Genomics informed design of a suite of real-time PCR assays for the specific detection of each Xylella fastidiosa subspecies

Abstract

Aims: Existing methods for the identification of the subspecies of Xylella fastidiosa are time-consuming which can lead to delays in diagnosis and the associated plant health response to outbreaks and interceptions. Methods and Results: Diagnostic markers were identified using a comparative genomics approach to allow fine differentiation of the very closely related subspecies. Five qPCR assays were designed to allow specific detection of X. fastidiosa subsp. fastidiosa, X. fastidiosa subsp. multiplex, X. fastidiosa subsp. pauca, X. fastidiosa subsp. morus and X. fastidiosa subsp. sandyi. All assays were validated according to the European and Mediterranean Plant Protection Organisation (EPPO) standard PM7/98(2). Conclusions: All of the assays were shown to be specific to the target subspecies and all the assays could be used to detect femtogram quantities of X. fastidiosa DNA. Significance and Impact of the Study: At present, diagnosing the subspecies of X. fastidiosa requires multiple conventional PCR assays (although only available for three of the five subspecies) or multi-locus sequence typing which takes several days. By comparison, the new assays provide a substantial reduction in the turnaround time for direct identification to the subspecies level in as little as 75 min.