Genetic diversity and symbiotic effectiveness of chickpea rhizobia strains

Abstract

Rhizobia are soil bacteria able to establish nitrogen-fixing symbioses with leguminous plants inside special root tissues, the nodules. These symbioses are of extreme importance in agriculture allowing many plant crops to be cultivated without the need for chemical fertilisers. In the present thesis work, the genetic diversity, phylogeny and symbiotic effectiveness (SE) of native rhizobia, able to nodulate chickpea (Citar arietinum L.), have been addressed. Forty-one chickpea rhizobia isolated from four different Portuguesa soils (Beja, Elvas, Elvas- ENMP and Évora) were phenotypically evaluated. Twenty-one isolates were further analysed and assigned to the genus Mesorhizobium based on their 16S rDNA sequentes. Highly diverse Mesorhizobium strains were identified, belonging to six different species groups: M ciceri and M mediterraneum, the expected known chickpea symbionts, M loti, M tianshanense, and two probably new species. A correlation was found between 16S rDNA species groups and origin of individual isolates. Rhizobial diversity of the forty-one isolates from natural populations was assessed by molecular methods, namely 16S rDNA restriction fragment length polymorphism (RFLP) analysis, plasmid profiles, direct amplified polymorphic DNA (DAPD) fingerprinting and SDS–PAGE analysis of protein profiles. Plasmid number of isolates ranged from zero to six and was found to be correlated with origin and with species groups. 16S rDNA RFLP, DAPD and protein profiles generated analogous clustering of the isolates, supporting results on 16S rDNA sequence based phylogeny of the subgroup of twenty-one isolates. DAPD analysis, a newly described PCR-based approach, proved to be the most discriminating approach in strain differentiation and can be used as a fast method to screen diversity in new isolates. Evaluation of genetic diversity by the four molecular methods showed different levels of heterogeneity in the natural populations. A higher genetic diversity was found in Elvas-ENMP and Beja populations. The SE determined for the twenty-one isolates as well as for the two chickpea microsymbionts, M ciceri and M mediterraneum, ranged from 4 to 84%. No correlation was depicted between SE and origin site of the isolates. However, Beja isolates show the highest mean. SE, and Elvas-ENMP isolates have the lowest mean SE. We detected no significant correlation between SE and species. A multilocus phylogenetic approach was used to confirm the molecular phylogeny of the subgroup of twenty-one chickpea rhizobia isolates. Phylogenetic analysis based on the intergenic spacer between 16S and 23S rRNA genes (ITS), the ATP synthase (atpD) or the DNA recombinase A (recA) sequences corroborated the 16S rDNA phylogeny and confirmed the existence of six distinct species groups among chickpea mesorhizobia. Further evidence is provided for supporting one of these evolutionary lineages as new species within the genus Mesorhizobium. Indeed, sequencing of another housekeeping protein coding gene, the glutamine synthetase I gene (glnA), from this new group of isolates, confirmed its separate position and assignment to a new species. The name M. lusitanum is proposed, with isolate 64b.-Beja as the type strain. A11 isolates from the M. lusitanum group showed a high symbiotic effectiveness (above 50%) and may be potentially useful field inoculants. Chickpea has been considered a restrictive host for nodulation by rhizobia. However, the present work, as well as other recent studies, have shown that several Mesorhizobium species may effectively nodulate chickpea. In order to investigate the relationships between symbiosis genes from different rhizobia species able to nodulate chickpea, the niƒH and nodC genes from the twenty-one Portuguesa chickpea rhizobia isolates were sequenced and used for phylogenetic studies. The phylogenies based on symbiosis genes showed that, regardless of their species affiliation, ali chickpea rhizobia isolates formed a single highly supported cluster, an evidence of lateral transfer of symbiosis genes across different species. Chickpea is confirmed as a non-promiscuous host. The six different rhizobia species, that nodulate chickpea, share common symbiosis genes, suggesting recognition of only a few Nod factors by chickpea. Further analysis of symbiosis genes, namely copy number and location, performed by Southern hybridisation of plasmid profiles suggests the presence of, at least, two symbiosis plasmids in some isolates. Moreover, we provide evidence for the existence of at least two copies of the nodC gene in three isolates (6b.-Beja, 29-Beja and EE-29-ENMP), which is uncommon and has not been reported before in mesorhizobia. Overall, this work has contributed to the study of diversity and evolutionary relationships among mesorhizobia and has presented further evidence of horizontal gene transfer among several species of the genus Mesorhizobium, being the first report on lateral gene transfer between chickpea mesorhizobia. It has changed the current view on chickpea microsymbionts, sinta it has revealed that several species of Mesorhizobium can nodulate chickpea, besides M. ciceri and M. mediterraneum. Furthermore, it has contributed to the better understanding of the symbiosis between chickpea and rhizobia at the molecular level.