B. Dreyfus

Characterization of tropical tree rhizobia and description of Mesorhizobium plurifarium sp. nov.

A collection of strains isolated from root nodules of Acacia species in Senegal was analysed previously by electrophoresis of total cell protein, auxanographic tests, rRNA-DNA hydridization, 16S rRNA gene sequencing, DNA base composition and DNA-DNA hybridization [de Lajudie, P., Willems, A., Pot, B. & 7 other authors (1994). Int J Syst Bacteriol 44, 715-733]. Strains from Acacia were shown to belong to two groups, Sinorhizobium terangae, and a so-called gel electrophoretic cluster U, which also included some reference strains from Brazil. Further taxonomic characterization of this group using the same techniques plus repetitive extragenic palindromic-PCR and nodulation tests is presented in this paper. Reference strains from Sudan and a number of new rhizobia isolated from nodules of Acacia senegal, Acacia tortilis subsp. raddiana and Prosopis juliflora in Senegal were included. As a result of this polyphasic approach, the creation of a new species, Mesorhizobium plurifarium, is proposed for a genotypically and phenotypically distinct group corresponding to the former cluster U and containing strains isolated from Acacia, Leucaena, Prosopis and Chamaecrista in West Africa (Senegal), East Africa (Sudan) and South America (Brazil). The type strain of Mesorhizobium plurifarium ORS 1032 has been deposited in the LMG collection as LMG 11892.

Allorhizobium undicola gen. nov., sp. nov., nitrogen-fixing bacteria that efficiently nodulate Neptunia natans in Senegal.

A group of nodule isolates from Neptunia natans, an indigenous stemnodulated tropical legume found in waterlogged areas of Senegal, was studied. Polyphasic taxonomy was performed, including SDS-PAGE of total proteins, auxanography using API galleries, host-plant specificity, PCR-RFLP of the internal transcribed spacer region between the 16S and the 23S rRNA coding genes, 16S rRNA gene sequencing and DNA-DNA hybridization. It was demonstrated that this group is phenotypically and phylogenetically separate from the known species of Rhizobium, Sinorhizobium, Mesorhizobium, Agrobacterium, Bradyrhizobium and Azorhizobium. Its closest phylogenetic neighbour, as deduced by 16S rRNA gene sequencing, is Agrobacterium vitis (96.2% sequence homology). The name Allorhizobium undicola gen. nov., sp. nov., is proposed for this group of bacteria, which are capable of efficient nitrogen-fixing symbiosis with Neptunia natans, and the type strain is ORS 992T (= LMG 11875T).

Stem Nodulation in Legumes: Diversity, Mechanisms, and Unusual Characteristics

Abstract Rhizobia can establish a nitrogen-fixing symbiosis with plants of the Leguminosae family. They elicit on their host plant the formation of new organs, called nodules, which develop on the ...

Nod factor thin-layer chromatography profiling as a tool to characterize symbiotic specificity of rhizobial strains: application to Sinorhizobium saheli, S. teranga, and Rhizobium sp. strains isolated from Acacia and Sesbania

Rhizobia isolated from Acacia or Sesbania belong to several taxonomic groups, including the newly described species Sinorhizobium sahelì, Sinorhizobium teranga, and the so-called cluster U. A collection of strains belonging to these different groups was analyzed in order to determine whether the host range of a strain could be correlated with various molecular nodulation determinants. Nodulation tests showed that, independently of their taxonomic position, all the strains isolated from the same plant genus exhibited a similar host range, which was different for Sesbania and Acacia isolates. The fact that S. teranga strains nodulate either Acacia or Sesbania led us to subdivide this species into biovars acaciae and sesbaniae. Thin-layer chromatography (TLC) analysis of the Nod factors synthesized by overproducing strains showed that (i) strains isolated from the same plant genus exhibited similar TLC profiles and (ii) profiles of Acacia and Sesbunia symbionts were easily distinguishable, Acacia strains producing, in particular, sulfated molecules. In contrast, no correlation could be established between the host range of a strain and its plasmid content, the nature of the nod gene inducers or the presence of DNA sequences homologous to specific nod genes. We thus propose that Nod factor TLC profiling may be used as an easy and powerful tool for the classification of rhizobial strains on the basis of their symbiotic properties.

Arbuscular Mycorrhizal Colonization and Nodulation Improve Flooding Tolerance in Pterocarpus officinalis jacq. Seedlings

Pterocarpus officinalis (Jacq.) seedlings inoculated with the arbuscular mycorrhizal fungus, Glomus intraradices, and the strain of Bradyrhizobium sp. (UAG 11A) were grown under stem-flooded or nonflooded conditions for 13xa0weeks after 4xa0weeks of nonflooded pretreatment under greenhouse conditions. Flooding of P. officinalis seedlings induced several morphological and physiological adaptive mechanisms, including formation of hypertrophied lenticels and aerenchyma tissue and production of adventitious roots on submerged portions of the stem. Flooding also resulted in an increase in collar diameter and leaf, stem, root, and total dry weights, regardless of inoculation. Under flooding, arbuscular mycorrhizas were well developed on root systems and adventitious roots compared with inoculated root systems under nonflooding condition. Arbuscular mycorrhizas made noteworthy contributions to the flood tolerance of P. officinalis seedlings by improving plant growth and P acquisition in leaves. We report in this study the novel occurrence of nodules connected vascularly to the stem and nodule and arbuscular mycorrhizas on adventitious roots of P. officinalis seedlings. Root nodules appeared more efficient fixing N2 than stem nodules were. Beneficial effect of nodulation in terms of total dry weight and N acquisition in leaves was particularly noted in seedlings growing under flooding conditions. There was no additive effect of arbuscular mycorrhizas and nodulation on plant growth and nutrition in either flooding treatment. The results suggest that the development of adventitious roots, aerenchyma tissue, and hypertrophied lenticels may play a major role in flooded tolerance of P. officinalis symbiosis by increasing oxygen diffusion to the submerged part of the stem and root zone, and therefore contribute to plant growth and nutrition.

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.

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.

Nodulated Tree Legumes and Their Symbiotic Bradyrhizobium in African and South-American Tropical Rainforests

Following the work of Doyle et al. (1997) on the rbcL gene, Wojciechowski et al. (2004) analysed the plastid matK genes of 330 legume species and brought new insight on clade organization within this family. Using the phylogenetic tree obtained by these latter authors, we superimposed on each plant species the taxonomic identity of its symbiotic N2-fixing partner (from the databank http://www.ncbi.nlm.nih.gov/). We also checked the nodulation status of each plant species (from http://www.ars-grin.gov/ ~sbmljw/cgi-bin/taxnodul.pl). Surprisingly, the bacterial genera are randomly distributed among the plant clades and the genus Bradyrhizobium is increasingly represented from the Milletioid to the Caesalpinioid clades.

A New Approach to Symbiotically Characterize Rhizobia, Based on NOD Factor Analysis

Rhizobia are currently classified using a combination of general taxonomic characters which now tend to exclude symbiotic properties as a taxonomic criterion, since symbionts from the same host plant are not always closely related, while very similar bacterial strains have different host ranges. The host range of rhizobia is however of great significance for agro-ecological studies, biodiversity analysis and plant-bacteria coevolution. Our aim is hence to develop alternative methods to nodulation tests for the symbiotic characterization of rhizobia.

Characterization of Tropical Isolates Nodulating Neptunia oleracea in Senegal

Neptunia oleracea is an annual aquatic stem-nodulated legume indigeneous of waterlogged places in Senegal. Bacterial isolates from nodules of N. oleracea can infect Medicago sativa roots but the nodules are not efficient. No information is available on the taxonomy of Neptunia specific bacteria.