Developing and testing a diagnostic probe for grape phylloxera applicable to soil samples.

Grape phylloxera, Daktulosphaira vitifoliae (Fitch) (Hemiptera Phylloxeridae) is a damaging pest of grapevines (Vitis spp.) around the world, and the management of this pest requires early detection of infestations. Here, we describe the development and validation of a sensitive DNA test for grape phylloxera that can be applied to soil. Species-specific primers were developed for grape phylloxera in the internal transcribed space region 2, and their specificity was confirmed after thorough screening by using a wide range of vineyard organisms and aphid genera. Preliminary testing of the detection limits of the grape phylloxera-specific primers was conducted using field-sourced soil types spiked with a known number of grape phylloxera. The assay was converted to a real-time polymerase chain reaction format (TaqMan MGB). This assay, in combination with DNA extraction from soil, can detect phylloxera crawlers added to soil. The assay was evaluated in the field at a recently detected grape phylloxera infestation site from the Yarra Valley in Victoria, Australia. The DNA assay proved to be substantially more sensitive than a standard ground survey for detecting grape phylloxera presence on vine roots in the infested vineyard. Moreover, unlike the ground survey, the assay provided quantitative information on grape phylloxera infestations, because grape phylloxera DNA concentrations in samples from vines closely matched the numbers of grape phylloxera crawlers collected with emergence traps placed at the base of vines. Unlike other detection techniques, the method can be applied at any time of the year, and it can be potentially modified to provide specific information on the virulence levels of the particular grape phylloxera genotypes responsible for any new infestations.