Pan-Genomic Analysis Permits Differentiation of Virulent and Non-virulent Strains of Xanthomonas arboricola That Cohabit Prunus spp. and Elucidate Bacterial Virulence Factors

Abstract

Xanthomonas arboricola is a plant-associated bacterial species that causes diseases on several plant hosts. One of the most virulent pathovars within this species is X. arboricola pv. pruni (Xap), the causal agent of bacterial spot disease of stone fruit trees and almond. Recently, a non-virulent Xap-look-a-like strain isolated from Prunus was characterized and its genome compared to pathogenic strains of Xap, revealing differences in the profile of virulence factors, such as the genes related to the type III secretion system (T3SS) and type III effectors (T3Es). The existence of this atypical strain arouses several questions associated with the abundance, the pathogenicity, and the evolutionary context of X. arboricola on Prunus hosts. After an initial characterization of a collection of Xanthomonas strains isolated from Prunus bacterial spot outbreaks in Spain during the past decade, six Xap-look-a-like strains, that did not clustered with the pathogenic strains of Xap according to a multi locus sequence analysis, were identified. Pathogenicity of these strains was analyzed and the genome sequences of two Xap-look-a-like strains, CITA 14 and CITA 124, non-virulent to Prunus spp., were obtained and compared to those available genomes of X. arboricola associated with this host plant. Differences were found among the genomes of the virulent and the Prunus non-virulent strains in several characters related to the pathogenesis process. Additionally, a pan-genomic analysis that included the available genomes of X. arboricola, revealed that the atypical strains associated with Prunus were related to a group of non-virulent or low virulent strains isolated from a wide host range. The repertoire of the genes related to T3SS and T3Es varied among the strains of this cluster and those strains related to the most virulent pathovars of the species, corylina, juglandis, and pruni. This variability provides information about the potential evolutionary process associated to the acquisition of pathogenicity and host specificity in X. arboricola. Finally, based in the genomic differences observed between the virulent and the non-virulent strains isolated from Prunus, a sensitive and specific real-time PCR protocol was designed to detect and identify Xap strains. This method avoids miss-identifications due to atypical strains of X. arboricola that can cohabit Prunus.

Lateral flow immunoassay for on-site detection of Xanthomonas arboricola pv. pruni in symptomatic field samples

“…arboricola pv. pruni in symptomatic samples, in which bacterial populations are typically higher than 10 6 CFU ml -1 [9, 34, 35]. This limit is similar to that reported with other lateral flow devices designed for the detection of bacterial pathogens, such as X .…”

Xanthomonas arboricola pv. pruni, causal agent of bacterial spot of stone fruits and almond: its genomic and phenotypic characteristics in the X. arboricola species context

“…Multilocus sequence analyses (MLSAs), based on the genes dnaK , fyuA , gyrB and rpoD (Garita‐Cambronero et al ., ; Young et al ., ), or on the partial sequences of atpD , efp and glnA (Boudon et al ., ; Fischer‐Le Saux et al ., ), have been useful in differentiating the pathovar pruni from the other pathovars of the species, confirming its phylogenetic proximity to the pathovars corylina and juglandis . These studies have concluded that these pathovars form monophyletic groups with a low genetic diversity amongst their strains, and must be considered as clonal complexes.…”

“…Moreover, new studies based on whole‐genome analysis, using either concatenated sequences of the core group of genes of X. arboricola (Cesbron et al ., ; Garita‐Cambronero et al ., ) or pangenomic analysis of at least 7074 protein coding sequences, established pathovar pruni as a monophyletic group; however, in contrast with the observations of MLSA, slight differences were observed in strains from different geographical origins. Despite this, the average nucleotide identity amongst all members of this pathovar was over 99% (Garita‐Cambronero et al ., ) (Fig. ; Table ).…”

“…Xanthomonas arboricola encompasses bacterial strains that cannot be assigned to any of the existing pathovars, although sometimes were initially wrongly assigned to an incorrect pathovar (Cesbron et al ., ; Garita‐Cambronero et al ., ). These bacteria may provide a source for the exchange of genetic material and may be involved in the evolution of pathogenic lineages (Jacques et al ., ).…”

Loop-Mediated Isothermal Amplification for the Detection of Xanthomonas arboricola pv. pruni in Peaches

“…Currently employed methods for the identification of X. arboricola pv. pruni are conventional PCR (Pagani, 2005), duplex PCR (Pothier et al, 2011), loop-mediated isothermal amplification (LAMP) detection (Bühlmann et al, 2013), lateral flow immunoassays (López-Soriano et al, 2017), and real-time PCR amplification (Garita-Cambronero et al, 2017; Palacio-Bielsa et al, 2011). Although efficient control of X. arboricola pv.…”