Genetic Diversity and Symbiotic Efficiency of Indigenous Common Bean Rhizobia in Croatia.

Nodule bacteria (rhizobia) in symbiotic associations with legumes enable considerable entries of biologically fixed nitrogen into soil. Efforts are therefore made to intensify the natural process of symbiotic nitrogen fixation by legume inoculation. Studies of field populations of rhizobia open up the possibility to preserve and probably exploit some indigenous strains with hidden symbiotic or ecological potentials. The main aim of the present study is to determine genetic diversity of common bean rhizobia isolated from different field sites in central Croatia and to evaluate their symbiotic efficiency and compatibility with host plants. The isolation procedure revealed that most soil samples contained no indigenous common bean rhizobia. The results indicate that the cropping history had a significant impact on the presence of indigenous strains. Although all isolates were found to belong to species Rhizobium leguminosarum, significant genetic diversity at the strain level was determined. Application of both random amplification of polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC- -PCR) methods resulted in similar grouping of strains. Symbiotic efficiency of indigenous rhizobia as well as their compatibility with two commonly grown bean varieties were tested in field experiments. Application of indigenous rhizobial strains as inoculants resulted in significantly different values of nodulation, seed yield as well as plant nitrogen and seed protein contents. The most abundant nodulation and the highest plant nitrogen and protein contents were determined in plants inoculated with R. leguminosarum strains S17/2 and S21/6. Although, in general, the inoculation had a positive impact on seed yield, differences depending on the applied strain were not determined. The overall results show the high degree of symbiotic efficiency of the specific indigenous strain S21/6. These results indicate different symbiotic potential of indigenous strains and confirmed the importance of rhizobial strain selection. These are the first studies of indigenous common bean rhizobia in Croatia that provide the basis for further characterization and selection of highly efficient indigenous strains and their potential use in agricultural practice and future research.

Biogeography of Sinorhizobium meliloti nodulating alfalfa in different Croatian regions.

Sinorhizobium meliloti is a nitrogen-fixing rhizobium symbiont of legumes, widespread in many temperate environments the high genetic diversity of which enables it to thrive as a symbiont of host legumes and free-living in soil. Soil type, together with geographic differences and host plant genotype, seem to be prominent factors in shaping rhizobial genetic diversity. While a large body of research supports the idea that the genetic structure of free-living microbial taxa exhibits a clear biogeographic pattern, few investigations have been performed on the biogeographic pattern of S. meliloti genotypes in a restricted geographic range. In the present study, a collection of 128 S. meliloti isolates from three different regions in Croatia was investigated to analyze the relationship between genetic diversity, geographic distribution, soil features and isolate phenotypes by using amplified fragment length polymorphism (AFLP) as a genome-wide scanning method. Results obtained led to the conclusion that the genotypes of isolates cluster according to the region of origin and that the differentiation of S. meliloti populations can be mainly ascribed to geographic isolation following an isolation-by-distance model, with a strong distance-decay relationship of genetic similarity with distance, in which local soil conditions are not the major component influencing the isolate phenotypes or their genomic differentiation.

Influence of different Sinorhizobium meliloti inocula on abundance of genes involved in nitrogen transformations in the rhizosphere of alfalfa (Medicago sativa L.).

Inoculation of leguminous seeds with selected rhizobial strains is practised in agriculture to ameliorate the plant yield by enhanced root nodulation and nitrogen uptake of the plant. However, effective symbiosis between legumes and rhizobia does not only depend on the capacity of nitrogen fixation but also on the entire nitrogen turnover in the rhizosphere. We investigated the influence of seed inoculation with two indigenous Sinorhizobium meliloti strains exhibiting different efficiency concerning plant growth promotion on nitrogen turnover processes in the rhizosphere during the growth of alfalfa. Quantification of six target genes (bacterial amoA, nirK, nirS, nosZ, nifH and archaeal amoA) within the nitrogen cycle was performed in rhizosphere samples before nodule formation, at bud development and at the late flowering stage. The results clearly demonstrated that effectiveness of rhizobial inocula is related to abundance of nifH genes in the late flowering phase of alfalfa. Moreover, other genes involved in nitrogen turnover had been affected by the inocula, e.g. higher numbers of amoA copies were observed during flowering when the more effective strain had been inoculated. However, the respective gene abundances differed overall to a greater extent between the three plant development stages than between the inoculation variants.