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Phenotypic and genotypic characterization of indigenous Sinorhizobium meliloti strains isolated from different regions in Croatia

The studying of rhizobial biodiversity opens up the possibility to preserve and maybe to exploit some indigenous strains with hidden symbiotic or ecological potential, particularly under unfavourable conditions. In order to improve the beneficial effect of alfalfa inoculation it is important to characterize the indigenous strains and to obtain information about actual composition of rhizobial field population. The main aim of the present study was to characterize indigenous rhizobia associated with alfalfa (Medicago sativa L.) in different regions of Croatia. The soil samples for rhizobial isolation were collected from three different regions in Croatia (Istrian peninsula, north-west Croatia and central Croatia). Greenhouse pot experiment was established in order to obtain nodules for isolation procedure. Due to better description of field sites, main physical and chemical characteristics were determined in all soil samples. Over 250 isolates were obtained from root nodules of alfalfa. To study their diversity and characterize them in relation to environmental conditions of their soils of origin, a polyphasic approach was used. Stress tolerance assays revealed significant variations in pH tolerance while almost all isolates showed similar tolerance to elevated salt concentrations and growth temperatures. Among all isolates recovered from different regions, significant number of isolates showed tolerance to acidic pH values with dominance of strains originating from Istrian region where 38% of isolates tolerated pH 4.5. The actual composition and genetic diversity of natural field population was studied by two PCR fingerprinting methods. PCR-RFLP of 16S rDNA revealed most of isolates to be closely related to S. meliloti. Cluster analysis of RFLP patterns obtained with RsaI, showed that none of the isolate was identical with S. medicae type strain. However, several isolates produced slightly different RFLP pattern from S. meliloti type strain and other isolates. Dendrogram derived from AFLP profiles revealed considerable genetic diversity among S. meliloti isolates. Only a few strains were identical or nearly identical to each other. The strains originated from the same region were mostly grouped within the same cluster. These results were confirmed by further analysis. Analysis of molecular variance (AMOVA) allowed relating the genetic structure of the symbiotic population to various factors, including location, soil pH and the type of vegetation. Majority of the factors considered were significant as source of genetic variation, indicating that these populations were structured according to their geographical location and environmental conditions. The certain number of isolates, showing unspecific rhizobial characteristics, were sequenced. The results confirmed the presence of other bacterial species within the nodules. Therefore, further investigations are needed for the assessment of hidden diversity within the nodules. Better understanding of rhizobial ecology and selection of locally adapted and genetically defined strains as well as their targeted application regarding the environmental conditions of certain production area is one among many approaches to improve the nitrogen fixation efficiency and crop productivity.

nitrogen fixation; rhizobia; Sinorhizobium meliloti; indigenous strains; stress tolerance; genetic diversity

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