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.     

Genotypic and symbiotic diversity of native rhizobia nodulating red pea (Lathyrus cicera L.) in Tunisia

Nodulation and genetic diversity of native rhizobia nodulating Lathyrus cicera plants grown in 24 cultivated and marginal soils collected from northern and central Tunisia were studied. L. cicera plants were nodulated and showed the presence of native rhizobia in 21 soils. A total of 196 bacterial strains were selected and three different ribotypes were revealed after PCR-RFLP analysis. The sequence analysis of the rrs and two housekeeping genes (recA and thrC) from 36 representative isolates identified Rhizobium laguerreae as the dominant (53%) rhizobia nodulating L. cicera. To the best of our knowledge, this is the first time that this species has been reported among wild populations of the rhizobia-nodulating Lathyrus genus. Twenty-five percent of the isolates were identified as R. leguminosarum and isolates LS11.5, LS11.7 and LS8.8 clustered with Ensifer meliloti. Interestingly, five isolates (LS20.3, LS18.3, LS19.10, LS1.2 and LS21.20) were segregated from R. laguerreae and clustered as a separate clade. These isolates possibly belong to new species. According to nodC and nodA phylogeny, strains of R. laguerreae and R. leguminosarum harbored the symbiotic genes of symbiovar viciae and clustered in three different clades showing heterogeneity within the symbiovar. Strains of E. meliloti harbored symbiotic genes of Clade V and induced inefficient nodules.     

Genetic Structure of Rhizobium leguminosarum biovar trifolii and viciae Populations Found in Two Oregon Soils under Different Plant Communities

An investigation was carried out to determine the genetic structure in soil populations of Rhizobium leguminosarum bv. trifolii and viciae at each of two Oregon sites (A and C) that were 1 km apart. Although the soils were similar, the plant communities were quite different because grazing by domestic animals had been allowed (site A) or prevented (site C). Analysis of allelic variation at 13 enzyme-encoding loci by multilocus enzyme electrophoresis delineated 202 chromosomal types (ETs) among a total of 456 isolates representing two populations of R. leguminosarum bv. trifolii (AT and CT) and two populations of R. leguminosarum bv. viciae (AV and CV). Regardless of their site of origin or biovar affiliation, isolates of the same ET were confirmed to be more closely related to each other than to isolates of other ETs by repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus sequences and the PCR technique. Despite the wide range in densities of the Rhizobium populations (<10² to >10⁵/g of soil), their overall genetic diversities were similar (mean genetic diversity, 0.45 to 0.51), indicating that low-density populations of soil-borne bacterial species are not necessarily of little genetic diversity. Linkage disequilibrium analysis revealed significant multilocus structure (nonrandom associations of alleles) within each of the four populations. From a combination of cluster and linkage disequilibrium analyses, a total of eight distinct groups of ETs were defined in the four populations. Two groups (I and III) contributed significant numbers of ETs and isolates to each population. The two populations of R. leguminosarum bv. viciae (AV and CV) exhibited similar genetic structures despite existing at different densities, in different plant communities, and in the presence (CV) or absence (AV) of their local Vicia hosts. In contrast, total linkage disequilibrium was partitioned differently in two biovar populations occupying the same soil (AV and AT), with disequilibrium in the latter being due entirely to the presence of group V.     

A hierarchical analysis of population genetic structure in Rhizobium leguminosarum bv. trifolii

Little is known about the population processes that shape the genetic diversity in natural populations of rhizobia. A sample of 912 Rhizobium leguminosarum biovar trifolii isolates were collected from naturalized red clover populations ( Trifolium pratense ) and analyzed for 15 allozyme loci to determine the levels and distribution of genetic diversity. Hierarchical analyses compared different sampling levels, geographical separation, and temporal separation. Total genetic diversity across all isolates was H = 0.426, with 57.6% of the total diversity found among isolates obtained from individual red clover plants. Relatively low genetic differentiation among populations and high differentiation among plants within populations was observed; this suggests that gene flow and founder effect act differently at geographical and local scales. Significant differences were observed in (i) allele frequencies among populations and among plants within populations, and (ii) the frequency distribution of the most widespread and the most abundant strains. When multilocus linkage disequilibrium was calculated, significant levels of disequilibrium were observed in the total sample and in three of the eight populations.     

Construction of an Acid-Tolerant Rhizobium leguminosarum Biovar Trifolii Strain with Enhanced Capacity for Nitrogen Fixation

Strain ANU1173 is an acid-tolerant Rhizobium leguminosarum biovar trifolii strain that is able to nodulate subterranean clover plants growing in agar culture at pH 4.4 At pH 6.5, its symbiotic effectiveness in association with Trifolium subterraneum cv. Mt. Barker was 80% relative to that of strain ANU794, a Sm^r derivative of the commercial inoculant R. leguminosarum bv. trifolii TA1. Strain ANU1173 contained four indigenous megaplasmids, the smallest of these being the symbiotic (Sym) plasmid. The critical pH requirement for growth of strain ANU1173 in laboratory media was shown not to be associated with this plasmid. When the Sym plasmid of strain ANU1173(pSym-1173) was mobilized into a Nod^- strain of R. leguminosarum bv. viciae, the plasmid conferred to the transconjugant a level of symbiotic effectiveness in association with T. subterraneum that was similar to that observed with ANU1173. The symbiotic effectiveness of strain ANU1173 was improved by first curing pSym-1173 (generating strain ANU1184) and replacing it with another R. leguminosarum bv. trifolii Sym plasmid, pBR1AN. Subterranean clover plants inoculated with strain ANU1184 (pBR1AN) exhibited a 35 or 53% increase in acetylene reduction activity and a 20 or 17% increase in dry weight when grown at pH 6.5 and pH 4.4, respectively, compared with plants inoculated with strain ANU1173 and grown under the same pH conditions. It was further shown that pBR1AN was stably maintained in strain ANU1184 under free-living and symbiotic conditions. These results indicate that it is possible to construct an acid-tolerant strain of R. leguminosarum bv. trifolii with an enhanced capacity for nitrogen fixation.     

Genetic Diversity among Rhizobium leguminosarum bv. Trifolii Strains Revealed by Allozyme and Restriction Fragment Length Polymorphism Analyses

Allozyme electrophoresis and restriction fragment length polymorphism (RFLP) analyses were used to examine the genetic diversity of a collection of 18 Rhizobium leguminosarum bv. trifolii, 1 R. leguminosarum bv. viciae, and 2 R. meliloti strains. Allozyme analysis at 28 loci revealed 16 electrophoretic types. The mean genetic distance between electrophoretic types of R. leguminosarum and R. meliloti was 0.83. Within R. leguminosarum, the single strain of bv. viciae differed at an average of 0.65 from strains of bv. trifolii, while electrophoretic types of bv. trifolii differed at a range of 0.23 to 0.62. Analysis of RFLPs around two chromosomal DNA probes also delineated 16 unique RFLP patterns and yielded genetic diversity similar to that revealed by the allozyme data. Analysis of RFLPs around three Sym (symbiotic) plasmid-derived probes demonstrated that the Sym plasmids reflect genetic divergence similar to that of their bacterial hosts. The large genetic distances between many strains precluded reliable estimates of their genetic relationships.     

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.     

Microsymbionts of Phaseolus vulgaris in acid and alkaline soils of Mexico

In order to investigate bean-nodulating rhizobia in different types of soil, 41 nodule isolates from acid and alkaline soils in Mexico were characterized. Based upon the phylogenetic studies of 16S rRNA, atpD, glnII, recA, rpoB, gyrB, nifH and nodC genes, the isolates originating from acid soils were identified as the phaseoli symbiovar of the Rhizobium leguminosarum-like group and Rhizobium grahamii, whereas the isolates from alkaline soils were defined as Ensifer americanum sv. mediterranense and Rhizobium radiobacter. The isolates of “R. leguminosarum” and E. americanum harbored nodC and nifH genes, but the symbiotic genes were not detected in the four isolates of the other two species. It was the first time that “R. leguminosarum” and E. americanum have been reported as bean-nodulating bacteria in Mexico. The high similarity of symbiotic genes in the Rhizobium and Ensifer populations showed that these genes had the same origin and have diversified recently in different rhizobial species. Phenotypic characterization revealed that the “R. leguminosarum” population was more adapted to the acid and low salinity conditions, while the E. americanum population preferred alkaline conditions. The findings of this study have improved the knowledge of the diversity, geographic distribution and evolution of bean-nodulating rhizobia in Mexico.     

Genomic insight into the taxonomy of Rhizobium genospecies that nodulate Phaseolus vulgaris

Due to the wide cultivation of bean (Phaseolus vulgaris L.), rhizobia associated with this plant have been isolated from many different geographical regions. In order to investigate the species diversity of bean rhizobia, comparative genome sequence analysis was performed in the present study for 69 Rhizobium strains mainly isolated from root nodules of bean and clover (Trifolium spp.). Based on genome average nucleotide identity, digital DNA:DNA hybridization, and phylogenetic analysis of 1,458 single-copy core genes, these strains were classified into 28 clusters, consistent with their species definition based on multilocus sequence analysis (MLSA) of atpD, glnII, and recA. The bean rhizobia were found in 16 defined species and nine putative novel species; in addition, 35 strains previously described as Rhizobium etli, Rhizobium phaseoli, Rhizobium vallis, Rhizobium gallicum, Rhizobium leguminosarum and Rhizobium spp. should be renamed. The phylogenetic patterns of symbiotic genes nodC and nifH were highly host-specific and inconsistent with the genomic phylogeny. Multiple symbiovars (sv.) within the Rhizobium species were found as a common feature: sv. phaseoli, sv. trifolii and sv. viciae in Rhizobium anhuiense; sv. phaseoli and sv. mimosae in Rhizobium sophoriradicis/R. etli/Rhizobium sp. III; sv. phaseoli and sv. trifolii in Rhizobium hidalgonense/Rhizobium acidisoli; sv. phaseoli and sv. viciae in R. leguminosarum/Rhizobium sp. IX; sv. trifolii and sv. viciae in Rhizobium laguerreae. Thus, genomic comparison revealed great species diversity in bean rhizobia, corrected the species definition of some previously misnamed strains, and demonstrated the MLSA a valuable and simple method for defining Rhizobium species.     

Genetic structure of the introduced and local populations of <Emphasis Type="Italic">Rhizobioum leguminosarum</Emphasis> in plant-soil systems

Comparative study of Rhizobium leguminosarum populations formed under the conditions of the Srednii Island (White Sea) demonstrated the introduced clover rhizobia (R. l. bv. trifolii) to be more variable than the aboriginal vetch/vetchling rhizobia (R. l. bv. viceae) in the chromosomal IGS locus, while being less variable in the plasmid-located symbiotic genes nodD and nifH. The analysis of these genes revealed the most pronounced differences between the clover and vetch/vetchling rhizobia populations. These differences, together with the results of ERIC-fingerprinting, indicated that the evolution of the clover rhizobia was mainly linked with the adaptation to local soil environment, and the evolution of the vetch/vetchling rhizobia, to the adaptation to various species of the host plants. High panmixia of R. leguminosarum population suggests its evolution to be based on the combinatory variability associated with the transfer of Sym-plasmids between R. l. bv. trifolii and R. l. bv. viceae, as well as with genomic rearrangements in the resulting recombinants.     

Large genotypic variation but small variation in N2 fixation among rhizobia nodulating red clover in soils of northern Scandinavia

AIMS: To analyse the symbiotic variations within indigenous populations of rhizobia nodulating red clover (Trifolium pratense L.) in soils of northern Norway and Sweden at different times of the growing season. METHODS AND RESULTS: A total of 431 nodule isolates sampled under field conditions in summer and autumn, were characterized genetically by targeting both chromosomal and symbiotic genes. The Enterobacterial Repetitive Intergenic Consensus polymerase chain reaction (PCR) fingerprinting of chromosomal DNA revealed considerable variation within the isolated populations that was more influenced by geographical origin than sampling time. Analysis of PCR amplified nodEF gene on the symbiotic plasmid by restriction fragment length polymorphism revealed a high proportion of nod types common to the two studied sites. The symbiotic efficiency of the isolates, representing both dominating and rare nodEF genotypes, showed high N(2) fixation rates in symbiosis with the host plant in a greenhouse experiment using the (15)N isotope dilution method. CONCLUSIONS: Effective N(2)-fixing strains of Rhizobium leguminosarum bv. trifolii nodulating red clover are common and genetically diverse in these northern Scandinavia soils. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information on the variability, stability and dynamics of resident populations of rhizobia nodulating red clover in Scandinavian soils which has practical implications for applying biological nitrogen fixation in subarctic plant production.     

Population structure of root nodulating Rhizobium leguminosarum in Vicia cracca populations at local to regional geographic scales

The genetic diversity and population structure of about 350 Rhizobium leguminosarum biovar viciae isolates from Vicia cracca were analysed. A hierarchical sampling design was used covering three regions, one region in Belgium and two in France, in which multiple local V. cracca populations were sampled. Rhizobium isolates were genotyped using RAPD and by sequencing two chromosomal housekeeping genes (glnII and recA) and one plasmid-borne gene (nodC). Twenty-six nodC types and sixty-seven chromosomal types were identified, many of which appeared to be regional or local endemics. We found strong genetic differentiation both among V. cracca populations that are separated by only a few kilometres, and among regions that are 50 to 350 km apart. Despite significant plasmid exchange, chromosomal and nod types were similarly structured among host populations and regions. We found two lineages of which one prevailed in the Belgian region while the other dominated the French regions. Although a significant correlation between genetic differentiation and geographic distance was found, it is deemed more likely that the observed biogeographic patterns are rather due to coevolutionary interactions and environmental pressures. Furthermore, the impact of recombination on the chromosomal differentiation was found to be considerable.     

Diversity and genetic structure of a natural population of Rhizobium leguminosarum bv. trifolii isolated from Trifolium subterraneum L.

A collection of 121 isolates of Rhizobium leguminosarum biovar (bv.) trifolii was obtained from root nodules of Trifolium subterraneum L. (subclover) plants growing in an established pasture. The collection consisted of a single isolate from each of 18 plants sampled from seven microplots. The following year, a further 28 and 27 isolates were collected from the first and seventh sampling points, respectively. Analysis of restriction fragment length polymorphisms (RFLPs) of both chromosomal and Sym (symbiotic) plasmid DNA and multilocus enzyme electrophoresis (MLEE) were used to assess the diversity, genetic relationships and structure of this population. Symbiotic effectiveness tests were used to examine the symbiotic phenotype of each isolate collected in the first year. Analysis of RFLPs of the first year isolates revealed 13 chromosomal types and 25 Sym plasmid types. Similar Sym plasmid types were grouped into 14 families containing 1–6 members. No new chromosomal types and six new Sym plasmid types were detected in the second year. The symbiotic effectiveness of the first year isolates of the same Sym plasmid type was similar. Significant differences in symbiotic effectiveness were detected between different Sym plasmid types in the same plasmid family. Representative isolates of each chromosomal type Sym plasmid type identified in the first year were analysed using multilocus enzyme electrophoresis. Mean genetic diversity per locus was high (0.559). Enzyme electrophoresis revealed 17 electrophoretic types (ETs). Ouster analysis of the enzyme data revealed large genetic diversity amongst the ETs. Strong linkage disequilibrium was observed for the population as a whole, i.e. clonal population structure, but significantly less disequilibrium was observed among a cluster of ETs suggesting that recombination occurred between ETs within the cluster. Our results revealed that a population of naturally occurring isolates of Rhizobium leguminosarum bv. trifolii can be genetically diverse and support the possibility that recombination plays a role in generating new genotypes.     

Arctic and subarctic soil populations of Rhizobium leguminosarum biovar trifolii nodulating three different clover species: Characterisation by diversity at chromosomal and symbiosis loci

Indigenous soil populations of Rhizobium leguminosarum biovar trifolii from Arctic and subarctic regions have been characterised with emphasis on chromosomal and symbiotic genes. Three clover species were used to trap rhizobia from soils along a latitudinal gradient from 78°N to 60°N in Norway. For the first time R. l. bv. trifolii was isolated from Svalbard at 78°N. Under the extreme conditions in the Arctic, rhizobia have survived as saprophytes and in symbiosis with clover legumes. The chromosomal diversity of the soil populations was mapped by rep-PCR. Separation of chromosomal types were strongly influenced by geographic origin. Symbiotic genes, the nodEF and nifDK IGS gene regions, were investigated by PCR-RFLP. The nifDK IGS were more conserved than the nodEF genes. Sym plasmids were widely distributed in different chromosomal types and across the latitudinal gradient.     

Genetic Characterization of a Novel Rhizobial Plasmid Conjugation System in Rhizobium leguminosarum bv. viciae Strain VF39SM

Rhizobium leguminosarum strain VF39SM contains two plasmids that have previously been shown to be self-transmissible by conjugation. One of these plasmids, pRleVF39b, is shown in this study to carry a set of plasmid transfer genes that differs significantly from conjugation systems previously studied in the rhizobia but is similar to an uncharacterized set of genes found in R. leguminosarum bv. trifolii strain WSM2304. The entire sequence of the transfer region on pRleVF39b was determined as part of a genome sequencing project, and the roles of the various genes were examined by mutagenesis. The transfer region contains a complete set of mating pair formation (Mpf) genes, a traG gene, and a relaxase gene, traA, all of which appear to be necessary for plasmid transfer. Experimental evidence suggested the presence of two putative origins of transfer within the gene cluster. A regulatory gene, trbR, was identified in the region between traA and traG and was mutated. TrbR was shown to function as a repressor of both trb gene expression and plasmid transfer.     

Genotypic and symbiotic diversity studies of rhizobia nodulating Acacia saligna in Tunisia reveal two novel symbiovars within the Rhizobium leguminosarum complex and Bradyrhizobium

Acacia saligna is an invasive alien species that has the ability to establish symbiotic relationships with rhizobia. In the present study, genotypic and symbiotic diversity of native rhizobia associated with A. saligna in Tunisia were studied. A total of 100 bacterial strains were selected and three different ribotypes were identified based on rrs PCR-RFLP analysis. Sequence analyses of rrs and four housekeeping genes (recA, atpD, gyrB and glnII) assigned 30 isolates to four putative new lineages and a single strain to Sinorhizobium meliloti. Thirteen slow-growing isolates representing the most dominant IGS (intergenic spacer) profile clustered distinctly from known rhizobia species within Bradyrhizobium with the closest related species being Bradyrhizobium shewense and Bradyrhizobium niftali, which had 95.17% and 95.1% sequence identity, respectively. Two slow-growing isolates, 1AS28L and 5AS6L, had B. frederekii as their closest species with a sequence identity of 95.2%, an indication that these strains could constitute a new lineage. Strains 1AS14I, 1AS12I and 6AS6 clustered distinctly from known rhizobia species but within the Rhizobium leguminosarum complex (Rlc) with the most closely related species being Rhizobium indicum with 96.3% sequence identity. Similarly, the remaining 11 strains showed 96.9 % and 97.2% similarity values with R. changzhiense and R. indicum, respectively. Based on nodC and nodA phylogenies and cross inoculation tests, these 14 strains of Rlc species clearly diverged from strains of Sinorhizobium and Rlc symbiovars, and formed a new symbiovar for which the name sv. “salignae” is proposed. Bacterial strains isolated in this study that were taxonomically assigned to Bradyrhizobium harbored different symbiotic genes and the data suggested a new symbiovar, for which sv. “cyanophyllae” is proposed. Isolates formed effective nodules on A. saligna.     

Core and symbiotic genes reveal nine Mesorhizobium genospecies and three symbiotic lineages among the rhizobia nodulating Cicer canariense in its natural habitat (La Palma,Canary Islands)

Cicer canariense is a threatened perennial wild chickpea endemic to the Canary Islands. In this study, rhizobia that nodulate this species in its natural habitats on La Palma (Canary Islands) were characterised. The genetic diversity and phylogeny were estimated by RAPD profiles, 16S-RFLP analysis and sequencing of the rrs, recA, glnII and nodC genes. 16S-RFLP grouped the isolates within the Mesorhizobium genus and distinguished nine different ribotypes. Four branches included minority ribotypes (3–5 isolates), whereas another five contained the predominant ribotypes that clustered with reference strains of M. tianshanense/M. gobiense/M. metallidurans, M. caraganae, M. opportunistum, M. ciceri and M. tamadayense. The sequences confirmed the RFLP groupings but resolved additional internal divergence within the M. caraganae group and outlined several potential novel species. The RAPD profiles showed a high diversity at the infraspecific level, except in the M. ciceri group. The nodC phylogeny resolved three symbiotic lineages. A small group of isolates had sequences identical to those of symbiovar ciceri and were only detected in M. ciceri isolates. Another group of sequences represented a novel symbiotic lineage that was associated with two particular chromosomal backgrounds. However, nodC sequences closely related to symbiovar loti predominated in most isolates, and they were detected in several chromosomal backgrounds corresponding to up to nine Mesorhizobium lineages. The results indicated that C. canariense is a promiscuous legume that can be nodulated by several rhizobial species and symbiotypes, which means it will be important to determine the combination of core and symbiotic genes that produce the most effective symbiosis.     

Rhizobium pisi sv. trifolii K3.22 harboring nod genes of the Rhizobium leguminosarum sv. trifolii cluster

The taxonomic status of the Rhizobium sp. K3.22 clover nodule isolate was studied by multilocus sequence analysis (MLSA) of 16S rRNA and six housekeeping chromosomal genes, as well as by a subsequent phylogenic analysis. The results revealed full congruence with the Rhizobium pisi DSM 30132^T core genes, thus supporting the same taxonomic position for both strains. However, the K3.22 plasmid symbiosis nod genes demonstrated high sequence similarity to Rhizobium leguminosarum sv. trifolii, whereas the R. pisi DSM 30132^Tnod genes were most similar to R. leguminosarum sv. viciae. The strains differed in the host range nodulation specificity, since strain K3.22 effectively nodulated red and white clover but not vetch, in contrast to R. pisi DSM 30132^T, which effectively nodulated vetch but was not able to nodulate clover. Both strains had the ability to form nodules on pea and bean but they differed in bean cultivar specificity. The R. pisi K3.22 and DSM 30132^T strains might provide evidence for the transfer of R. leguminosarum sv. trifolii and sv. viciae symbiotic plasmids occurring in natural soil populations.     

Highly Diverse Endophytic and Soil Fusarium oxysporum Populations Associated with Field-Grown Tomato Plants

The diversity and genetic differentiation of populations of Fusarium oxysporum associated with tomato fields, both endophytes obtained from tomato plants and isolates obtained from soil surrounding the sampled plants, were investigated. A total of 609 isolates of F. oxysporum were obtained, 295 isolates from a total of 32 asymptomatic tomato plants in two fields and 314 isolates from eight soil cores sampled from the area surrounding the plants. Included in this total were 112 isolates from the stems of all 32 plants, a niche that has not been previously included in F. oxysporum population genetics studies. Isolates were characterized using the DNA sequence of the translation elongation factor 1α gene. A diverse population of 26 sequence types was found, although two sequence types represented nearly two-thirds of the isolates studied. The sequence types were placed in different phylogenetic clades within F. oxysporum, and endophytic isolates were not monophyletic. Multiple sequence types were found in all plants, with an average of 4.2 per plant. The population compositions differed between the two fields but not between soil samples within each field. A certain degree of differentiation was observed between populations associated with different tomato cultivars, suggesting that the host genotype may affect the composition of plant-associated F. oxysporum populations. No clear patterns of genetic differentiation were observed between endophyte populations and soil populations, suggesting a lack of specialization of endophytic isolates.     

Improvement of biological nitrogen fixation in Egyptian winter legumes through better management of Rhizobium

The diversity of rhizobia nodulating common bean ( Phaseolus vulgaris), berseem clover (Trifolium alexanderinum) and lentil (Lens culinaris) was assessed using several characterization techniques, including nitrogen fixation efficiency, intrinsic antibiotic-resistance patterns (IAR), plasmid profiles, serological markers and rep-PCR fingerprinting. Wide diversity among indigenous rhizobial populations of the isolates from lentil, bean and clover was found. Strikingly, a large percentage of the indigenous rhizobial population was extremely poor at fixing nitrogen. This emphasizes the need to increase the balance of highly efficient strains within the rhizobial population. Use of high-quality inocula strains that survive and compete with other less-desired and less-efficient N2-fixing rhizobia represents the best approach to increase biological nitrogen fixation of the target legume. In field-grown lentils, the inoculant strains were not able to outcompete the indigenous rhizobia and the native lentil rhizobia occupied 76–88% of the total nodules formed on inoculated plants. Nitrogen fixation by lentils, estimated using the ¹⁵N isotope dilution technique, ranged between 127 to 139 kg ha-1 in both inoculated and un-inoculated plants. With berseem clover, the inoculant strains were highly competitive against indigenous rhizobia and occupied 52–79% of all nodules. Inoculation with selected inocula improved N2 fixation by clover from 162 to 205 kg ha-1 in the three cuts as compared with 118 kg ha-1 in the un-inoculated treatment. The results also indicated the potential for improvement of N2 fixation by beans through the application of efficient N2-fixing rhizobia.