Effect of arbuscular mycorrhiza establishment on the genetic structure and diversity of bacteria in the rhizosphere of Medicago truncatula

National audienceThe rhizosphere is the volume of soil which is influenced by plant root activity. Plant roots release 20% of the photosynthates into the rhizosphere. This process sustains the development of an active free living microflora in the rhizosphere compared to the bulk soil. Plants establish also symbiotic associations with soil microorganisms. Arbuscular mycorrhizas (AM) are mutualistic symbioses between soil fungi and plant roots. AM contribute to plant adaptation to low fertility environments. AM are established by 80% of terrestrial plant families including major crop plants. AM are also ancient symbiosis (400 million years). AM are known to be non-specific associations. However, the plant community is affected by the diversity of AM fungi, and reciprocally we have recently shown that the genetic diversity of AM fungi differ according to the plant species. These observations are consistent by the long joint evolution of plants and AM fungi. The evolution of plants and AM fungi is expected not to have occurred independently from the rhizosphere microflora leading to the emergence of bacteria adapted to mycorrhizal roots, these bacteria being able to influence AM establishment. To test this hypothesis, the genetic structures of bacterial communities associated with Medicago truncatula J5 (Myc+/Nod+) and its symbiosis-defective mutants TRV48 (Myc+/Nod-) and TRV25 (Myc-/Nod-) were compared as follows. DNA was extracted directly from the rhizosphere. Bacterial DNA polymorphism was assessed by an A-RISA (Automated-Ribosomal Intergenic Spacer Analysis) DNA fingerprint. The obtained fingerprints represented the genetic structure of bacterial communities. The genetic structures of bacterial communities were compared by analyzing A-RISA fingerprints with a Principal Component Analysis (PCA). Molecular markers explaining the differences in the genetic structures of the communities associated with M. truncatula genotypes were cloned and sequenced and the corresponding bacterial groups were identified. The cloned markers were further used to design a DNA probe. The probe was used to screen bacterial isolates from mycorrhizal and non-mycorrhizal roots and identify those harboring the sequences of cloned markers. Diversity of isolates showing a positive hybridization signal with the DNA probe was characterized by BOX-PCR. The genetic structure of bacterial communities associated with mycorrhizal and non-mycorrhizal genotypes differed significantly. Phylogenetic analysis of the molecular markers explaining these differences indicated that they belong to Comamonadaceae, Oxalobacteriaceae, and Rubrivivax sub-group. The isolates harboring sequences highly similar to that of the cloned markers were clustered in 25 groups on the basis of their BOX-PCR fingerprints, indicating a high diversity of these isolates. The distribution of the isolates in the different BOX-PCR groups differed significantly upon their origin (mycorrhizal/non-mycorrhizal roots). The effects of isolates representative of the genetic diversity described here above are currently being evaluated to test the value of our initial hypothesis