Diversity in symbiotic specificity of cowpea rhizobia indigenous to Zimbabwean soils

Tropical cowpea rhizobia are often presumed to be generally promiscuous but poor N fixers. This study was conducted to evaluate symbiotic interactions of 59 indigenous rhizobia isolates (49 of them from cowpea (Vigna unguiculata)), with up to 13 other (mostly tropical) legume species. Host ranges averaged 2.4 and 2.3 legume species each for fast- and slow-growing isolates respectively compared to 4.3 for slow-growing reference cowpea strains. An average of 22% and 19% of fast- and slow-growing cowpea isolates respectively were effective on each of 12 legume species tested. We conclude that the indigenous cowpea rhizobia studied have relatively narrow host ranges. The ready nodulation of different legumes in tropical soils appears due to the diversity of indigenous symbiotic genotypes, each consisting of subgroups compatible with a limited number of legume species.     

Nodulation of legume trees from South East Brazil

Summary An extended survey of nodulation of legume trees from South-East Brazilian forests was conducted. Six new species from the Caesalpinioideae, 23 from the Mimosoideae and 27 from the Papilionoideae are reported to have nodules. Nitrogenase activity (acetylene reduction) was tested for all nodules and rhizobia were isolated from the most active.     

CHROMOSOME NUMBERS IN THE LEGUMINOSAE II: AFRICAN SPECIES,INCLUDING PHYLETIC INTERPRETATIONS

Turner , B. L., and O. S. Fearing . (U. Texas, Austin.) Chromosome numbers in the Leguminosae. II. African species, including phyletic interpretations. Amer. Jour. Bot. 46(1) : 49-57. Illus. 1959.—Chromosome numbers for 30 African legume species have been reported. These include first reports for 28 taxa, including 12 genera (Bolusanthus, Calpurnia, Melolobium, Lessertia, Sulherlandia, Colophospermum, Guibourtia, Burkea, Julbernardia, Schotia, Piliostigma and Swartzia). The counts are discussed with respect to those previously reported for related groups, and this chromosomal information was used to construct hypothetical phyletic lines at the tribal level within the subfamilies Papilionoideae and Caesalpinioideae. A phyletic scheme for the Leguminosae (excluding the Mimosoideae) based on this evidence from chromosome studies is presented. Notable departures from previously suggested phyletic treatments include: (1) Suggestion for inclusion of genera of the Galegeae and Hedysareae with base numbers of x = 10 and 11 with the Phaseoleae and Dalbergieae. (2) Derivation of the Papilionoideae through caesalpinoid prototypes, possibly from Swartzia-like ancestors. (3) Recognition of several very old chromosomal lines stemming from the subfamily Caesalpinioideae, and the suggestion that parts of the tribes Sclerolobieae, Cynometreae, Swartzieae and Sophoreae are, perhaps, more closely related to each other and to the Papilionoideae than they are to the remaining caesalpinoid tribal lines.     

Studies on fast and slow growing Rhizobium spp. nodulating Cqjanus cajan and Cicer arietinum

Fast and slow growing Rhizobium spp. isolated from Cajanus cajan and Cicer arietinum were compared in terms of colony characteristics, utilisation of carbon sources, acid production, symbiotic effectiveness and nodulating competitiveness. Fast growing isolates from C. cajan and C. arietinum formed 3–6 mm diameter colonies on yeast-extract mannitol agar after 4 days and were unlike the slow growers which produced colonies of c. 1 mm diameter after 7–10 days at 28 °C. The fast growing Rhizobium spp. from C. cajan utilised a wider range of carbon sources than the slow growing isolates from this host. Fast and slow growing strains from C. arietinum were able to utilise most of the carbon sources tested suggesting that the slow growers possessed glycolytic pathways similar to those in other fast growing species of Rhizobium. In culture, slow growing isolates from C. cajan produced a near-neutral to alkaline reaction (pH 66·7-5) whereas the fast growers from this host and both fast and slow growing isolates from C. arietinum produced an acidic reaction (pH 4·4–5·6). These data are discussed in the context of Norris' (1965) evolutionary concept of the Leguminosae. Under glassshouse conditions, fast and slow growing strains isolated from C. cajan and C. arietinum were equally effective on their respective hosts. In competition with slow growing rhizobia, half of the fast growers formed more than 70% of the nodules on C. cajan grown in sand. In all but one instance similar results were obtained when plants were grown in soil. With C. arietinum grown in sand or soil, all fast growing isolates from this host formed more than 85% of the nodules in competition with slow growing strains.     

Novel associations between rhizobial populations and legume species within the genera Lathyrus and O xytropis grown in the temperate region of China

Fifty rhizobial isolates of Lathyrus and Oxytropis collected from northern regions of China were studied in their genotypic characterization based upon analyses of ARDRA, 16S-23S IGS PCR-RFLP, TP-RAPD, MLEE, sequences of 16S rDNA gene and housekeeping genes of atpD, recA and glnII. The results demonstrated that most of the Lathyrus rhizobia belonged to Rhizobium and most of the Oxytropis rhizobia belonged to Sinorhizobium. A novel group of Rhizobium sp. I and S. meliloti were identified as the main microsymbionts respectively associated with Lathyrus and Oxytropis species in the collection area, which were new associations between rhizobia and the mentioned hosts. This study also provides new evidence for biogeography of rhizobia. Supported by the National Program for Basic S&T Platform Construction (Grant No. 2005DKA21201-1), the National Natural Science Foundation of China (Grant No. 30670001), and the National Basic Research Program of China (Grant No. 2006CB100206)     

我国豆科树种结瘤调查(续)

据国内资料报道,我国豆科树种约94属763种,已调查过结瘤状况的有含羞草亚科的123种(包括近几年来从国外引种的54种)、蝶形花亚科的211种和苏木亚科的70种,其中可以结瘤的有含羞草亚科的116种、蝶形花亚科的207种和苏木亚科的18种。含羞草亚科中的2种、蝶形花亚科中的3种均有结瘤和不结瘤的报道。     

我国豆科树种结瘤调查

据国内资料报道,我国豆科树种约94属763种,已调查过结瘤状况的有含羞草亚科的123种(包括近几年来从国外引种的54种)、蝶形花亚科的211种和苏木亚科的70种,其中可以结瘤的有含羞草亚科的116种、蝶形花亚科的207种和苏木亚科的18种。含羞草亚科中的2种、蝶形花亚科中的3种均有结瘤和不结瘤的报道。     

Determination of Host Specificity of Cowpea Miscellany Rhizobium spp. by nodABC-lacZ Fusion

Cowpea miscellany group of rhizobia are generally broad host range. Transconjugants of these cowpea rhizobial isolates having nodABC-lacZ fusion were monitored for flavonoid/root exudate-induced activation of the nod genes in terms of β-galactosidase activity, thus determining the potential host range of the rhizobial isolates. Received: 17 November 1997 / Accepted: 23 December 1997     

The large mimosoid genus Inga Mill. (tribe Ingeae,Caesalpinioideae) is nodulated by diverse Bradyrhizobium strains in its main centers of diversity in Brazil

Inga (Caesalpinioideae) is the type genus of the Ingeae tribe in the mimosoid clade. It comprises about 300 species, all trees or treelets, and has an exclusively neotropical distribution, with Brazil as its main center of diversity. In this study, we analyzed the diversity of 40 strains of rhizobia isolated from root nodules collected from ten species of Inga belonging to different types of vegetation in Brazil. Sequences of their housekeeping genes (dnaK, recA, rpoB, gyrB and glnII), 16S rRNA genes, internal transcribed spacer (ITS) regions, as well as their symbiosis-essential genes (nodC and nifH) were used to characterize them genetically. The ability of the rhizobia to form nodules on Inga spp., and on the promiscuous legume siratro (Macroptilium atropurpureum) was also evaluated. A multilocus sequence analysis (MLSA) combined with an analysis of the ITS region showed that the isolates were distributed into four main groups (A-D) within the large genus Bradyrhizobium. Analysis of the nodC and nifH genes showed that the isolates formed a separate branch from all described species of Bradyrhizobium, except for B. ingae. Most of the tested isolates formed nodules on siratro and all isolates tested nodulated Inga spp. Our results suggest a unique co-evolutionary history of Bradyrhizobium and Inga and demonstrate the existence of potential new species of microsymbionts nodulating this important and representative genus of leguminous tree from the Caesalpinioideae mimosoid clade.     

Diverse genotypes of Bradyrhizobium nodulate herbaceous Chamaecrista (Moench) (Fabaceae,Caesalpinioideae) species in Brazil

The genus Chamaecrista comprises more than 330 species which are mainly distributed across tropical America, especially in Brazil (256 spp.), the main center of radiation. In this study, nodulation of herbaceous Chamaecrista species that are commonly found growing in different vegetation types in the north eastern Brazilian state of Bahia was assessed together with the diversity of rhizobia isolated from their root nodules. Genetic characterization of the isolates was performed using molecular markers to examine the phylogeny of their “core” (16S rRNA, ITS, recA, glnII, dnaK and gyrB) and symbiosis-related (nifH, nodC) genomes. Nodule morphology, anatomy and ultrastructure were also examined, as was the capacity of the isolates to form nodules on Chamaecrista desvauxii and siratro (Macroptilium atropurpureum). Analysis of 16S rRNA gene sequences demonstrated that the isolates belonged to seven clusters within the genus Bradyrhizobium, and more detailed analyses using sequences of the ITS region and concatenated housekeeping genes grouped the Chamaecrista rhizobia by vegetation type and plant species. These analyses also suggested some potentially novel Bradyrhizobium species, which was corroborated by analyses of their nifH and nodC sequences, as these formed separated branches from all Bradyrhizobium type strains. All the 47 strains tested produced effective nodules on C. desvauxii but none on siratro. Chamaecrista nodules are herein described for the first time in detail: they are indeterminate and structurally similar to others described in the Caesalpinioideae, with infection threads in the invasion and nitrogen fixation zones, and with both infected and uninfected (interstitial) cells in the nitrogen fixation zone.     

Invalidity of the Concept of Slow Growth and Alkali Production in Cowpea Rhizobia

A total of 103 rhizobial strains representing the cowpea miscellany and Rhizobium japonicum were studied with regard to growth rate, glucose metabolic pathways, and pH change in culture medium. Doubling times ranged from 1.4 ± 0.04 to 44.1 ± 5.2 h; although two populations of “fast-growing” and “slow-growing” rhizobia were noted, they overlapped and were not distinctly separated. Twenty-four strains which had doubling times of less than 8 h all showed NADP-linked 6-phosphogluconate dehydrogenase (6-PGD) activity, whereas only one slow-growing strain (doubling time, 10.8 ± 0.9 h) of all those tested showed 6-PGD activity. Doubling times among fast growers could not be explained solely by the presence or absence of 6-PGD activity (r² = 0.14) because the tricarboxylic acid cycle and the Emden-Meyerhoff-Parnas pathway were operative in both 6-PGD-positive and 6-PGD-negative strains. Growth rate and pH change were unrelated to each other. Fast- or slow-growing strains were not associated with any particular legume species or group of species from which they were originally isolated, with the exception of Stylosanthes spp., all nine isolates of which were slow growers. We conclude that 6-PGD activity is a more distinctive characteristic among physiologically different groups of rhizobia than doubling times and that characterization of the cowpea rhizobia as slow-growing alkali producers is an invalid concept.     

黄土高原根瘤菌数值分类及DNA-DNA杂交

黄土高原位于我国内陆,气候比较干旱,生长的植被较少,水土流失严重,而有些豆科植物如锦鸡儿(Caragana sinica)、苦豆子(Sophora alopecuroides)、甘草(Glycyrrhiza uralensis)、苦马豆(Swainsoniasalsula)、洋槐(Robinia pseudoacacia)等却能很好生长.这些豆科植物的生长,在防风固沙、保持水土、绿化环境、作为饲用牧草等方面起着很重要的作用.但人们对于黄土高原野生豆科植物根瘤菌的研究尚很少.为此,作者在地处黄土高原的陕西、宁夏及甘肃的部分地区进行了广泛的根瘤菌资源调查.在此基础上,对分离的部分菌株进行了数值分类和DNA同源性分析.     

Insight into the evolutionary history of symbiotic genes of Robinia pseudoacacia rhizobia deriving from Poland and Japan

The phylogeny of symbiotic genes of Robinia pseudoacacia (black locust) rhizobia derived from Poland and Japan was studied by comparative sequence analysis of nodA, nodC, nodH, and nifH loci. In phylogenetic trees, black locust symbionts formed a branch of their own suggesting that the spread and maintenance of symbiotic genes within Robinia pseudoacacia rhizobia occurred through vertical transmission. There was 99–100% sequence similarity for nodA genes of Robinia pseudoacacia nodulators, 97–98% for nodC, and 97–100% for nodH and nifH loci. A considerable sequence conservation of sym genes shows that the symbiotic apparatus of Robinia pseudoacacia rhizobia might have evolved under strong host plant constraints. In the nodA and nodC gene phylograms, Robinia pseudoacacia rhizobia grouped with Phaseolus sp. symbionts, although they were not closely related to our isolates based on 16S rRNA genes, and with Mesorhizobium amorphae. nifH gene phylogeny of our isolates followed the evolutionary history of 16S rDNA and Robinia pseudoacacia rhizobia grouped with Mesorhizobium genus species. Nodulation assays revealed that Robinia pseudoacacia rhizobia effectively nodulated their native host and also Amorpha fruticosa and Amorpha californica resulting in a significant enhancement of plant growth. The black locust root nodules are shown to be of indeterminate type.     

Nodulation of certain legumes of the genus Crotalaria by the new species Methylobacterium

We studied a collection of 126 rhizobial isolates from eight species of Crotalaria (C. comosa, C. glaucoides, C. goreensis, C. hyssopifolia, C. lathyroides, C. perrottetii, C. podocarpa, and C. retusa) growing in Senegal. Nodulation and nitrogen-fixation tests on nine Crotalaria species revealed two specificity groups within the genus Crotalaria. Group I consists of plants solely nodulated by very specific fast-growing strains. Group II plants are nodulated by slow-growing strains similar to promiscuous Bradyrhizobium spp. strains already reported to nodulate many tropical legumes. SDS-PAGE studies showed that slow-growing strains grouped with Bradyrhizobium while fast-growing strains constituted a homogeneous group distinct from all known rhizobia. Amplified ribosomal DNA restriction analysis (ARDRA) of 10 representative strains of this group using four restriction enzymes showed a single pattern for each enzyme confirming the high homogeneity of group I. The 16S rDNA sequence analysis revealed that this specific group belonged to the genus Methylobacterium, thus constituting a new branch of nodulating bacteria.     

Genetic Diversity of Rhizobia Nodulating Arachis hypogaea L. in Central Argentinean Soils

The diversity of thirty-nine isolates from peanut plants growing at fourteen different sites in the Argentinean province of Córdoba was examined by rep-PCR, RFLP of PCR amplified 16S rRNA gene and complete sequencing of ribosomal genes. The genomic analysis of the peanut isolates indicated that each group encompasses heterogeneity among their members, having distinct rep fingerprints and 16S rRNA alleles. Complete sequencing of 16S rRNA demonstrated that native peanut rhizobia from Córdoba soils representative of the slow and fast growers are phylogenetically related to Bradyrhizobium japonicum and Bradyrhizobium sp. and Rhizobium giardinii and R. tropici species, respectively. The nodC gene sequence analysis showed phylogenetic similarity between fast grower peanut symbionts and Rhizobium tropici.     

Phylogenetic multilocus sequence analysis of indigenous slow-growing rhizobia nodulating cowpea (Vigna unguiculata L.) in Greece

Cowpea (Vigna unguiculata) is a promiscuous grain legume, capable of establishing efficient symbiosis with diverse symbiotic bacteria, mainly slow-growing rhizobial species belonging to the genus Bradyrhizobium. Although much research has been done on cowpea-nodulating bacteria in various countries around the world, little is known about the genetic and symbiotic diversity of indigenous cowpea rhizobia in European soils. In the present study, the genetic and symbiotic diversity of indigenous rhizobia isolated from field-grown cowpea nodules in three geographically different Greek regions were studied. Forty-five authenticated strains were subjected to a polyphasic approach. ERIC-PCR based fingerprinting analysis grouped the isolates into seven groups and representative strains of each group were further analyzed. The analysis of the rrs gene showed that the strains belong to different species of the genus Bradyrhizobium. The analysis of the 16S-23S IGS region showed that the strains from each geographic region were characterized by distinct IGS types which may represent novel phylogenetic lineages, closely related to the type species of Bradyrhizobium pachyrhizi, Bradyrhizobium ferriligni and Bradyrhizobium liaoningense. MLSA analysis of three housekeeping genes (recA, glnII, and gyrB) showed the close relatedness of our strains with B. pachyrhizi PAC48^T and B. liaoningense USDA 3622^T and confirmed that the B. liaoningense-related isolate VUEP21 may constitute a novel species within Bradyrhizobium. Moreover, symbiotic gene phylogenies, based on nodC and nifH genes, showed that the B. pachyrhizi-related isolates belonged to symbiovar vignae, whereas the B. liaoningense-related isolates may represent a novel symbiovar.     

Nodulation of legumes from the Thar desert of India and molecular characterization of their rhizobia

Aims

To survey the occurrence of nodulated legumes in the arid and semi-arid areas of Western Rajasthan and to characterize their associated symbiotic bacteria.

Methods

Herbaceous annual species were excavated whole, while tree species were studied as seedlings in the field or as trap plants in pot experiments. Nodules were examined by microscopy to confirm their effectiveness and to determine their internal structure. Bacteria isolated from the nodules were authenticated on their original hosts and were identified on the basis of 16S rRNA sequencing. Phylogenetic trees were inferred using the neighbour-joining method.

Results

We studied 35 of more than 50 species of native legume reported from these areas. Legumes are drought escaping (annual species), drought tolerant perennials or trees possessing deep root systems and other adaptations to arid conditions. Nodulation was recorded in all members of the Papilionoideae and Mimosoideae, but only one species of Caesalpinioideae. Internal structure of nodules varied within these groups, especially with respect to the presence or absence of uninfected cells in the infected region. Full 16S rRNA gene sequencing revealed that the nodules harboured a range of nodulating bacteria belonging to the genera Sinorhizobium, Rhizobium and Bradyrhizobium, within which they formed separate sub clades.

Conclusions

This study extends the range of legumes known to grow and nodulate in semi-arid regions, and provides information about their endosymbionts.     

Partial 16S rRNA gene sequence diversity and numerical taxonomy of slow growing pigeonpea (Cajanus cajan L Millsp) nodulating rhizobia

An investigation was carried out to determine the diversity of 30 isolates of slow growing pigeonpea nodulating rhizobia based on variations in partial sequences of the 16S rRNA gene and numerical analysis of 80 phenotypic traits. Phylogenetic analysis using molecular sequences of 23 isolates showed that ARPE1 separated from the other isolates at an average distance of >14% divergence level. The other isolates were all within 5% divergence from each other but separated into four main groups, with group 1 containing 16 of the 23 isolates. Comparisons to sequences of reference strains revealed that the group 1 isolates were phylogenetically closely related to the slow growing soybean nodulating rhizobia belonging to Bradyrhizobium elkanii, although only three of these isolates were able to nodulate soybean. Numerical analysis of phenotypic data of 19 isolates showed that 14 isolates clustered together in one branch of the phenogram, which included the group 1, group 2 and group 4 isolates from the phylogenetic analysis. The group 3 isolates were highly variable in the phenogram with similarity levels lower than 50% among these isolates.     

A phylogeny of the chloroplast gene rbcL in the Leguminosae: taxonomic correlations and insights into the evolution of nodulation

Phylogenetic analysis of the chloroplast-encoded rbcL gene in Leguminosae are consistent with previous hypotheses in suggesting that the family as a whole is monophyletic, but that only two of its three subfamilies are natural. The earliest dichotomies in the family appear to have involved tribes Cercideae or Cassieae (subtribe Dialiinae), followed by Detarieae/ Macrolobieae, all of which are members of subfamily Caesalpinioideae. The remainder of the family is divided into two clades: (1) Mimosoideae and the caesalpinioid tribes Caeasalpinieae and Cassieae (subtribes Ceratoniinae and Cassiinae); (2) Papilionoideae. Basal groups within Papilionoideae are, as expected, elements of the grade tribes Sophoreae and Swartzieae. Major clades within Papilionoideae include: (1) a Genistoid Alliance comprising Genisteae, Crotalarieae, Podalyrieae, Thermopsideae, Euchresteae, and also some Sophoreae; (2) a clade marked by the absence of one copy of the chloroplast inverted repeat, with which are associated Robinieae. Loteae, and some Sophoreae; (3) Phaseoleae, Desmodieae. Psoraleeae, and most Millettieae, a group also marked by presence of pseudoracemose inflorescences; and (4) a well-supported clade comprising Aeschynomeneae, Adesmieae, and some Dalbergieae. Nodulation is most parsimoniously optimized on the rbcL strict consensus tree as three parallel gains, occurring in Papilionoideae, the caesalpioioid ancestors of Mimosoideae, and in the genus Chamaecrista (Caesalpinieae: Cassieae).     

Characterization of rhizobia fromLeucaena

Seventy-six rhizobia were isolated from the nodules ofLeucaena plants of various genotypes growing in a wide range of soil types and climatic regions. The isolates were fast-growing and acid-producing. In establishing a serological grouping for the isolates, the intrinsic antibiotic resistance (IAR) patterns to low concentrations of eight antibiotics was helpful for selecting the strains for immunization purposes. Eight distinct somatic serogroups ofLeucaena rhizobia were identified by using strain-specific fluorescent antibodies. The results indicated that use of serological markers is a more specific technique than IAR pattern for strain identification. Strains from some different serogroups had the same IAR patterns. The immunofluorescence cross-reactions ofLeucaena rhizobia serogroups among themselves and with other species of fast- and slow-growing rhizobia were very low. Sero-grouping is ideal for use in further ecological studies in field inoculation trials.