Multiple DNA Markers for Identification of Xanthomonas arboricola pv. juglandis Isolates and its Direct Detection in Plant Samples

Abstract

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.

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

“…Particularly, isolates from MLSA cluster I, which included 29 isolates, were divided into four sub-groups when hybridization patterns were considered. The genotyping appraisal of a considerable number of isolates confirm the utility of these markers for the identification of different Xaj lineages, as suggested previously (27). Furthermore, these molecular markers have shown to be useful as genetic tools on the characterization of all xanthomonads population found on walnut trees.…”

“…mainly isolated from asymptomatic material (bud and catkin samples) showing the highest genetic variation. To further elucidate the genetic diversity of all 131 isolates, dot blot hybridization assays using Xaj -specific DNA markers XAJ1 to XAJ9 (27), allowed to identify eighteen distinct hybridization patterns (Fig. 2), and distinguish isolates within the same MLSA cluster (Fig.…”

“…Sample preparation for bacterial isolation was carried out differently for symptomatic and asymptomatic material: i) for symptomatic leaves, fruits and branches, plant tissues adjacent to necrotic areas were first excised using a sterile scalpel; ii) for asymptomatic buds and catkins, either single terminal buds, axillary bud groups or catkins groups of the same branch were excised also using a sterile scalpel according sampling procedures previously described (7, 8). Bacterial isolation was carried out as procedure detailed in Fernandes et al (27). One to three isolates were selected per sample and stored at −80°C at the Portuguese Collection of Phytopathogenic Bacteria (CPBF - Colecção Portuguesa de Bactérias Fitopatogénicas, Oeiras, Portugal).…”

“…Dot blot assays were performed as described in a previous study using nine Xaj specific DNA markers (XAJ1 to XAJ9) (27). Briefly, 100 ng of bacterial DNA were bound to a nylon hybridization transfer membrane (Roche Diagnostics GmbH, Basel, Switzerland) using a Bio-Dot microfiltration unit (Bio-Rad, Hercules, CA).…”

“…This study aimed to characterize the genetic diversity of xanthomonads isolates obtained from walnut trees over a three-year period, across distinct bioclimatic regions and taking into account different walnut cultivars, production regimes (orchards vs. isolated trees), host walnut tree and plant organs. Multilocus sequence analysis (MLSA) and dot blot hybridization patterns of nine Xaj -specific markers (27), were used to determine the genetic diversity of isolates. These data were complemented by monitoring the presence/absence of four informative type III effector genes (T3E) observed to differentiate non-pathogenic and pathogenic Xaj clusters (20) and by pathogenicity tests of representative lineages.…”