Morphological and genotypic features of Xanthomonas arboricola pv. juglandis populations from walnut groves in Romagna region, Italy

Seventy-seven Xanthomonas arboricola pv. juglandis isolates, originating from a small region (Romagna, Italy) within 4 years, were phenotypically typed, in order to study their population features. Assessment of phenotypes resulted in the identification of three different groups of morphotypes, in the assessment of different virulence on walnut fruitlets, and in the evidence that all isolates were able to grow on Mannitol-glutamate-yeast agar containing 50 ppm of copper sulphate. Moreover, several isolates showed to be highly copper resistant in vitro, up to 500 ppm. Forty-one isolates, selected considering year/origin of plant material and phenotypic features, were molecularly studied by rep-PCR fingerprinting using BOXA1R primer. These strains showed a clear intra-pathovar variation by the presence of eight different haplotypes. Twenty isolates, representative of different BOX profile, were studied by means of variable number of tandem repeats (VNTR) on the locus TR5b. Such analysis highlighted five different sequence types. Eight polymorphic strains on this flanking region isolated in between 2007 and 20 and one isolated in 2010 were subject to multilocus sequence analysis (MLSA) using atpD, dnaK, efP, fyuA, glnA, gyrB, and rpoD housekeeping genes. A diversity level in Italian isolates was highlighted in the same range as in reference strains from a worldwide origin. Finally, the gene cluster copLAB presence was confirmed for all isolates. In this study, the high phenotype and genotype variability inside Xanthomonas arboricola pv. juglandis, was explained by the different origin of the propagation material. Information provided in this study on an Italian Xanthomonas arboricola pv. juglandis collection allows a better understanding of the walnut bacterial blight epidemiology.