Protecting grapevines in stress environments: Early detection of grape phylloxera infestation through identification of chemical biomarkers
Creators: name: Benheim, Devin A.
Publisher: name: La Trobe
Year: 2023

description: Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Molecular Sciences, Faculty of Science, Technology &amp; Engineering, La Trobe University, Bundoora.<br><br>Grapevine phylloxera (Daktulosphaira vitifoliae Fitch) is a destructive, root feeding insect pests that is difficult to detect and is a major biosecurity pest which causes economic damage to ungrafted European grapevines Vitis vinifera L. Early detection of this pest would enable rapid implementation of quarantine procedures and rootstock replanting and is vital to ensure the sustainability and profitability of the viticulture industry both in Australia and internationally. This dissertation explores an untargeted metabolomics approach to early detection of grape phylloxera by studying biochemical changes in the metabolome of leaf material of V. vinifera in response to infestation. LC-MS and NMR analytical platforms were applied to leaf samples acquired under both glasshouse and field conditions. Metabolic profiling using LC-MS was conducted on V. vinifera cv. Shiraz independently infested with two endemic high-virulence genetic strains of grape phylloxera, G1 and G4, and uninfested grapevines grown under glasshouse conditions. Statistical discrimination between treatments (infested vs. uninfested) was observed from 4 weeks post-infestation. Several unidentified metabolites were detected which were differentially regulated in infested grapevines. No discrimination was observed between G1 and G4-infested grapevines, tentatively suggesting that a grape phylloxera-induced stress response in grapevines may be independent of grape phylloxera genetic strain. These results also indicated a measurable metabolic response to phylloxera infestation in relation to the relative abundance of active life stages of grape phylloxera on the roots at the time of sampling. A second glasshouse trial compared the metabolic profile of G1-infested V. vinifera cv. Chardonnay with nutrient stressed, water stressed and unstressed grapevines over a 14-week period using LC-MS. Statistical discrimination between grape phylloxera-infested grapevines, uninfested controls and nutrient-stressed grapevines was achieved at 14 weeks post-infestation, with partial separation of phylloxera-infested from water stressed grapevines. Nineteen statistically significant metabolites were identified. Results principally indicated induction of both phenylpropanoid and shikimate pathways and the production of carotenoid oxidation products in response to grape phylloxera infestation. A field study of G1-infested mature V. vinifera cv. Chardonnay in a commercial vineyard over a single growing season was conducted and analysed using NMR and LC-MS techniques. Statistical analysis of LC-MS data showed separation of phylloxera-infested from uninfested grapevines at both the peak aboveground first instar abundance and two months prior. Analysis confirmed induction of the phenylpropanoid pathway in response to grape phylloxera infestation. Statistical discrimination of treatments was observed in NMR data of aqueous extracts of leaf material, with differential regulation of carbohydrates, amino acids and phenylpropanoid products. Overall, quercetin was identified as the main biomarker of grape phylloxera infestation as quercetincontaining flavonoid metabolites were observed up-regulated by LC-MS and NMR in infested grapevines under both glasshouse and field conditions. Further validation is recommended in order to develop a robust early detection system for grapevine phylloxera using biochemical markers.