We analyzed the genetic diversity of 531 Sinorhizobium meliloti strains isolated from nodules of Medicago sativa cultivars in two different Italian soils during 4 years of plant growth. The isolates were analyzed for DNA polymorphism with the random amplified polymorphic DNA method. The populations showed a high level of genetic polymorphism distributed throughout all the isolates, with 440 different haplotypes. Analysis of molecular variance allowed us to relate the genetic structure of the symbiotic population to various factors, including soil type, alfalfa cultivar, individual plants within a cultivar, and time. Some of these factors significantly affected the genetic structure of the population, and their relative influence changed with time. At the beginning of the experiment, the soil of origin and, even more, the cultivar significantly influenced the distribution of genetic variability of S. meliloti. After 3 years, the rhizobium population was altered; it showed a genetic structure based mainly on differences among plants, while the effects of soil and cultivar were not significant. Alfalfa (Medicago sativa) and its symbiont Sinorhizobium meliloti have a long history of coexistence and coevolution. In every region where alfalfa has been cultivated for centuries, the natural nodulating population of S. meliloti plays a major role in satisfying the nitrogen requirements of the plants. Thus, it is important to investigate the genetic structure of natural populations of S. meliloti and their dynamics in relation to the host plant.In recent years, the use of molecular techniques has stimulated the development of rapid and simple methods for characterizing natural microbial populations. Studies utilizing restriction fragment length polymorphism-PCR, multilocus enzyme electrophoresis, 16S ribosomal DNA analysis, repetitive extragenic palindromic-PCR, and DNA reassociation (1, 2, 4-7, 12, 15, 17) have revealed extensive genetic variability of microbial communities in natural soil. This variability has been widely investigated in the Rhizobiaceae (2,5,10,16,19), and there is some evidence of genetic exchange in populations (18). In previous work, we exploited random amplified polymorphic DNA (RAPD) techniques, combined with a powerful statistical analysis (analysis of molecular variance [AMOVA]), to describe the genetic structure of natural S. meliloti populations (8, 9). Greater understanding of the genetic structure and dynamics of indigenous populations of S. meliloti would be of great agricultural interest in view of the influence that an established or varying bacterial population can have on the nodulation efficiency of particular plant cultivars.Little is known about the evolution of natural bacterial populations through the years in relation to a host plant. Bromfield et al. (1) showed that the M. sativa cultivar had an influence on the frequency of certain phage types in a natural S. meliloti population. Rooney-Varga et al. (12) described a seasonal modification of a natural population of sulfate-redu...

We investigated the genetic diversity of 96 Rhizobium meliloti strains isolated from nodules of four Medicago sativa varieties from distinct geographic areas and planted in two different northern Italian soils. The 96 isolates, which were phenotypically indistinguishable, were analyzed for DNA polymorphism with the following three methods: (i) a randomly amplified polymorphic DNA (RAPD) method, (ii) a restriction fragment length polymorphism (RFLP) analysis of the 16S-23S ribosomal operon spacer region, and (iii) an RFLP analysis of a 25-kb region of the pSym plasmid containing nod genes. Although the bacteria which were studied constituted a unique genetic population, a considerable level of genetic diversity was found. The new analysis of molecular variance (AMOVA) method was used to estimate the variance among the RAPD patterns. The results indicated that there was significant genetic diversity among strains nodulating different varieties. The AMOVA method was confirmed to be a useful tool for investigating the genetic variation in an intraspecific population. Moreover, the data obtained with the two RFLP methods were consistent with the RAPD results. The genetic diversity of the population was found to reside on the whole bacterial genome, as suggested by the RAPD analysis results, and seemed to be distributed on both the chromosome and plasmid pSym.

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