Differing courses of genetic evolution of Bradyrhizobium inoculants as revealed by long-term molecular tracing in Acacia mangium plantations.

Introducing nitrogen-fixing bacteria as an inoculum in association with legume crops is a common practice in agriculture. However, the question of the evolution of these introduced microorganisms remains crucial, both in terms of microbial ecology and agronomy. We explored this question by analyzing the genetic and symbiotic evolution of two Bradyrhizobium strains inoculated on Acacia mangium in Malaysia and Senegal 15 and 5 years, respectively, after their introduction. Based on typing of several loci, we showed that these two strains, although closely related and originally sampled in Australia, evolved differently. One strain was recovered in soil with the same five loci as the original isolate, whereas the symbiotic cluster of the other strain was detected with no trace of the three housekeeping genes of the original inoculum. Moreover, the nitrogen fixation efficiency was variable among these isolates (either recombinant or not), with significantly high, low, or similar efficiencies compared to the two original strains and no significant difference between recombinant and nonrecombinant isolates. These data suggested that 15 years after their introduction, nitrogen-fixing bacteria remain in the soil but that closely related inoculant strains may not evolve in the same way, either genetically or symbiotically. In a context of increasing agronomical use of microbial inoculants (for biological control, nitrogen fixation, or plant growth promotion), this result feeds the debate on the consequences associated with such practices.

Genetic diversity of symbiotic Bradyrhizobium elkanii populations recovered from inoculated and non-inoculated Acacia mangium field trials in Brazil.

Acacia mangium is a legume tree native to Australasia. Since the eighties, it has been introduced into many tropical countries, especially in a context of industrial plantations. Many field trials have been set up to test the effects of controlled inoculation with selected symbiotic bacteria versus natural colonization with indigenous strains. In the introduction areas, A. mangium trees spontaneously nodulate with local and often ineffective bacteria. When inoculated, the persistence of inoculants and possible genetic recombination with local strains remain to be explored. The aim of this study was to describe the genetic diversity of bacteria spontaneously nodulating A. mangium in Brazil and to evaluate the persistence of selected strains used as inoculants. Three different sites, several hundred kilometers apart, were studied, with inoculated and non-inoculated plots in two of them. Seventy-nine strains were isolated from nodules and sequenced on three housekeeping genes (glnII, dnaK and recA) and one symbiotic gene (nodA). All but one of the strains belonged to the Bradyrhizobium elkanii species. A single case of housekeeping gene transfer was detected among the 79 strains, suggesting an extremely low rate of recombination within B. elkanii, whereas the nodulation gene nodA was found to be frequently transferred. The fate of the inoculant strains varied depending on the site, with a complete disappearance in one case, and persistence in another. We compared our results with the sister species Bradyrhizobium japonicum, both in terms of population genetics and inoculant strain destiny.

Genetic diversity and distribution of Bradyrhizobium and Azorhizobium strains associated with the herb legume Zornia glochidiata sampled from across Senegal.

Herb legumes have great potential for rehabilitation of semi-arid degraded soils in Sahelian ecosystems as they establish mutualistic symbiosis with N(2)-fixing rhizobia. A phylogenetic analysis was performed for 78 root nodule bacteria associated with the common Sahelian herb legume Zornia glochidiata Reichb ex DC in Senegal. Based on ITS (rDNA16S-23S) and recA sequences, these strains were shown to belong to the two genera Bradyrhizobium and Azorhizobium. Strains of this latter, although frequent, formed small and ineffective nodules and suggested a parasitism rather than a symbiotic association. A potential negative effect of Azorhizobium on Zornia growth was tested for when inoculated alone or in association with a Bradyrhizobium strain. Bradyrhizobium isolates were distributed in four groups. Groups A and B were two sister clades in a larger monophyletic group also including Bradyrhizobium liaoningense, Bradyrhizobium yuanmingense, and Bradyrhizobium japonicum. Strains of cluster D fell in a sister clade of the photosynthetic Bradyrhizobium sp. group, including ORS278, whereas group C appeared to be divergent from all known Bradyrhizobium clusters. Amplified fragment length polymorphism (AFLP) clustering was congruent with ITS and recA phylogenies, but displayed much more variability. However, within the main Bradyrhizobium clades, no obvious relationship could be detected between clustering and geographical origin of the strains. Each sub-cluster included strains sampled from different locations. Conversely, Azorhizobium strains showed a tendency in the phylogeny to group together according to the site of sampling. The predominance of ineffective Azorhizobium strains in the nodules of Zornia roots, the large Bradyrhizobium genetic diversity and the geographical genetic diversity pattern are explored.

Nitrogen-fixing nodules from rose wood legume trees (Dalbergia spp.) endemic to Madagascar host seven different genera belonging to alpha- and beta-Proteobacteria.

Although legume biodiversity is concentrated in tropical regions, the majority of studies on legume nodulating bacteria (LNB) are focused on cultivated leguminous plants from temperate regions. However, recent works on tropical regions tend to indicate that the actual diversity of LNB is largely underestimated. In this study, we report the isolation and characterization of 68 nitrogen-fixing root nodule bacteria collected from eight endemic tree species of Dalbergia in Madagascar. The isolates were characterized by (i) restriction fragment length polymorphism (RFLP) analysis of 16S-IGS rDNA, (ii) 16S rDNA gene sequencing and (iii) nodulation tests. Results revealed a wide diversity of bacteria present in the nodules of Dalbergia. Among the 68 isolated bacteria, 65 belonged to Bradyrhizobium, Mesorhizobium, Rhizobium, Azorhizobium and Phyllobacterium from the alpha-class of Proteobacteria, and three isolates belonged to Burkholderia and Ralstonia from the beta-class of Proteobacteria. Our results also show for the first time that a strain belonging to the Burkholderia cepacia complex is able to induce efficient nodules on a legume plant.

Medicago-Sinorhizobium symbiotic specificity evolution and the geographic expansion of Medicago.

The legume genus Medicago interacts with soil bacteria commonly referred to as rhizobia, in a nitrogen fixing symbiosis. We analysed the diversity of symbiotic association specificity among the two organisms, and its evolution in the plant genus. Nitrogen fixation tests and molecular phylogenetic reconstructions revealed that the genus Medicago includes more symbiotic specificity groups than previously suggested and that plant specificity is highly unstable and has repeatedly switched along the diversification of this genus. A phylogenetic analysis including geographical data shows that bacterial geographical diversity distribution has a strong influence on the geographic distribution of plant species and their ability to colonize new areas. Multiple other modifications of specificity occurred along the diversification of the genus, presumably due to selection for specialization to a single bacterial biovar. Codivergence between plants and bacteria may also have taken place.

The last common ancestor of Sarcolaenaceae and Asian dipterocarp trees was ectomycorrhizal before the India-Madagascar separation, about 88 million years ago.

Phylogenetic studies comparing the Dipterocarpaceae and the Sarcolaenaceae, a tree family endemic to Madagascar, have shown that the Sarcolaenaceae share a common ancestor with Asian dipterocarps. This suggests that Asian dipterocarps drifted away from Madagascar with the India-Seychelles landmass and then dispersed through Asia. Although all dipterocarps examined so far have been found to be ectomycorrhizal, the ectomycorrhizal status of Sarcolaenaceae had not been investigated. Here we establish the ectomycorrhizal status of Sarcolaenaceae using histological and molecular methods. This indicates that the common ancestor of the Sarcolaenaceae and Asian dipterocarps was ectomycorrhizal, at least before the separation of the Madagascar-India landmass, 88 million years ago.

The spatial structure of sexual and cytonuclear polymorphism in the gynodioecious Beta vulgaris ssp. maritima: I/ at a local scale.

We have analyzed the spatial distribution of the sex phenotypes and of mitochondrial, chloroplast, and nuclear markers within two gynodioecious populations of Beta vulgaris ssp. maritima. Within both populations, sexual phenotype variation is controlled mainly by the cytoplasmic genotype, although in one study population a joint polymorphism of cytonuclear factors is clearly involved. In spite of contrasts in the ecology (mainly due to different habitats), a clear common feature in both populations is the highly patchy distribution of cytoplasmic haplotypes, contrasting with the wide distribution of nuclear diversity. This high contrast between cytoplasmic vs. nuclear spatial structure may have important consequences for the maintenance of gynodioecy. It provides opportunities for differential selection since nuclear restorer alleles are expected to be selected for in the presence of their specific cytoplasmic male sterile (CMS) type, but to be neutral (or selected against if there is a cost of restoration) in the absence of their CMS type. Selective processes in such a cytonuclear landscape may explain the polymorphism we observed at restorer loci for two CMS types.

Molecular phylogenetic approach for studying life-history evolution: the ambiguous example of the genus Medicago L.

We present a molecular phylogeny including most species of the genus Medicago L. (Fabaceae). Based on the consensus of the 48 most parsimonious trees, life-history and mating-system characters are mapped, and a putative history of the genus is suggested. The most parsimonious reconstruction suggests an ancestral annual and selfing state, and recurrent evolution towards perenniality and outcrossing. Based on theoretical predictions and classical hypotheses of the history of the genus, different assumptions about the ancestral state and different weighting schemes of evolution between the character states are made. Assuming an outcrossing, perennial ancestral state (partly supported by morphological features) does not fundamentally change the reconstruction. To meet theoretical expectations, various weighting schemes favouring evolution towards annuality and selfing are applied. Influence and validity of such weighting schemes are discussed with regard to other studies.

Evolution of annual species of the genus Medicago: a molecular phylogenetic approach.

We performed a molecular phylogenetic study based on the nuclear ribosomal internal and external transcribed spacer (ITS and ETS). Thirty-one annual Medicago species were included in the study, representing more than half of the genus and 85% of the annuals of the genus. Major incongruences were found between phylogenetic relationships and morphological classification of the genus. Morphological and cytological traits were mapped onto the phylogeny. The most parsimonious reconstruction suggested an ancestral spiny state and a recurrent transition from spiny to spineless state. From the ancestral state of 2n=16, three loss events of chromosomes must have occurred leading to the same specific number of 14 chromosomes whereas species having 30 chromosomes form a monophyletic clade.

Ribosomal External and Internal Transcribed Spacers: Combined Use in the Phylogenetic Analysis of Medicago (Leguminosae)

We have estimated the potential phylogenetic utility of the ribosomal external transcribed spacer (ETS) from the nuclear ribosomal region. The ETS was sequenced from 13 annual Medicago (Fabaceae) species upstream a highly conserved motive which was found among many different organisms. In the genus Medicago, the ETS was found to evolve 1.5 times faster than the internal transcribed spacer and to be 1.5 times more informative. Reduced ribosomal maturation process constraints on ETS are proposed to explain the different evolutionary rates between the two spacers. Maximal phylogenetic resolution and support was obtained when the two spacers were analyzed together. No incongruence between the two spacers was found and ETS appears to be a valuable source of information for solidifying ITS plant phylogeny. The phylogeny obtained in Medicago suggests that none of the three subsections included in the study is monophyletic.