Developing and Testing a Diagnostic Probe for Grape Phylloxera Applicable to Soil Samples

Abstract

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.

Direct measurement of roots in soil for single and mixed species using a quantitative DNA-based method

“…Using procedures that are widely used to measure pathogen inoculum levels in soil (Herbert et al 2008;Ophel-Keller et al 2008), DNA extracted directly from soil can be assessed quantitatively for specific plant species using qPCR with species-specific Taqman® probes designed across the internal transcribed spacer region (ITS) of ribosomal RNA genes. In the original report, DNA was extracted from soil samples up to 0.5 kg, which enables more representative sampling of roots in the field and removes the need to mill large root-soil samples before sub-sampling.…”

Biology and management of the redheaded pasture cockchaferAdoryphorus couloni(Burmeister) (Scarabaeidae: Dynastinae) in Australia: a review of current knowledge

“…DNA probes have been developed for a range of soil pathogens and rootfeeding insects (Herbert et al 2008), and potentially could be adapted as a field-based method for early detection of A. couloni and to enable it to be distinguished from other scarabs present in the soil. A DNA approach to surveillance and detection of A. couloni could result in improved sensitivity and allow more rapid implementation of management options.…”

“…There are a range of evolving detection methods for other root-feeding invertebrate pests, including spectrochemical fingerprinting of vegetation and use of soil-based DNA probes (Apan et al 2005;Renzullo et al 2006;Tucker et al 2007;Herbert et al 2008) that could potentially be adapted for A. couloni. However, there is also a need to develop biophysical descriptors that directly indicate the potential attractiveness of given soil types and susceptibility of pasture species to cockchafer infestation.…”

Accounting for spatially heterogeneous conditions in local‐scale surveillance strategies: case study of the biosecurity insect pest, grape phylloxera (Daktulosphaira vitifoliae (Fitch))

“…Vineyard managers are already encouraged to collect EM38 data for other aspects of grapevine production, so there should be little increased cost . Several new methods for early phylloxera detection are currently being developed and trialled, including chemical and pigment fingerprinting of metabolites as biomarkers in leaves, a phylloxera‐specific DNA probe, emergence traps and, more recently, the use of unmanned aerial vehicles and sniffer dogs . The efficiency of all these new methods has the potential to be improved by spatially targeted surveillance designs like those considered in this study.…”