Detection of grape phylloxera on grapevine roots with diagnostic polymerase chain reaction methods targeted to the internal transcribed space region 2 nuclear gene

Background and AimsPhylloxera (Daktulosphaira vitifoliaeFitch) is one of the most economically important, obligate parasitic insect pests of the global grape industry. It is vital to establish a rapid, accurate and efficient method to detect phylloxera on grapevine roots and thereby minimise its spread. The objective of this research is to determine if diagnostic polymerase chain reaction (PCR) methods targeted to the nuclear internal transcribed space region 2 (ITS2) genes can detect phylloxera on grapevine roots. Methods and ResultsFirst, ITS2 gene region PCR primers specifically designed for amplification of grape phylloxera were trialled in standard PCR amplifications and with real-time quantitative PCR (RT-qPCR) technology to test the validity and sensitivity of the primers. The primers amplified the ITS2 region gene of Chinese phylloxera lineages, and no amplification products were observed when the primers were applied to DNA extracted from root-knot nematode and grapevine roots. Phylloxera DNA at a concentration of 1pg/L and as few as five first instar phylloxera nymphs per 0.1g of root bark were detected with these primers, which indicated that the method was highly sensitive to the presence of phylloxera. Finally, the sensitivity of the diagnostic PCR analysis and of microscopy for detection of phylloxera on roots was compared; the PCR methods were more sensitive. ConclusionsThe method of RT-qPCR targeted to the ITS2 gene region has the potential to routinely detect grape phylloxera on infested grapevine roots. Significance of the StudyThe diagnostic PCR methodology can be applied to phylloxera quarantine of grapevine root material and help prevent the spread of phylloxera through grapevine transfer.