Diversity and phenotypic analyses of salt- and heat-tolerant wild bean Phaseolus filiformis rhizobia native of a sand beach in Baja California and description of Ensifer aridi sp. nov.

In northern Mexico, aridity, salinity and high temperatures limit areas that can be cultivated. To investigate the nature of nitrogen-fixing symbionts of Phaseolus filiformis, an adapted wild bean species native to this region, their phylogenies were inferred by MLSA. Most rhizobia recovered belong to the proposed new species Ensifer aridi. Phylogenetic analyses of nodC and nifH show that Mexican isolates carry symbiotic genes acquired through horizontal gene transfer that are divergent from those previously characterized among bean symbionts. These strains are salt tolerant, able to grow in alkaline conditions, high temperatures, and capable of utilizing a wide range of carbohydrates and organic acids as carbon sources for growth. This study improves the knowledge on diversity, geographic distribution and evolution of bean-nodulating rhizobia in Mexico and further enlarges the spectrum of microsymbiont with which Phaseolus species can interact with, including cultivated bean varieties, notably under stressed environments. Here, the species Ensifer aridi sp. nov. is proposed as strain type of the Moroccan isolate LMR001T (= LMG 31426T; = HAMBI 3707T) recovered from desert sand dune.

Genetic diversity patterns and functional traits of Bradyrhizobium strains associated with Pterocarpus officinalis Jacq. in Caribbean islands and Amazonian forest (French Guiana).

Pterocarpus officinalis Jacq. is a legume tree native to the Caribbean islands and South America growing as a dominant species in swamp forests. To analyze (i) the genetic diversity and (ii) the symbiotic properties of its associated nitrogen-fixing soil bacteria, root nodules were collected from P. officinalis distributed in 16 forest sites of the Caribbean islands and French Guiana. The sequencing of the 16S-23S ribosomal RNA intergenic spacer region (ITS) showed that all bacteria belonged to the Bradyrhizobium genus. Bacteria isolated from insular zones showed very close sequence homologies with Bradyrhizobium genospecies V belonging to the Bradyrhizobium japonicum super-clade. By contrast, bacteria isolated from continental region displayed a larger genetic diversity and belonged to B. elkanii super-clade. Two strains from Puerto Rico and one from French Guiana were not related to any known sequence and could be defined as a new genospecies. Inoculation experiments did not show any host specificity of the Bradyrhizobium strains tested in terms of infectivity. However, homologous Bradyrhizobium sp. strain-P. officinalis provenance associations were more efficient in terms of nodule production, N acquisition, and growth than heterologous ones. The dominant status of P. officinalis in the islands may explain the lower bacterial diversity compared to that found in the continent where P. officinalis is associated with other leguminous tree species. The specificity in efficiency found between Bradyrhizobium strains and host tree provenances could be due to a coevolution process between both partners and needs to be taken in consideration in the framework of rehabilitation plantation programs.

Radiation of the Nod-independent Aeschynomene relies on multiple allopolyploid speciation events.

• The semi-aquatic legumes belonging to the genus Aeschynomene constitute a premium system for investigating the origin and evolution of unusual symbiotic features such as stem nodulation and the presence of a Nod-independent infection process. This latter apparently arose in a single Aeschynomene lineage. But how this unique Nod-independent group then radiated is not yet known. • We have investigated the role of polyploidy in Aeschynomene speciation via a case study of the pantropical A. indica and then extended the analysis to the other Nod-independent species. For this, we combined SSR genotyping, genome characterization through flow cytometry, chromosome counting, FISH and GISH experiments, molecular phylogenies using ITS and single nuclear gene sequences, and artificial hybridizations. • These analyses demonstrate the existence of an A. indica polyploid species complex comprising A. evenia (C. Wright) (2n = 2x = 20), A. indica L. s.s. (2n = 4x = 40) and a new hexaploid form (2n = 6x = 60). This latter contains the two genomes present in the tetraploid (A. evenia and A. scabra) and another unidentified genome. Two other species, A. pratensis and A. virginica, are also shown to be of allopolyploid origin. • This work reveals multiple hybridization/polyploidization events, thus highlighting a prominent role of allopolyploidy in the radiation of the Nod-independent Aeschynomene.

Evolution of symbiosis in the legume genus Aeschynomene.

Legumes in the genus Aeschynomene form nitrogen-fixing root nodules in association with Bradyrhizobium strains. Several aquatic and subaquatic species have the additional capacity to form stem nodules, and some of them can symbiotically interact with specific strains that do not produce the common Nod factors synthesized by all other rhizobia. The question of the emergence and evolution of these nodulation characters has been the subject of recent debate. We conducted a molecular phylogenetic analysis of 38 different Aeschynomene species. The phylogeny was reconstructed with both the chloroplast DNA trnL intron and the nuclear ribosomal DNA ITS/5.8S region. We also tested 28 Aeschynomene species for their capacity to form root and stem nodules by inoculating different rhizobial strains, including nodABC-containing strains (ORS285, USDA110) and a nodABC-lacking strain (ORS278). Maximum likelihood analyses resolved four distinct phylogenetic groups of Aeschynomene. We found that stem nodulation may have evolved several times in the genus, and that all Aeschynomene species using a Nod-independent symbiotic process clustered in the same clade. The phylogenetic approach suggested that Nod-independent nodulation has evolved once in this genus, and should be considered as a derived character, and this result is discussed with regard to previous experimental studies.

Nodulation of Crotalaria podocarpa DC. by Methylobacterium nodulans displays very unusual features.

Crotalaria are plants of the Fabaceae family whose nodulation characteristics have been little explored despite the recent discovery of their unexpected ability to be efficiently nodulated in symbiosis with bacteria of the genus Methylobacterium. It has been shown that methylotrophy plays a key role in this unusual symbiotic system, as it is expressed within the nodule and as non-methylotroph mutants had a depleting effect on plant growth response. Within the nodule, Methylobacterium is thus able to obtain carbon both from host plant photosynthesis and from methylotrophy. In this context, the aim of the present study was to show the histological and cytological impacts of both symbiotic and methylotrophic metabolism within Crotalaria podocarpa nodules. It was established that if Crotalaria nodules are multilobed, each lobe has the morphology of indeterminate nodules but with a different anatomy; that is, without root hair infection or infection threads. In the fixation zone, bacteroids display a spherical shape and there is no uninfected cell. Crotalaria nodulation by Methylobacterium displayed some very unusual characteristics such as starch storage within bacteroid-filled cells of the fixation zone and also the complete lysis of apical nodular tissues (where bacteria have a free-living shape and express methylotrophy). This lysis could possibly reflect the bacterial degradation of plant wall pectins through bacterial pectin methyl esterases, thus producing methanol as a substrate, allowing bacterial multiplication before release from the nodule.

Multi-host ectomycorrhizal fungi are predominant in a Guinean tropical rainforest and shared between canopy trees and seedlings.

The diversity of ectomycorrhizal (ECM) fungi on adult trees and seedlings of five species, Anthonotha fragrans, Anthonotha macrophylla, Cryptosepalum tetraphyllum, Paramacrolobium coeruleum and Uapaca esculenta, was determined in a tropical rain forest of Guinea. Ectomycorrhizae were sampled within a surface area of 1600 m(2), and fungal taxa were identified by sequencing the rDNA Internal Transcribed Spacer region. Thirty-nine ECM fungal taxa were determined, of which 19 multi-hosts, 9 single-hosts and 11 singletons. The multi-host fungi represented 92% (89% when including the singletons in the analysis) of the total abundance. Except for A. fragrans, the adults of the host species displayed significant differentiation for their fungal communities, but their seedlings harboured a similar fungal community. These findings suggest that there was a potential for the formation of common mycorrhizal networks in close vicinity. However, no significant difference was detected for the δ(13)C and δ(15)N values between seedlings and adults of each ECM plant, and no ECM species exhibited signatures of mixotrophy. Our results revealed (i) variation in ECM fungal diversity according to the seedling versus adult development stage of trees and (ii) low host specificity of ECM fungi, and indicated that multi-host fungi are more abundant than single-host fungi in this forest stand.

Nickel resistance determinants in bradyrhizobium strains from nodules of the endemic New Caledonia legume Serianthes calycina.

Bradyrhizobium strains, isolated in New Caledonia from nodules of the endemic legume Serianthes calycina growing in nickel-rich soils, were able to grow in the presence of 15 mM NiCl2. The genomes of these strains harbored two Ni resistance determinants, the cnr and nre operons. By constructing a cnrA mutant, we demonstrated that the cnr operon determines the high nickel resistance in Bradyrhizobium strains.

Legumes symbioses: absence of Nod genes in photosynthetic bradyrhizobia.

Leguminous plants (such as peas and soybeans) and rhizobial soil bacteria are symbiotic partners that communicate through molecular signaling pathways, resulting in the formation of nodules on legume roots and occasionally stems that house nitrogen-fixing bacteria. Nodule formation has been assumed to be exclusively initiated by the binding of bacterial, host-specific lipochito-oligosaccharidic Nod factors, encoded by the nodABC genes, to kinase-like receptors of the plant. Here we show by complete genome sequencing of two symbiotic, photosynthetic, Bradyrhizobium strains, BTAi1 and ORS278, that canonical nodABC genes and typical lipochito-oligosaccharidic Nod factors are not required for symbiosis in some legumes. Mutational analyses indicated that these unique rhizobia use an alternative pathway to initiate symbioses, where a purine derivative may play a key role in triggering nodule formation.

Genetic diversity of Acacia seyal Del. rhizobial populations indigenous to Senegalese soils in relation to salinity and pH of the sampling sites.

The occurrence and the distribution of rhizobial populations naturally associated to Acacia seyal Del. were characterized in 42 soils from Senegal. The diversity of rhizobial genotypes, as characterized by polymerase chain reaction restriction fragment length polymorphism (RFLP) analysis of 16S-23S rDNA, performed on DNA extracted from 138 nodules resulted in 15 clusters. Results indicated the widespread occurrence of compatible rhizobia associated to A. seyal in various ecogeographic areas. However, the clustering of rhizobial populations based on intergenic spacer (IGS) RFLP profiles did not reflect their geographic origin. Four genera were discriminated on the basis of 16S rRNA gene sequences of the strains representative for the IGS-RFLP profiles. The majority of rhizobia associated to A. seyal were affiliated to Mesorhizobium and Sinorhizobium 64 and 29%, respectively, of the different IGS-RFLP profiles. Our results demonstrate the coexistence inside the nodule of plant-pathogenic non-N(2)-fixing Agrobacterium and Burkholderia strains, which induced the formation of ineffective nodules, with symbiotic rhizobia. Nodulation was recorded in saline soils and/or at low pH values or in alkaline soils, suggesting adaptability of natural rhizobial populations to major ecological environmental stress and their ability to establish symbiotic associations within these soil environments. These results contribute to the progressing research efforts to uncover the biodiversity of rhizobia and to improve nitrogen fixation in agroforestry systems in sub-Saharan Africa.

Genetic diversity of ectomycorrhizal Basidiomycetes from African and Indian tropical rain forests.

Ectomycorrhizal (ECM) fungi have a worldwide distribution. However, the ecology of tropical ECM fungi is poorly documented, limiting our understanding of the symbiotic associations between tropical plants and fungi. ECM Basidiomycete diversity was investigated for the first time in two tropical rain forests in Africa (Western Upper Guinea) and in Asia (Western Ghats, India), using a fragment of the mitochondrial large subunit rRNA gene to type 140 sporocarps and 54 ectomycorrhizas. To evaluate taxonomic diversity, phylogenetic analyses were performed, and 40 sequences included from identified European specimens were used as taxonomic benchmarks. Five clades were recovered corresponding to six taxonomic groups: boletoids, sclerodermatoids, russuloids, thelephoroids, and a clade grouping the Amanitaceae and Tricholomataceae families. Our results revealed that the Russulaceae species display a great diversity with several putative new species, especially in Guinea. Other taxonomic issues at family/section levels are also briefly discussed. This study provides preliminary insights into taxonomic diversity, ECM status, and biogeographic patterns of ECM fungi in tropical two rain forest ecosystems, which appear to be as diverse as in temperate and boreal forests.

Diverse bacteria associated with root nodules of spontaneous legumes in Tunisia and first report for nifH-like gene within the genera Microbacterium and Starkeya.

We characterized 34 endophytic bacterial isolates associated to root nodules collected from spontaneous legumes in the arid zone of Tunisia by 16S rDNA polymerase chain reaction (PCR)-restriction fragment length polymorphism, whole cell protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 16S rDNA and 16S-23S rDNA internal transcribed spacer sequencing. Phylogenetically, these isolates belong to the branches containing the genera Inquilinus, Bosea, Rhodopseudomonas, Paracraurococcus, Phyllobacterium, Ochrobactrum, Starkeya, Sphingomonas, Pseudomonas, Agromyces, Microbacterium, Ornithinicoccus, Bacillus, and Paenibacillus. These strains did not induce any nodule formation when inoculated on the wide host spectrum legume species M. atropurpureum (Siratro) and no nodA gene could be amplified by PCR. However, nifH sequences, most similar to those of Sinorhizobium meliloti, were detected within strains related to the genera Microbacterium, Agromyces, Starkeya and Phyllobacterium.

Spatial distribution of ectomycorrhizal Basidiomycete Russula subsect. Foetentinae populations in a primary dipterocarp rainforest.

The spatial distribution of basidiocarps of the ectomycorrhizal Basidiomycete Russula subsect. Foetentinae was assessed in a primary forest in the Western Ghâts (India) dominated by the ectomycorrhizal tree species Vateria indica and Dipterocarpus indicus. Over a 7,700-m(2) sampling area, both trees and basidiocarps of Russula subsect. Foetentinae were mapped during the first month of the 2002 rainy season. First-order spatial analysis revealed that the distribution of the 45 collected carpophores was highly aggregated, with 60% of all basidiocarps located at a distance lower than 1 m from the nearest one. The genetic structure of the Russula subsect. Foetentinae population was studied by inter-simple sequence repeat polymorphism analysis using three primers. Eighteen of the 45 genotypes were represented by single basidiocarps. Twenty-seven basidiocarps were identified as belonging to 11 genets or separated ramets. Five genets were small, with diameters ranging from 0.5 to 5 m. The six others were large, with a diameter ranging from 31 m to a maximum measured distance of 70 m. In spite of the lack of data concerning the reproductive biology of this species, the presence of large genets suggests that mature stands may shelter well-spread underground mycelium, crucial for durable interaction with plant partner.

Role of methylotrophy during symbiosis between Methylobacterium nodulans and Crotalaria podocarpa.

Some rare leguminous plants of the genus Crotalaria are specifically nodulated by the methylotrophic bacterium Methylobacterium nodulans. In this study, the expression and role of bacterial methylotrophy were investigated during symbiosis between M. nodulans, strain ORS 2060T, and its host legume, Crotalaria podocarpa. Using lacZ fusion to the mxaF gene, we showed that the methylotroph genes are expressed in the root nodules, suggesting methylotrophic activity during symbiosis. In addition, loss of the bacterial methylotrophic function significantly affected plant development. Indeed, inoculation of M. nodulans nonmethylotroph mutants in C. podocarpa decreased the total root nodule number per plant up to 60%, decreased the whole-plant nitrogen fixation capacity up to 42%, and reduced the total dry plant biomass up to 46% compared with the wild-type strain. In contrast, inoculation of the legume C. podocarpa with nonmethylotrophic mutants complemented with functional mxa genes restored the symbiotic wild phenotype. These results demonstrate the key role of methylotrophy during symbiosis between M. nodulans and C. podocarpa.

Occurrence of nodulation in unexplored leguminous trees native to the West African tropical rainforest and inoculation response of native species useful in reforestation.

Despite the abundance and diversity of timber tree legumes in the West African rainforest, their ability to form nitrogen-fixing nodules in symbiosis with rhizobia, and their response to rhizobial inoculation, remain poorly documented. In the first part of this study the occurrence of nodulation was determined in 156 leguminous species growing in six natural forest areas in Guinea, mostly mature trees. In the second part, an in situ experiment of rhizobial inoculation was performed on eight selected tree species belonging to three genera: Albizia, Erythrophleum and Millettia. Of the 97 plant species and 14 genera that had never been examined before this study, 31 species and four genera were reported to be nodulated. After 4 months of growing in a nursery and a further 11 months after transplantation of plants to the field, we observed a highly significant (P < 0.001) and positive effect of inoculation with Bradyrhizobium sp. strains on the growth of the eight tree species tested. The importance of determining the nodulation ability of unexplored local trees and subsequently using this information for inoculation in reforestation programmes was demonstrated.

Methylobacterium nodulans sp. nov., for a group of aerobic, facultatively methylotrophic, legume root-nodule-forming and nitrogen-fixing bacteria.

Data on 72 non-pigmented bacterial strains that specifically induce nitrogen-fixing root nodules on the legume species Crotalaria glaucoides, Crotalaria perrottetii and Crotalaria podocarpa are reviewed. By SDS-PAGE analysis of total protein patterns and by 16S rRNA PCR-RFLP, these strains form a homogeneous group that is separate from other legume root-nodule-forming bacteria. The 16S rRNA gene-based phylogeny indicates that these bacteria belong to the genus Methylobacterium. They can grow on C(1) compounds such as methanol, formate and formaldehyde but not methylamine as sole carbon source, and carry an mxaF gene, encoding methanol dehydrogenase, which supports their methylotrophic metabolism. Presence of a nodA nodulation gene, and ability to nodulate plants of Crotalaria species and to fix nitrogen are features that separate the strains currently included in this group from other members of the genus Methylobacterium. The present study includes additional genotypic and phenotypic characterization of this novel Methylobacterium species, i.e. nifH gene sequence, morphology, physiology, enzymic and carbon source assimilation tests and antibiotic resistance. The name Methylobacterium nodulans sp. nov. (type strain, ORS 2060(T)=CNCM I 2342(T)=LMG 21967(T)) is proposed for this group of root-nodule-forming bacteria.

Characterisation of wild legume nodulating bacteria (LNB) in the infra-arid zone of Tunisia.

We report on the isolation and the characterization of nitrogen-fixing root nodule bacteria isolated from natural legumes in a region of South Tunisia corresponding to the infra-arid climatic zone. A collection of 60 new bacterial root nodule isolates were obtained from 19 legume species belonging to the genera Acacia, Anthyllis, Argyrolobium, Astragalus, Calycotome, Coronilla, Ebenus, Genista, Hedysarum, Hippocrepis, Lathyrus, Lotus, Medicago, Ononis. The isolates were characterised by (1) comparative 16S ARDRA using 7 enzymes, (2) total cell protein SDS-PAGE analysis and (3) 16S rDNA sequencing. The results show that these isolates are diverse and belong to the genera Rhizobium, Sinorhizobium, Mesorhizobium and Bradyrhizobium. Bradyrhizobium were further characterised by 16S-23S rDNA IGS sequencing. Surprisingly strains nodulating Astragalus cruciatus, Lotus creticus and Anthyllis henoniana were identified as Rhizobium galegae, a species recorded only as endosymbiont of Galega officinalis and G. orientalis in northern regions so far.

Two distinct crt gene clusters for two different functional classes of carotenoid in Bradyrhizobium.

Aerobic photosynthetic bacteria possess the unusual characteristic of producing different classes of carotenoids. In this study, we demonstrate the presence of two distinct crt gene clusters involved in the synthesis of spirilloxanthin and canthaxanthin in a Bradyrhizobium strain. Each cluster contains the genes crtE, crtB, and crtI leading to the common precursor lycopene. We show that spirilloxanthin is associated with the photosynthetic complexes, while canthaxanthin protects the bacteria from oxidative stress. Only the spirilloxanthin crt genes are regulated by light via the control of a bacteriophytochrome. Despite this difference in regulation, the biosyntheses of both carotenoids are strongly interconnected at the level of the common precursors. Phylogenetic analysis suggests that the canthaxanthin crt gene cluster has been acquired by a lateral gene transfer. This acquisition may constitute a major selective advantage for this class of bacteria, which photosynthesize only under conditions where harmful reactive oxygen species are generated.

Bacteriophytochrome controls photosystem synthesis in anoxygenic bacteria.

Plants use a set of light sensors to control their growth and development in response to changes in ambient light. In particular, phytochromes exert their regulatory activity by switching between a biologically inactive red-light-absorbing form (Pr) and an active far-red-light absorbing form (Pfr). Recently, biochemical and genetic studies have demonstrated the occurrence of phytochrome-like proteins in photosynthetic and non-photosynthetic bacteria--but little is known about their functions. Here we report the discovery of a bacteriophytochrome located downstream from the photosynthesis gene cluster in a Bradyrhizobium strain symbiont of Aeschynomene. The synthesis of the complete photosynthetic apparatus is totally under the control of this bacteriophytochrome. A similar behaviour is observed for the closely related species Rhodopseudomonas palustris, but not for the more distant anoxygenic photosynthetic bacteria of the genus Rhodobacter, Rubrivivax or Rhodospirillum. Unlike other (bacterio)phytochromes, the carboxy-terminal domain of this bacteriophytochrome contains no histidine kinase features. This suggests a light signalling pathway involving direct protein-protein interaction with no phosphorelay cascade. This specific mechanism of regulation may represent an important ecological adaptation to optimize the plant-bacteria interaction.

Phenotypic and genotypic diversity of rhizobia nodulating Pterocarpus erinaceus and P. lucens in Senegal.

A total of fifty root nodules isolates of fast-growing and slow growing rhizobia from Pterocarpus ennaceus and Pterocarpus lucens respectively native of sudanean and sahelian regions of Senegal were characterized. These isolates were compared to representative strains of known rhizobial species. Twenty-two new isolates were slow growers and twenty-eight were fast growers. A polyphasic approach was performed including comparative total protein sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) profile analysis; 16S rDNA and 16S-23S rDNA intergenic spacer (IGS) sequence analysis. By SDS-PAGE the slow growing isolates grouped in one major cluster containing reference strains of Bradyrhizobium sp. including strains isolated in Africa, in Brazil and in New Zealand. Most of the fast-growing rhizobia grouped in four different clusters or were separate strains related to Rhizobium and Mesorhizobium strains. The 16S rDNA and 16S-23S rDNA IGS sequences analysis showed accurately the differentiation of fast growing rhizobia among the Rhizobium and Mesorbizobium genospecies. The representative strains of slow growing rhizobia were identified as closely related to Bradyrbizobium elkanii and Bradyrhizobium japonicum. Based on 16S rDNA sequence analysis, one slow growing strain (ORS199) was phylogenetically related to Bradyrbizobium sp. (Lupinus) and Blastobacter denitrificans. This position of ORS 199 was not confirmed by IGS sequence divergence. We found no clear relation between the diversity of strains, the host plants and the ecogeographical origins.