecular data identified the fungus as Stemphylium vesicarium. When inoculated onto parsley leaves, the isolates caused symptoms that were identical to those seen in the field; the same fungus was recovered from test plants, thus completing Koch's postulates. Additional inoculation experiments demonstrated that 10 of 11 tested flat leaf and curly parsley cultivars were susceptible. The parsley isolates also caused small leaf spots on other Apiaceae family plants (carrot and celery) but not on leek, onion, spinach, and tomato. Isolates caused brown lesions to form when inoculated onto pear fruit but only when the fruit tissue was wounded. Using a freeze-blotter seedborne pathogen assay, parsley seed was found to have a low incidence (0.25%) of S. vesicarium. When inoculated onto parsley leaves, three of four isolates from seed caused the same leaf spot disease. This is the first documentation of a foliar parsley disease caused by S. vesicarium. The occurrence of S. vesicarium on parsley seed indicates that infested seed may be one source of initial inoculum. Based on the negative results in the host range experiments, it appears that this parsley pathogen differs from the S. vesicarium that causes disease on leek, garlic, onion, and pear fruit.

Development and application of a multilocus sequence analysis method for the identification of genotypes within genus Bradyrhizobium and for establishing nodule occupancy of soybean (Glycine max L. Merr).

A multilocus sequence typing (MLST) method based on allelic variation of seven chromosomal loci was developed for characterizing genotypes (GT) within the genus Bradyrhizobium. With the method, 29 distinct multilocus GT were identified among 190 culture collection soybean strains. The occupancy of 347 nodules taken from uninoculated field-grown soybean plants also was determined. The bacteroid GT were either the same as or were closely related to GT identified among strains in the culture collection. Double-nodule occupancy estimates of 2.9% were much lower than values published based on serology. Of the 347 nodules examined, 337 and 10 were occupied by Bradyrhizobium japonicum and B. elkanii, respectively. The collection strains within the species B. japonicum and B. elkaniialso were compared with Bradyrhizobium cultures from other legumes. In many cases, the observed GT varied more according to their geographic origin than by their trap hosts of isolation. In other cases, there were no apparent relationships with either the legume or geographic source. The MLST method that was developed should be a useful tool in determining the influence of geographic location, temperature, season, soil type, and host plant cultivar on the distribution of GT of Bradyrhizobium spp.

Evidence of an American origin for symbiosis-related genes in Rhizobium lusitanum.

Randomly amplified polymorphic DNA (RAPD) analysis was used to investigate the diversity of 179 bean isolates recovered from six field sites in the Arcos de Valdevez region of northwestern Portugal. The isolates were divided into 6 groups based on the fingerprint patterns that were obtained. Representatives for each group were selected for sequence analysis of 4 chromosomal DNA regions. Five of the groups were placed within Rhizobium lusitanum, and the other group was placed within R. tropici type IIA. Therefore, the collection of Portuguese bean isolates was shown to include the two species R. lusitanum and R. tropici. In plant tests, the strains P1-7, P1-1, P1-2, and P1-16 of R. lusitanum nodulated and formed nitrogen-fixing symbioses both with Phaseolus vulgaris and Leucaena leucocephala. A methyltransferase-encoding nodS gene identical with the R. tropici locus that confers wide host range was detected in the strain P1-7 as well as 24 others identified as R. lusitanum. A methyltransferase-encoding nodS gene also was detected in the remaining isolates of R. lusitanum, but in this case the locus was that identified with the narrow-host-range R. etli. Representatives of isolates with the nodS of R. etli formed effective nitrogen-fixing symbioses with P. vulgaris and did not nodulate L. leucocephala. From sequence data of nodS, the R. lusitanum genes for symbiosis were placed within those of either R. tropici or R. etli. These results would support the suggestion that R. lusitanum was the recipient of the genes for symbiosis with beans from both R. tropici and R. etli.

Evidence that the exoH gene of Sinorhizobium meliloti does not appear to influence symbiotic effectiveness with Medicago truncatula 'Jemalong A17'.

The purpose of this study was to identify strains of Sinorhizobium meliloti that formed either an effective or completely ineffective symbiosis with Medicago truncatula L. 'Jemalong A17' and, subsequently, to determine whether differences existed between their exoH genes. Sinorhizobium meliloti TII7 and A5 formed an effective and ineffective symbiosis with M. truncatula 'Jemalong A17', respectively. Using a multilocus sequence typing method, both strains were shown to have chromosomes identical with S. meliloti Rm1021 and RCR2011. The 2260-bp segments of DNA stretching from the 3' end of exoI through open reading frames of hypothetical proteins SM_b20952 and SM_b20953 through exoH into the 5' end of exoK in strains TII7 and Rm1021 differed by a single nucleotide at base 127 of the hypothetical protein SM_b20953. However, the derived amino acid sequences of the exoH genes of effective TII7, ineffective A5, and strain Rm1021 were shown to be identical with each other. Therefore, it would seem unlikely that the gene product of exoH is directly involved with the low efficiency of a symbiosis of strain Rm1021 with M. truncatula 'Jemalong A17'. Complementation or complete genome sequence analyses involving strains TII7 and A5 might be useful approaches to investigate the molecular bases for the differential symbiotic response with M. truncatula 'Jemalong A17'.

Application of multilocus sequence typing to study the genetic structure of megaplasmids in medicago-nodulating rhizobia.

A multilocus sequence typing (MLST) analysis was used to examine the genetic structure and diversity within the two large extrachromosomal replicons in Medicago-nodulating rhizobia (Sinorhizobium meliloti and Sinorhizobium medicae). The allelic diversity within these replicons was high compared to the reported diversity within the corresponding chromosomes of the same strains (P. van Berkum et al., J. Bacteriol. 188:5570-5577, 2006). Also, there was strong localized linkage disequilibrium (LD) between certain pSymA loci: e.g., nodC and nifD. Although both of these observations could be explained by positive (or diversifying) selection by plant hosts, results of tests for positive selection did not provide consistent support for this hypothesis. The strong LD observed between the nodC and nifD genes could also be explained by their close proximity on the pSymA replicon. Evidence was obtained that some nodC alleles had a history of intragenic recombination, while other alleles of this locus had a history of intergenic recombination. Both types of recombination were associated with a decline in symbiotic competence with Medicago sativa as the host plant. The combined observations of LD between the nodC and nifD genes and intragenic recombination within one of these loci indicate that the symbiotic gene region on the pSymA plasmid has evolved as a clonal segment, which has been laterally transferred within the natural populations.

Rhizobia from Lanzarote, the Canary Islands, that nodulate Phaseolus vulgaris have characteristics in common with Sinorhizobium meliloti isolates from mainland Spain.

The stable, low-molecular-weight (LMW) RNA fractions of several rhizobial isolates of Phaseolus vulgaris grown in the soil of Lanzarote, an island of the Canary Islands, were identical to a less-common pattern found within Sinorhizobium meliloti (assigned to group II) obtained from nodules of alfalfa and alfalfa-related legumes grown in northern Spain. The P. vulgaris isolates and the group II LMW RNA S. meliloti isolates also were distinguishable in that both had two conserved inserts of 20 and 46 bp in the 16S-23S internal transcribed spacer region that were not present in other strains of S. meliloti. The isolates from P. vulgaris nodulated bean but not Medicago sativa, while those recovered from Medicago, Melilotus, and Trigonella spp. nodulated both host legumes. The bean isolates also were distinguished from those of Medicago, Melilotus, and Trigonella spp. by nodC sequence analysis. The nodC sequences of the bean isolates were most similar to those reported for S. meliloti bv. mediterranense and Sinorhizobium fredii bv. mediterranense (GenBank accession numbers DQ333891 and AF217267, respectively). None of the evidence placed the bean isolates from Lanzarote in the genus Rhizobium, which perhaps is inconsistent with seed-borne transmission of Rhizobium etli from the Americas to the Canaries as an explanation for the presence of bean-nodulating rhizobia in soils of Lanzarote.

Evidence from internally transcribed spacer sequence analysis of soybean strains that extant Bradyrhizobium spp. are likely the products of reticulate evolutionary events.

The internally transcribed spacer (ITS) sequences of several members within each of 17 soybean bradyrhizobial serogroups were determined to establish whether the regions within all members of each serogroup were identical. The rationale was to provide a sequence-based alternative to serology. The objective also was to link the extensive older literature on soybean symbiosis based on serology with ITS sequence data for more recent isolates from both soybean and other legumes nodulated by rhizobia within the genus Bradyrhizobium. With the exception of serogroup 31 and 110 strains, sequence identity was established within each serogroup. Variation ranged from 0 to 23 nucleotides among serogroup 31 strains, and the regions in the type strains USDA 31 (serogroup 31) and USDA 130 (serogroup 130) were identical. Sequence identity was established among most strains within serogroup 110. The exceptions were USDA 452 and USDA 456, which had ITS sequences that were identical with those of the serotype 124 strain, USDA 124. Perhaps this would imply that USDA 452, USDA 456, and serogroup 31 strains are members of rhizobial lineages resulting from genetic exchange and homologous recombination events. This conclusion would be supported by the construction of a phylogenetic network from the ITS sequence alignment implying that the genomes of extant members of the genus Bradyrhizobium are likely the products of reticulate evolutionary events. A pairwise homoplasy index (phi or Phi(w)) test was used to obtain further evidence for recombination. The ITS sequences of USDA 110 and USDA 124 were more divergent (53 nucleotides) than this region between the type strain Bradyrhizobium japonicum USDA 6(T) and the proposed species Bradyrhizobium yuanmingense (28 nucleotides) and Bradyrhizobium liaoningense (48 nucleotides). Therefore, support for assigning discrete species boundaries among these three proposed species appears limited, considering the evidence for recombination, the narrow divergence of the ITS sequence, and their relative placement on the phylogenetic network.

Stable low molecular weight RNA profiling showed variations within Sinorhizobium meliloti and Sinorhizobium medicae nodulating different legumes from the alfalfa cross-inoculation group.

Four different low molecular weight (LMW) RNA profiles, designated I-IV, among 179 isolates from Medicago, Melilotus and Trigonella species growing in a field site in Northern Spain were identified. From sequence analysis of the 16S rRNA, atpD and recA genes as well as DNA-DNA hybridization analysis with representatives of each LMW RNA profile it was evident that isolates with LMW RNA profiles I and II belonged to Sinorhizobium meliloti and those displaying profiles III and IV to Sinorhizobium medicae. Therefore, two distinct LMW RNA electrophoretic mobility profiles were found within each of these two species. Collectively, LMW RNA profiles I and II (identified as S. meliloti) were predominant in Melilotus alba, Melilotus officinalis and Medicago sativa. Profiles III and IV (identified as S. medicae) were predominant in Melilotus parviflora, Medicago sphaerocarpa, Medicago lupulina and Trigonella foenum-graecum. All the four LMW RNA profiles were identified among isolates from Trigonella monspelliaca nodules. These results revealed a different specificity by the hosts of the alfalfa cross-inoculation group towards the two bacterial species found in this study.

Chromosomal and symbiotic relationships of rhizobia nodulating Medicago truncatula and M. laciniata.

Multilocus sequence typing (MLST) is a sequence-based method used to characterize bacterial genomes. This method was used to examine the genetic structure of Medicago-nodulating rhizobia at the Amra site, which is located in an arid region of Tunisia. Here the annual medics Medicago laciniata and M. truncatula are part of the natural flora. The goal of this study was to identify whether distinct chromosomal groups of rhizobia nodulate M. laciniata because of its restricted requirement for specific rhizobia. The MLST analysis involved determination of sequence variation in 10 chromosomal loci of 74 isolates each of M. laciniata and M. truncatula. M. truncatula was used as a control trap host, because unlike M. laciniata, it has relatively unrestricted rhizobial requirements. Allelic diversity among the plasmid nodC alleles in the isolates was also determined. The 148 isolates were placed into 26 chromosomal sequence types (STs), only 3 of which had been identified previously. The rhizobia of M. laciniata were shown to be part of the general Medicago-nodulating population in the soil because 99.95% of the isolates had chromosomal genotypes similar to those recovered from M. truncatula. However, the isolates recovered from M. laciniata were less diverse than those recovered from M. truncatula, and they also harbored an unusual nodC allele. This could perhaps be best explained by horizontal transfer of the different nodC alleles among members of the Medicago-nodulating rhizobial population at the field site. Evidence indicating a history of lateral transfer of rhizobial symbiotic genes across distinct chromosomal backgrounds is provided.

Rhizobium giardinii is the microsymbiont of Illinois bundleflower (Desmanthus illinoensis (Michx.) Macmillan) in midwestern prairies.

Illinois bundleflower (Desmanthus illinoensis (Michx.) Macmillan) has potential as a grain and forage legume for the American Midwest. Inoculant-quality rhizobia for this legume have been identified but not previously characterized. Rhizobia trapped from 20 soils in the natural range of the Illinois bundleflower had characteristics that placed them overwhelmingly within the species Rhizobium giardinii, one of the few occasions this species has been recovered from legumes, raising questions on the biogeography and spread of midwestern prairie rhizobia.

Multilocus sequence typing as an approach for population analysis of Medicago-nodulating rhizobia.

Multilocus sequence typing (MLST), a sequence-based method to characterize bacterial genomes, was used to examine the genetic structure in a large collection of Medicago-nodulating rhizobial strains. This is the first study where MLST has been applied in conjunction with eBURST analysis to determine the population genetic structure of nonpathogenic bacteria recovered from the soil environment. Sequence variation was determined in 10 chromosomal loci of 231 strains that predominantly originated from southwest Asia. Genetic diversity for each locus ranged from 0.351 to 0.819, and the strains examined were allocated to 91 different allelic profiles or sequence types (STs). The genus Medicago is nodulated by at least two groups of rhizobia with divergent chromosomes that have been classified as Sinorhizobium meliloti and Sinorhizobium medicae. Evidence was obtained that the degree of genetic exchange among the chromosomes across these groups is limited. The symbiosis with Medicago polymorpha of nine strains placed in one of these groups, previously identified as S. medicae, ranged from ineffective to fully effective, indicating that there was no strong relationship between symbiotic phenotype and chromosomal genotype.

Proposal for combining Bradyrhizobium spp. (Aeschynomene indica) with Blastobacter denitrificans and to transfer Blastobacter denitrificans (Hirsch and Muller, 1985) to the genus Bradyrhizobium as Bradyrhizobium denitrificans (comb. nov.).

The symbiotic bradyrhizobia of Aeschynomene indica and the aquatic budding bacterium Blastobacter denitrificans have much in common and this study broadens the characters that are shared between the two. The 23S rRNA gene sequences of the bradyrhizobial isolates were most similar to each other and to the sequence of Bl. denitrificans. Evidence for the presence of photosynthetic genes in the genome of Bl. denitrificans was obtained by PCR using primers to the conserved M subunit (pufM) of the photosynthetic reaction center present in purple sulfur and purple nonsulfur bacteria. The deduced amino acid sequences of the partial PufM protein of Bl. denitrificans and the corresponding sequences obtained from the bradyrhizobial isolates were identical. Both the bradyrhizobial isolates and the type strain of Bl. denitrificans shared the ability to propagate by budding, demonstrated by electron microscopy. Even though many interspecific characters were shared among the bradyrhizobial isolates including Bl. denitrificans, it was evident from Amplified Fragment Length Polymorphism (AFLP) analysis that genomic variation existed among the collection that was examined. Variation among bradyrhizobial isolates and Bl. denitrificans also was established in carbon and nitrogen source utilization and the ability to grow at elevated temperature. Based on these results and previously reported evidence it is suggested that the type strain for Bl. denitrificans and the bradyrhizobial isolates from nodules of A. indica belong to a common group of bacteria. Therefore, it is proposed that they be combined into the genus Bradyrhizobium and that LMG 8443 be transferred to this genus as the type strain for B. denitrificans.

Ethiopian soils harbor natural populations of rhizobia that form symbioses with common bean ( Phaseolus vulgaris L.).

The diversity and taxonomic relationships of 83 bean-nodulating rhizobia indigenous to Ethiopian soils were characterized by PCR-RFLP of the internally transcribed spacer (ITS) region between the 16S and 23S rRNA genes, 16S rRNA gene sequence analysis, multilocus enzyme electrophoresis (MLEE), and amplified fragment-length polymorphism. The isolates fell into 13 distinct genotypes according to PCR-RFLP analysis of the ITS region. Based on MLEE, the majority of these genotypes (70%) was genetically related to the type strain of Rhizobium leguminosarum. However, from analysis of their 16S rRNA genes, the majority was placed with Rhizobium etli. Transfer and recombination of the 16S rRNA gene from presumptively introduced R. etli to local R. leguminosarum is a possible theory to explain these contrasting results. However, it seems unlikely that bean rhizobia originating from the Americas (or Europe) extensively colonized soils of Ethiopia because Rhizobium tropici, Rhizobium gallicum, and Rhizobium giardinii were not detected and only a single ineffective isolate of R. etli that originated from a remote location was identified. Therefore, Ethiopian R. leguminosarum may have acquired the determinants for nodulation of bean from a low number of introduced bean-nodulating rhizobia that either are poor competitors for nodulation of bean or that failed to survive in the Ethiopian environment. Furthermore, it may be concluded from the genetic data presented here that the evidence for separating R. leguminosarum and R. etli into two separate species is inconclusive.