Characterization and phylogenetic diversity of Allorhizobium vitis isolated from grapevine in Morocco.

AIMS: Crown gall, a phytobacteriosis characterized by the formation of tumours on plant roots was observed in recently planted vineyards of the Meknes region (Morocco). The objective of this research was to analyse the diversity of pathogenic agrobacteria isolated from grapevine in Morocco. METHODS AND RESULTS: Eighty-two isolates from 11 affected vineyards were characterized by recA sequencing and were found to belong to Agrobacterium tumefaciens genomospecies G1, G4 or G7, Rhizobium rhizogenes, and to Allorhizobium vitis. Only the All. vitis isolates appeared to be pathogenic on tomato and multilocus sequence analysis phylogenetic analyses revealed a weak genetic diversity, with the definition of only four genomic groups. Definition of the All. vitis genomic groups correlated with specific pathogenic traits: indeed, genomic groups differed with respect to the severity of hypersensitive response symptoms on tobacco leaves, the intensity of necrotic response on grapevine explants and opine profiles. Both vitopine and octopine were detected by UHPLC in tumours induced by isolates of three genomic groups, an opine signature scarcely ever reported. CONCLUSIONS: Allorhizobium vitis is the only causative agent of crown gall on grape in Morocco, pathogenic isolates can be separated into four genomic groups. SIGNIFICANCE AND IMPACT OF THE STUDY: This study of recently crown-gall-infested vineyards demonstrated that All. vitis is the only causative agent and revealed the presence of nonpathogenic Agrobacterium strain within tumours. Moreover, as the genetic diversity of the All. vitis isolates is relatively narrow, this study lays the basis for further analyses on the evolution of the disease, on the dissemination of the pTi and more globally on the fate of the different genomic groups in this newly colonized environment.

Rapid and accurate species and genomic species identification and exhaustive population diversity assessment of Agrobacterium spp. using recA-based PCR.

Agrobacteria are common soil bacteria that interact with plants as commensals, plant growth promoting rhizobacteria or alternatively as pathogens. Indigenous agrobacterial populations are composites, generally with several species and/or genomic species and several strains per species. We thus developed a recA-based PCR approach to accurately identify and specifically detect agrobacteria at various taxonomic levels. Specific primers were designed for all species and/or genomic species of Agrobacterium presently known, including 11 genomic species of the Agrobacterium tumefaciens complex (G1-G9, G13 and G14, among which only G2, G4, G8 and G14 still received a Latin epithet: pusense, radiobacter, fabrum and nepotum, respectively), A. larrymoorei, A. rubi, R. skierniewicense, A. sp. 1650, and A. vitis, and for the close relative Allorhizobium undicola. Specific primers were also designed for superior taxa, Agrobacterium spp. and Rhizobiaceace. Primer specificities were assessed with target and non-target pure culture DNAs as well as with DNAs extracted from composite agrobacterial communities. In addition, we showed that the amplicon cloning-sequencing approach used with Agrobacterium-specific or Rhizobiaceae-specific primers is a way to assess the agrobacterial diversity of an indigenous agrobacterial population. Hence, the agrobacterium-specific primers designed in the present study enabled the first accurate and rapid identification of all species and/or genomic species of Agrobacterium, as well as their direct detection in environmental samples.