Improving reliability of PCR diagnostics for Xylophilus ampelinus by metagenome-informed circumscription of the target taxon

Xylophilus ampelinus is a xylematic bacterium causing bacterial blight of grapevine, a disease regarded as a potential threat for viticulture in several countries. Currently, PCR detection is pivotal in diagnostic protocols due to the bacterium's infrequent occurrence in the field and the technical advantages of PCR. Recent metagenomic studies have unveiled diversity in its taxonomic domain, unknown when the most widely used assays for the detection of X. ampelinus infections were developed. In particular, PCR assays relying on highly conserved sequence regions, such as those surrounding ribosomal RNA genes, may be substituted with more specific PCR assays. In this study, we first investigated the diversity of detectable grapevine endophytes related to but different from X. ampelinus and delineated the genotaxonomic boundaries of the species in relation to the known (meta)genomes of closely related bacteria. Then, by exploiting the wealth of genomes now available for bacteria classified in the Burkholderiales, we devised several sets of primers targeting only X. ampelinus and its closest relatives. These primers were employed to (i) genotaxonomically circumscribe the grapevine endophyte species related to but distinguishable from X. ampelinus and (ii) develop a robust multiplex PCR assay expected to be specific for the species X. ampelinus based on in vitro and in silico evidence. The adoption of the multiplex PCR assay presented here is expected to reduce the risk of false positives in the diagnosis of bacterial blight of grapevine.