Comprehensive diversity assessment of walnut-associated xanthomonads reveal the occurrence of distinct Xanthomonas arboricola lineages and of a new species (Xanthomonas euroxanthea) within the same tree

Xanthomonas arboricola pv. juglandis (Xaj) is the aetiological agent of walnut diseases causing economic losses on walnut production worldwide. This phytopathogen is spread around the world where walnuts are produced and has a considerable genetic diversity. Using a comprehensive sampling methodology, focusing on factors that could influence the diversity of walnut-colonizing Xaj in Portugal, this work provides new insights on xanthomonad populations on walnut. Genetic diversity was assessed by multilocus sequence analysis (MLSA) and dot blot hybridization patterns on 131 Xanthomonas isolates obtained from 64 walnut trees considering epidemiological metadata such as year of isolation, distinct bioclimatic regions, production regimes, and host-related features. The results showed that the majority of isolates were split into 17 lineages of Xaj, while the other isolates clustered in four MLSA groups that did not include Xaj strains. These four groups were represented by three lineages of X. arboricola, and 11 lineages of Xanthomonas spp., including strains assigned to the recently proposed new species Xanthomonas euroxanthea. Furthermore, distinct Xaj, X. arboricola, and Xanthomonas spp. were isolated from the same walnut tree, suggesting possible genetic admixture within the same host. Phylogenetic analysis through geoBurst revealed the high diversity of these Xanthomonas spp. populations. Assessment of type III effector genes gave the indication that some Xanthomonas spp. strains were nonpathogenic on walnut, with the exception for X. euroxanthea CPBF 424. Altogether, these findings add to the thorough characterization of walnut-associated xanthomonads in Portugal, providing a comprehensive snapshot of the current diversity that could contribute to risk assessment analysis and improve phytosanitary control.