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

Abstract

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.

DNA Markers for Detection and Genotyping of Xanthomonas euroxanthea

“…It is important to investigate the distribution and role played by X. euroxanthea and the closely related X. arboricola within the plant hosts that they frequently co-colonize [12]. Therefore, it is essential to develop a reliable and efficient method for the accurate detection and identification of X. euroxanthea strains and its differentiation from X. arboricola [12].…”

“…In fact, multiplex-PCR and dot-blot hybridization assays showed that three X. arboricola pv. juglandis specific DNA markers (XAJ1, XAJ6 and XAJ8) were absent from X. euroxanthea strains CPBF 367, CPBF 424 T and CPBF 426, which formed a distinct MLSA cluster [12,14].…”

“…More strikingly is that X. arboricola strains were also isolated from all the mentioned plant species, suggesting that both X. euroxanthea and X. arboricola share the same host plants, including the same plant specimen, which raises questions regarding co-colonization and niche-specific adaptations [7,8,12]. Still, taking into consideration the recent taxonomic refinement of X. arboricola species and the unearthing of six new X. euroxanthea strains (2949, 2955, 2957, 2974, 3640 and F2) [11], it is foreseeable the identification of additional X. euroxanthea isolates from plant species that have passed unnoticed so far.…”

“…A robust method to detect X. euroxanthea while undoubtedly distinguishing it from X. arboricola has not been described so far. Furthermore, while several genotyping techniques have been used to assess the diversity of phytosanitary regulated Xanthomonas, as recently reviewed [13], the data currently available regarding genotyping of X. euroxanthea is scarce and limited to MLSA studies performed on Xanthomonas isolated from walnut trees [12].…”

Trends in Molecular Diagnosis and Diversity Studies for Phytosanitary Regulated Xanthomonas

“…These nonpathogenic strains, also sometimes referred to as look-alikes of Xanthomonas pathogens, have been isolated from a wide diversity of organs and plant species, such as seeds, buds, leaves from legumes, cereals, and fruit trees [23][24][25][26][27]. Non-pathogenic strains are interspersed with pathogenic ones at an infraspecific level, especially within the X. arboricola and X. euroxanthea species [11,[28][29][30], which implies that highly specific detection tools are required to avoid confusion that could have harmful socio-economic consequences. The coexistence of pathogenic and non-pathogenic Xanthomonas strains in the same host plant largely increased the estimated diversity in the genus [6,19,24,[30][31][32], with events such as recombination and horizontal gene transfer contributing to the pathogen diversification across the different pathosystems [28,[33][34][35][36][37].…”