This project conducted research directed at developing tools, technologies, and practices to improve the integrated pest management (IPM) of phylloxera in Australia. The three-year program of research has delivered outputs that will be of significant benefit to growers, industry, and national plant biosecurity. These include new tools for the in-field detection of phylloxera, robust screening assays to improve rootstock selection and safeguard Australia against the spread of virulent phylloxera strains , scientifically validated disinfestation procedures, new knowledge in phylloxera temperature tolerance, and a review of phylloxera biocontrol.
This project aimed to develop tools, technologies and practices to improve the integrated pest management (IPM) of phylloxera in Australia. Research fell into five broad themes that are seen as being crucial to preventing the spread of this pest: (i) enhancing early detection systems, (ii) providing region- and genotype-specific rootstock recommendations, (iii) developing improved quarantine protocols, (iv) addressing key knowledge gaps on the biology of phylloxera endemic strains, and (v) re-evaluating biocontrol options. The three-year program of research has delivered outputs that will be of significant benefit to growers, industry and national plant biosecurity.
New and improved tools for early detection
The potential for using electronic noses to detect phylloxera. A commercially available electronic nose (e- nose) was evaluated for its ability to identify phylloxera-infested vine roots across different rootstocks, during early and late stages of infestation. The device failed to detect differences in leaf odours of healthy and phylloxera-infested vines. For root odours, there was evidence that the e-nose might be capable of identifying infested roots, but the level of accuracy was not acceptable for a detection tool and there were major practical limitations in the use of the e-nose (sample preparation, testing environments, sensors consistency). To explore the potential for developing future e-nose with finely tuned sensors, gas chromatography-mass spectrometry (GC-MS) analysis was conducted on leaves and excised roots of infested and phylloxera-free plants. GC-MS analysis revealed small variations in the concentrations of multiple root volatiles induced by the presence of phylloxera, which differed according to rootstock and phylloxera strain. However, no “key volatiles” indicative of the presence of phylloxera were identified, and results suggest root volatiles are unlikely to be reliably used to predict the infestation status of vines. The volatiles identified might, however, provide insight into the biochemical basis of rootstock resistance.
An industry perspective on sniffer dogs. To better understand the views of vignerons, biosecurity officers and researchers regarding the use of sniffer dogs in phylloxera detection, an online survey in which participants were asked eight multiple choice questions addressing practicality, feasibility, priority, and costs, was conducted. Out of 54 respondents, only 29% thought that sniffer dogs would likely be adopted for phylloxera detection, and 73% of respondents felt there were biosecurity concerns in using sniffer dogs. The outcome of this study triggered a stop-go milestone and led to a variation, removing further sniffer dog research from the project.
LAMP, an exciting new tool for in-field phylloxera detection and identification. LAMP is a portable molecular technology that Agriculture Victoria has recently developed for rapid and accurate diagnostics of phylloxera. The current study focused on validating and applying LAMP for in-field use. Surveillance trapping was conducted using “bucket traps” and trapped insects (including phylloxera) were processed as “crude” whole specimens, which were contaminated by plant tissue and soil. Our field-optimised methods clearly demonstrated the success of LAMP for presence/absence of phylloxera. We further evaluated the LAMP surveillance approach for detecting phylloxera on root samples dug up from infested vineyards and tested in the field. Our methods successfully detected phylloxera in eighteen positive vine samples.
LAMP appears accurate and sensitive enough to accurately diagnostics to be carried out without entomological expertise, hence could save considerable training and diagnostic time and effort. Further research should focus on incorporating LAMP protocols into larger-scale visual surveys, particularly during rezoning undertakings.
Enabling strain-related rootstock selection
Uncovering susceptibility of 5C Teleki. Six genetically diverse phylloxera strains (G1, G4, G19, G20, G30, and G38) were screened for resistance against rootstock, 5C Teleki. With the exception of G1, five strains caused nodosities on vines in pots, implying that these strains will damage 5C Teleki vines. Compared with Vitis vinifera (which is highly susceptible to phylloxera), reproduction of strains G19, G20 and G30 on excised roots of 5C Teleki was, however, low. Results demonstrated that 5C Teleki is tolerant to specific strains, and insects can survive and develop to reproductive adults, implying a hidden risk of phylloxera dispersal.
Superclone G38 screening and in-field studies. Field trials in a mixed-rootstock block infested by the virulent “superclone” G38 showed rootstocks 101-14 and Schwarzmann were susceptible to this strain. Standardised laboratory and glasshouse screening assays performed using the same rootstocks also indicated susceptibility to this strain. The study was important in that it validated current screening assays in terms of predicting resistance / susceptibility of rootstocks in vineyards. That rootstocks may be becoming susceptible to G38 is particularly worrying given additional findings in this project that indicate that this genotype may be able to reproduce sexually.
Should we be worried about phylloxera genetic diversity in the King Valley? Phylloxera has been known in the King Valley since the early 1990’s, where previous studies found only a single strain (G4) present on vine roots. An updated and more detailed survey was conducted to better understand the genetic variation present. A total of 1400 phylloxera were collected from 18 blocks and 764 genotyped. The study revealed that the amount of genetic diversity present in the King Valley is considerably higher than previously thought, with many novel genotypes present. A total of 36 genotypes were identified, with 4 strains known from previous work and 32 new strains. A potential explanation for the high diversity is that sexual reproduction is occurring. If this is the case it is of considerable importance, as sexual reproduction of phylloxera could significantly increase the risk of resistance adaptation and long- range dispersal (reproductive winged forms) occurring in the field. Strong associations of strains with specific rootstocks were not found, and vines were frequently found to have more than one strain of phylloxera on their roots. Currently, there is a single PIZ in northeastern Victoria. Our new data has revealed several groups of genetically related phylloxera strains which appear geographically localized to regions within this PIZ. This evidence, together with the cluster of new (possibly sexually reproducing) variants appearing in the King Valley, might warrant a re-evaluation of the single northeastern PIZ to reduce the risk of novel strains being introduced into new areas. These results show that a larger state- wide survey is needed to update strain-specific distribution maps for other PIZs across Victoria, particularly where the genotypes are still unknown.
Are there alternative treatments for disinfestation of footwear? Household bleach is currently recommended for disinfecting footwear and small hand-held tools against phylloxera, but this product is unfavourable for use by growers. Off-the-shelf products were reviewed and scored using a matrix that looked at availability, safety and practicality. Twenty-two products were then tested in laboratory assays using first instar G4 phylloxera in 30 or 60 second immersions, with or without a follow-on water rinse. Products that were effective were further screened across genetically diverse strains (G1, G19, G20, G30 and G38). Only Dettol (5% active ingredient Chloroxylenol) achieved 100% mortality against the six phylloxera strains tested and was deemed suitable for use in a practical setting. We can therefore recommend Dettol as a suitable substitute to bleach for disinfestation of footwear and handheld tools.
Validating fermentation treatments as a disinfestation protocol. Currently, the endorsed procedure in the National Phylloxera Management Protocols for movement of grape products to complete at least three days (72 hours) of fermentation to ensure disinfestation of phylloxera (Procedure D). The efficacy of this procedure was evaluated under laboratory conditions testing five phylloxera strains in red and wine ferments, with and without the addition of yeast, for 6, 12, 24, 48 and 72 hours. 100% mortality was achieved across all phylloxera strains when subjected to yeast fermentation of grape products lasting 48 and 72 hours. However, phylloxera survived in ferments without yeast at 48 and 72 hour time points. The current protocols are therefore only valid when yeast is added to wine ferments. Fermentation protocols in the NPMP require a review in light of these findings. New protocols should be developed and evaluated to determine mortality in naturally fermented products.
Phylloxera biology – knowledge gaps
The effect of temperature on phylloxera survival. A better understanding of phylloxera strain-specific temperature tolerance and survival is important for effective quarantine and could help with predictions for phylloxera distribution under climate change. The effect of temperature on phylloxera survival, development and reproduction was studied in laboratory assays, using temperatures of 18, 22, 26 and 30°C. Survival was evaluated on excised roots (i.e. with a feeding site), and also removed from roots (without a food source) under dry and wet conditions. Results suggest that the threshold temperature for phylloxera to establish a feeding site (hence assimilate nutrients, survive and develop to adulthood) by the Australian phylloxera strains is <30°C, and that the optimal temperature to maintain a feeding site and develop to adulthood is >18°C. Survival at lower temperatures while phylloxera is not attached to a vine root was found to be longer than previously thought, with G20 strain surviving for up to 29 days at 18°C. This finding has implications for managing movement of contractor workers or vineyard machinery from PIZs to exclusion zones.
Re-evaluating biocontrol options
Drawing on expert advice from North America, Europe and Australia, an extensive review on phylloxera biocontrol evaluated approaches including the potential release of exotic natural enemies from the pest’s native range, commercially-reared predatory insects, the application of insect killing microorganisms such as nematodes and fungi, and practices to enhance native predators in vineyards. The review found 16 predatory species, of which only two attack root- feeding phylloxera. An exotic predatory syrphid fly is particularly promising and warrants further investigation. Targeted field surveys in eastern USA could search for native predators and pathogens. In Australia, predators in vineyards could be supported through habitat manipulation for conservation biocontrol. There is also considerable scope to further investigate the use of entomopathogenic nematodes and fungi as biological pesticides.