Detecting Xylella fastidiosa, a grape bacteriosis agent (Pierce disease) applicability evaluation of molecular methods used in international and domestic practice

Abstract

The grape bacteriosis agent (Pierce disease), namely, Xylella fastidiosa, represents a quarantine disease that is absent on the territory of the Eurasian Economic Union. For its efficient detection, the methods should be harmonised with international and local protocols; the import substitution of the recommended reagents should be considered. Five PCR tests were optimised using domestically produced compounds, and their main efficiency criteria, as per the EPPO standard 7/098 (4), were determined during studies carried out at the FSBI VNIIKR. In addition, a commercial kit, “Phytoscreen” (LLC “Syntol”, Russia), was validated. The results of the applicability assessment revealed a high sensitivity of real-time PCR (102 CFU/ml) following Harper et al. (2010), Li et al. (2013) and Ouyang et al. (2013), high sensitivity (no false positive or non-specific reactions were detected) and 100 % precision in experiment and site precision. These tests will be included in the guidelines for detecting X. fastidiosa as a screening test. It should be noted that the sensitivity of PCR, as per Li et al. (2013), using the 5x MasCFE TaqMIX -2025 reaction mixture (“Dialat Ltd.”, Russia), was improved relative to the data reported in TPS (2018). In addition, real-time PCR based on Francis et al. (2006) and classical PCR, as per Minsavage et al. (1994), exhibit an analytical sensitivity of 100 % at target concentrations of 103 and 104 CFU/ml, respectively. All real-time PCR showed selectivity for lavender extract, excluding the commercial Phytoscreen kit, which was characterised by inhibition of amplification.