Phytostimulatory effect of Rhizobium and Plant Growth Promoting Rhizobacteria in common bean (Phaseolus vulgaris L.) interaction
Torres Gutiérrez, Roldán
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The symbiosis between plants of the Leguminosae family and prokaryotic partners is typically characterized by the formation of specialized organs, called nodules, on plant roots or stems that are invaded by the specific microsymbionts. These include the well-known alpha- proteobacterial group of Rhizobiaceae containing the genera Rhizobium, Bradyrhizobium, Sinorhizobium (Ensifer), Mesorhizobium, Azorhizobium, and Allorhizobium, collectively referred as rhizobia. Legumes play a crucial role in sustainable agriculture. Symbiotic nitrogen fixation (SNF) through interaction between legumes and rhizobia, contributes to nitrogen (N) nutrition of most legumes and legume cropping systems. Common bean (Phaseolus vulgaris L.) is the most important legume for direct human consumption worldwide and particularly in many parts of Latin America and Africa. However, the application of SNF in common bean in the field is often low compared to the nitrogen fixing capacity of beans under optimal conditions and as compared to the amounts of nitrogen fixed by other legumes. The aim of our study is to identify, quantify and enhance the phytostimulatory effect of the interplay between Rhizobium, bean genotypes and plant growth promoting rhizobacteria (PGPR) under different growth conditions and to contribute to the understanding of the molecular mechanisms involved in the Rhizobium-bean interaction. To reach this objective, combinations of Rhizobium-PGPR were evaluated under different growth conditions in Cuba using two local bean genotypes. The nodulation and plant growth parameters were significantly stimulated with the combination of Rhizobium-Azospirillum and Rhizobium-Azotobacter under pot experiment condition, as well as in a field trial. Variations among genotypes were observed for growth parameters and yield in a second field trial. The combination Rhizobium-Azospirillum and the fertilizer treatments showed the best result in yield for ICA Pijao beans, while for BAT-304 beans the best result was obtained with the single Rhizobium inoculation. Secondly, the morphological and genetic characterization of bacterial isolates from Cuban bean fields, as well as the phenotypic characterization of Cuban Rhizobium isolates under controlled and field conditions, demonstrate the biodiversity of beneficial microbes in the common bean rhizosphere and the stimulatory effect of compatible interactions between common bean genotypes and Rhizobium strains. The genetic characterization of isolated bacterial strains form Cuban soils using 16S rDNA sequencing revealed 8 groups of bacteria belonging to the genera: Agrobacterium, Rhizobium, Ochrobactrum, Sphingomonas, Stenotrophomonas, Bacillus, Brevibacillus and Paenibacillus. In nodule samples, 37.5% of isolates were 100% similar to Agrobacterium tumefaciens or Rhizobium species. This study allowed the identification of two species of Rhizobium isolates (Rhizhobium etli and Rhizobium tropici) in nodule samples. In nodulation tests Agrobacterium isolates were unable to nodulate the original host. The phenotypic characterization showed the stimulation of nodulation parameters and the N fixation through the native Rhizobium isolates at early stage of common bean plants. Under field trial conditions, the nodulation, growth parameters and yield were stimulated significantly for ICA Pijao as compared with BAT-304 upon inoculation with the isolated Rhizobium strains. Furthermore, genes differentially expressed during the bean root interaction with Rhizobium etli CNPAF512, infection with Fusarium solani f. sp. phaseoli and a control respectively, were identified using the cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) technique. In silico analysis was used to determine the differential expression profiles of transcript derived fragments (TDFs). Several TDFs were isolated, cloned, sequenced and the obtained DNA sequences were compared with sequences in the GenBank database. The sequences retrieved revealed homology with genes encoding stress/defense and cell metabolism functions for Rhizobium treatments, as well as stress/defense functions for the Fusarium condition. The results outlined in this study demonstrate the potential of selection for efficient associations among bean genotypes, rhizobia and plant growth promoting rhizobacteria in order to achieve the increase of SNF in common bean under local agro-ecosystems, as well as increase our insight of the molecular dialogue in common bean-rhizobia interaction. However, these studies should be expanded using more bean genotypes and bacterial combinations in different environmental conditions, in order to provide recommendations to farmers.
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