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.

Analysis of pFQ31, a 8551-bp cryptic plasmid from the symbiotic nitrogen-fixing actinomycete Frankia.

The actinomycete Frankia has never been transformed genetically. To favour the development of Frankia cloning vectors, we have fully sequenced the Frankia alni pFQ31 cryptic plasmid and performed analyses to characterise its coding and non-coding regions. This plasmid is 8551 bp-long and contains 72% G+C. Computer-assisted analyses identified 18 open reading frames (ORFs). These ORFs show a synonymous codon usage different from the one of Frankia chromosomal genes, suggesting an evolutionary bias linked to the nature of the replicon or a horizontal transfer. Three ORFs were found to encode genes likely to be involved in plasmid replication and stability: parFA (partition protein), ptrFA (transcriptional repressor of the GntR family) and repFA (initiation of replication). DNA signatures of a replication origin were identified in the ptrFA-repFA intergenic region. These structural motifs are similar to those observed among origins of iteron-containing plasmids replicating via a θ mode.

Analysis of Frankia evolutionary radiation using glnII sequences.

Using a glnII (encoding glutamine synthetase II) PCR selective screening, a Frankia ACN14a gene library clone was isolated. A derived glnII-hybridising 2.7-kb HindIII subclone was characterised. Identities of 95% and 93% were observed, respectively, with the corresponding Frankia CpI1 glnI and glnII regions. A variable segment of the glnII region was selected, PCR amplified from various Frankia genomes, sequenced, and used to investigate phylogenetic relationships within the genus. glnII phylogenetic inferences are well-resolved and allowed us to deduce evolutionary trends among Frankia. Frankia radiation seems to begin with a diversification according to the ability or not to infect actinorhizal plants. The infective strains are divided into two clusters matching plant-colonising specificities.