Xanthomonas arboricola pv. juglandis (Xaj) is the etiological agent of walnut (Juglans regia L.) bacterial blight (WBB), and has been associated to other walnut emerging diseases, namely brown apical necrosis (BAN) and vertical oozing canker (VOC), altogether severely affecting the walnut production worldwide. Despite the research efforts carried out to disclose Xaj genetic diversity, reliable molecular methods for rapid identification of Xaj isolates and culture-independent detection of Xaj in infected plant samples are still missing. In this work, we propose nine novel specific DNA markers (XAJ1 to XAJ9) selected by dedicated in silico approaches to identify Xaj isolates and detect these bacteria in infected plant material. To confirm the efficacy and specificity of these markers, dot blot hybridization was carried out across a large set of xanthomonads. This analysis, which confirmed the pathovar specificity of these markers, allowed to identify four broad-range markers (XAJ1, XAJ4, XAJ6, and XAJ8) and five narrow-range markers (XAJ2, XAJ3, XAJ5, XAJ7, and XAJ9), originating 12 hybridization patterns (HP1 to HP12). No evident relatedness was observed between these hybridization patterns and the geographic origin from which the isolates were obtained. Interestingly, four isolates that clustered together according the gyrB phylogenetic analysis (CPBF 1507, 1508, 1514, and 1522) presented the same hybridization pattern (HP11), suggesting that these nine markers might be informative to rapidly discriminate and identify different Xaj lineages. Taking into account that a culture-independent detection of Xaj in plant material has never been described, a multiplex PCR was optimized using markers XAJ1, XAJ6, and XAJ8. This triplex PCR, besides confirming the dot blot data for each of the 52 Xaj, was able to detect Xaj in field infected walnut leaves and fruits. Altogether, these nine Xaj-specific markers allow conciliating the specificity of DNA-detection assays with typing resolution, contributing to rapid detection and identification of potential emergent and acutely virulent Xaj genotypes, infer their distribution, disclose the presence of this phytopathogen on potential alternative host species and improve phytosanitary control.

Xanthomonas campestris pathovars are widely distributed throughout the globe and have a broad host range, causing severe economic losses in the food and ornamental crucifers markets. Using an approach based on multilocus sequence typing, phylogenetic diversity and population structure of a set of 75 Portuguese and other Xanthomonas campestris isolates from several cruciferous hosts were assessed. Although this population displayed a major clonal structure, neighbour‐net phylogenetic analysis highlighted the presence of recombinational events that may have driven the ecological specialization of X. campestris with different host ranges within the Brassicaceae family. A high level of genetic diversity within and among X. campestris pathovars was also revealed, through the establishment of 46 sequence types (STs). This approach provided a snapshot of the global X. campestris population structure in cruciferous host plants, correlating the existing pathovars with three distinct genetic lineages. Phylogenetic relationships between the founder genotype and remaining isolates that constitute the X. campestris pv. campestris population were further clarified using goeBURST algorithm. Identification of an intermediate link between X. campestris pv. campestris and X. campestris pv. raphani provided new insights into the mechanisms driving the differentiation of both pathovars. Wide geographic distribution of allelic variants suggests that evolution of X. campestris as a seedborne pathogen was not shaped by natural barriers. However, as Portuguese isolates encompass 26 unique STs and this country is an important centre of domestication of Brassica oleracea crops, a strong case is made for its role as a diversification reservoir, most probably through host–pathogen coevolution.

BackgroundBacterial spot-causing xanthomonads (BSX) are quarantine phytopathogenic bacteria responsible for heavy losses in tomato and pepper production. Despite the research on improved plant spraying methods and resistant cultivars, the use of healthy plant material is still considered as the most effective bacterial spot control measure. Therefore, rapid and efficient detection methods are crucial for an early detection of these phytopathogens.MethodologyIn this work, we selected and validated novel DNA markers for reliable detection of the BSX Xanthomonas euvesicatoria (Xeu). Xeu-specific DNA regions were selected using two online applications, CUPID and Insignia. Furthermore, to facilitate the selection of putative DNA markers, a customized C program was designed to retrieve the regions outputted by both databases. The in silico validation was further extended in order to provide an insight on the origin of these Xeu-specific regions by assessing chromosomal location, GC content, codon usage and synteny analyses. Primer-pairs were designed for amplification of those regions and the PCR validation assays showed that most primers allowed for positive amplification with different Xeu strains. The obtained amplicons were labeled and used as probes in dot blot assays, which allowed testing the probes against a collection of 12 non-BSX Xanthomonas and 23 other phytopathogenic bacteria. These assays confirmed the specificity of the selected DNA markers. Finally, we designed and tested a duplex PCR assay and an inverted dot blot platform for culture-independent detection of Xeu in infected plants.SignificanceThis study details a selection strategy able to provide a large number of Xeu-specific DNA markers. As demonstrated, the selected markers can detect Xeu in infected plants both by PCR and by hybridization-based assays coupled with automatic data analysis. Furthermore, this work is a contribution to implement more efficient DNA-based methods of bacterial diagnostics.

20Xanthomonas arboricola pv. juglandis (Xaj) is the etiological agent of walnut diseases 21 affecting leaves, fruits, branches and trunks. Although this phytopathogen is widely 22 spread in walnut producing regions and has a considerable genetic diversity, there is 23 still a poor understanding of its epidemic behaviour. To shed some light on the 24 epidemiology of these bacteria, 131 Xanthomonas isolates obtained from 64 walnut 25 trees were included in this study considering epidemiological metadata such as year of 26 isolation, bioclimatic regions, walnut cultivars, production regimes, host walnut 27 specimen and plant organs. Genetic diversity was assessed by multilocus sequence 28 analysis (MLSA) and dot blot hybridization patterns obtained with nine Xaj-specific 29 DNA markers (XAJ1 -XAJ9). The results showed that Xanthomonas isolates grouped 30 in ten distinct MLSA clusters and in 18 hybridization patterns (HP). The majority of 31 isolates (112 out of 131) were closely related with X. arboricola strains of pathovar 32 juglandis as revealed by MLSA (clusters I to VI) and hybridize with more than five Xaj-33 specific markers. Nineteen isolates clustered in four MLSA groups (clusters VII to X) 34 which do not include Xaj strains, and hybridize to less than five markers. Taking this 35 data together, was possible to distinguish 17 lineages of Xaj, three lineages of X. 36 arboricola and 11 lineages of Xanthomonas sp. Some Xaj lineages appeared to be 37 widely distributed and prevalent across the different bioclimatic regions and apparently 38 not constrained by the other features considered. Assessment of type III effector genes 39 and pathogenicity tests revealed that representative lineages of MLSA clusters VII to X 40 were nonpathogenic on walnut, with exception for strain CPBF 424, making this 41 bacterium particularly appealing to address Xanthomonas pathoadaptations to walnut. 42 43 3 IMPORTANCE Xanthomonas arboricola pv. juglandis is one of the most serious 44threats of walnut trees. New disease epidemics caused by this phytopathogen has been a 45 big concern causing high economic losses on walnut production worldwide. Using a 46 comprehensive sampling methodology to disclose the diversity of walnut infective 47 Xanthomonas, we were able to identify a genetic diversity higher than previously 48 reported and generally independent of bioclimatic regions and the other epidemiological 49 features studied. Furthermore, co-colonization of the same plant sample by distinct 50 Xanthomonas strains were frequent and suggested a sympatric lifestyle. The extensive 51 sampling carried out resulted in a set of non-arboricola Xanthomonas sp. strains, 52 including a pathogenic strain, therefore diverging from the nonpathogenic phenotype 53 that have been associated to these atypical strains, generally considered to be 54 commensal. This new strain might be particularly informative to elucidate novel 55 pathogenicity traits and unveil pathogenesis evolution within walnut infective 56 xanthomonads. Beyond extending ...