Rapid and Efficient Methods to Isolate, Type Strains and Determine Species of Agrobacterium spp. in Pure Culture and Complex Environments

Agrobacterium are Alphaproteobacteria common in most soils that closely interact with plants in two respects. Firstly, and as a general trait of the whole taxon, they are rhizospheric bacteria saprophytically living in the root environment (i.e. rhizosphere) of numerous plants. Rhizospheric interactions are generally considered to be of commensal type with no detrimental effect to the plant, but in most instances they are likely beneficial to plants. For evident agronomic purposes it is worthwhile to explore whether agrobacteria are themselves plant growth-promoting rhizobacteria (PGPR) or not. However, this requires an expert determination of the Agrobacterium taxonomy. Indeed our current investigations suggest that only some agrobacterial species from the abundant soil Agrobacterium guild are selected in the rhizosphere of a given plant. Secondly, but only when they harbor a dispensable Ti plasmid (i.e. tumor inducing plasmid), agrobacteria are plant pathogens able to cause the crown gall disease to most dicots and gymnosperms and some monocots (Pitzscke & Hirt, 2010). Ti plasmids are conjugative and can easily spread in indigenous soil agrobacteria. As a result transconjugant agrobacteria become in turn pathogenic, contributing both to disease spread and perennial soil contamination. An epidemiological survey of crown gall thus also requires expert determination of the Agrobacterium taxonomy. A set of biochemical and molecular methods were thus set up to facilitate the taxonomic assessment of agrobacteria either in pure culture or directly from complex environments such as soils, rhizospheres or tumours. After a presentation of the present state of Agrobacterium taxonomy, this work provides efficient methods to : i) isolate agrobacteria from complex environments thanks to elective media; ii) determine the Agrobacterium genus status of newly isolated strains on the basis of a minimal set of biochemical tests; iii) determine species and type novel isolates of Agrobacterium by sequence analysis of relevant marker genes; iv) determine amplicon content and genome architecture; v) detect the presence of Agrobacterium and Ti or Ri plasmid directly in complex environments by PCR using selected primers and metagenomic DNA extracted from whole bacterial communities.