Development and validation of X-ComEC qPCR, a novel assay for accurate universal detection of both Xylella fastidiosa and Xylella taiwanensis

Abstract Xylella fastidiosa is a devastating plant pathogenic bacteria known for its broad host range, in contrast to the related species Xylella taiwanensis , which is only known to cause disease in Asian pears. Despite the potential threats they pose to Australian agriculture, diagnostic assays capable of detecting both Xylella species are scarce. Bridging this critical gap, this study presents the development of the X- ComEC qPCR assay that targets a genus-specific DNA sequence, enabling accurate generic detection of all Xylella species. Benchmarking this novel qPCR assay against other published Xylella qPCR assays demonstrated its superior performance. The X- ComEC qPCR assay stands out as the only assay that can accurately detect both X. fastidiosa and X. taiwanensis without cross-reactivity with related bacteria. We have also carried out a comprehensive inter-laboratory test performance study, which demonstrated that the X- ComEC qPCR and the qPCR described by Harper et al. (Development of LAMP and real-time PCR methods for the rapid detection of Xylella fastidiosa for quarantine and field applications; erratum 2013) are highly robust and ready to use in Australia. Combining these two assays into a duplex qPCR enables simultaneous detection and species-level identification of X. fastidiosa and X. taiwanensis. The findings of this study have been incorporated into the Australian National Diagnostic Protocol for Xylella detection, arming diagnostic laboratories with critical knowledge to combat these globally significant pathogens. Overall, the collaborative and systematic approach employed in this study provides a model for developing and validating assays for all plant pathogens.