Genotyping and epidemiological metadata provides new insights into population structure of Xanthomonas isolated from walnut trees

ABSTRACT Xanthomonas arboricola pv. juglandis ( Xaj ) is the etiological agent of walnut diseases affecting leaves, fruits, branches and trunks. Although this phytopathogen is widely spread in walnut producing regions and has a considerable genetic diversity, there is still a poor understanding of its epidemic behaviour. To shed some light on the epidemiology of these bacteria, 131 Xanthomonas isolates obtained from 64 walnut trees were included in this study considering epidemiological metadata such as year of isolation, bioclimatic regions, walnut cultivars, production regimes, host walnut specimen and plant organs. Genetic diversity was assessed by multilocus sequence analysis (MLSA) and dot blot hybridization patterns obtained with nine Xaj -specific DNA markers (XAJ1 – XAJ9). The results showed that Xanthomonas isolates grouped in ten distinct MLSA clusters and in 18 hybridization patterns (HP). The majority of isolates (112 out of 131) were closely related with X. arboricola strains of pathovar juglandis as revealed by MLSA (clusters I to VI) and hybridize with more than five Xaj -specific markers. Nineteen isolates clustered in four MLSA groups (clusters VII to X) which do not include Xaj strains, and hybridize to less than five markers. Taking this data together, was possible to distinguish 17 lineages of Xaj , three lineages of X. arboricola and 11 lineages of Xanthomonas sp. Some Xaj lineages appeared to be widely distributed and prevalent across the different bioclimatic regions and apparently not constrained by the other features considered. Assessment of type III effector genes and pathogenicity tests revealed that representative lineages of MLSA clusters VII to X were nonpathogenic on walnut, with exception for strain CPBF 424, making this bacterium particularly appealing to address Xanthomonas pathoadaptations to walnut. IMPORTANCE Xanthomonas arboricola pv. juglandis is one of the most serious threats of walnut trees. New disease epidemics caused by this phytopathogen has been a big concern causing high economic losses on walnut production worldwide. Using a comprehensive sampling methodology to disclose the diversity of walnut infective Xanthomonas , we were able to identify a genetic diversity higher than previously reported and generally independent of bioclimatic regions and the other epidemiological features studied. Furthermore, co-colonization of the same plant sample by distinct Xanthomonas strains were frequent and suggested a sympatric lifestyle. The extensive sampling carried out resulted in a set of non- arboricola Xanthomonas sp. strains, including a pathogenic strain, therefore diverging from the nonpathogenic phenotype that have been associated to these atypical strains, generally considered to be commensal. This new strain might be particularly informative to elucidate novel pathogenicity traits and unveil pathogenesis evolution within walnut infective xanthomonads. Beyond extending the present knowledge about walnut infective xanthomonads, this study might contribute to provide a methodological framework for phytopathogen epidemiological studies, still largely disregarded.