Integrated management of grapevine phylloxera: Phase II

Grapevine phylloxera is Australia’s most damaging viticulture pest. Management practices aimed at preventing the spread of phylloxera outside defined quarantine boundaries incorporate a suite of management tools and strategies including i) effective surveillance and diagnostics, ii) availability of resistant rootstocks, iii) validated disinfestation protocols, iv) comprehensive understanding of the insect’s ecology, and v) sustainable management options for growers. This project aimed to improve the industry’s ability to sustainably manage grapevine phylloxera by building on the previous research under these five broad themes.

Effective surveillance and diagnostics: Current surveillance practices for phylloxera rely on a time- and resource-limited “grid approach” that involves visually inspecting the roots of vines. Using this method it is, however, difficult to provide assuredness of the insect’s absence when no individuals are detected following a surveillance event.

Field surveys were conducted in an infested vineyard to develop simulation models that could improve confidence in declarations of phylloxera absence. These models have been provided to Biosecurity, Agriculture Victoria, as an interactive spreadsheet where users can enter surveillance details (e.g. vineyard size, survey method, season) and be provided with an estimate of the probability of detecting phylloxera and the time needed to survey the vineyard.

Data from the field surveys were also used in a side-by-side comparison of three detection methods; visual inspection of roots, LAMP tests on emergence trap samples, and LAMP assays on root samples. Regardless of season, LAMP tests on root samples was the most effective surveillance method, followed by visual inspections and then LAMP tests on emergence trap samples. A combination of both LAMP tests on root samples and visual inspections captured over 90% of all positive detections. Incorporating LAMP tests on root samples is thus likely to benefit surveillance efforts for phylloxera by improving the probability of detecting the pest, improving confidence in declarations of absence (i.e. when LAMP tests are negative, and no physical specimen is detected), and providing overlapping layers of protection against one detection method failing.

Additionally, data collected from the field surveys was used to determine if the season (summer or autumn) or the experience of the surveyor (expert or beginner) had an impact on the probability of detecting phylloxera and the time needed to survey the vineyard. All three detection methods were significantly more effective in autumn compared to summer and experienced surveyors took significantly less time to detect phylloxera on vine roots compared to beginners. Biosecurity agencies could improve the probability of detecting phylloxera and reduce the time needed to conduct vineyard surveys by prioritising vineyard surveys in autumn and ensuring at least some of the surveyors have more than one year of experience.

The relative effectiveness of the different methods (visual inspections, LAMP tests on root samples, and LAMP tests on emergence trap samples) in winter (outside of the peak phylloxera activity) was compared. All three detection methods were significantly less effective in winter but were still able to detect the pest. The possibility of off-season surveys could be beneficial for biosecurity efforts by increasing the surveillance window and allowing surveys to be conducted at the first sign of infestation regardless of season.

Improved selection of resilient rootstocks: Grafting European vine varieties onto American rootstock is recognized as the best viticultural practice to safeguard vineyards from phylloxera-induced damage. Ongoing rootstock screening trials are essential in managing grapevine phylloxera, allowing growers to make informed replanting decisions based on the prevalent phylloxera genotype/s on or surrounding their vineyard.

This project assessed the susceptibility of two new rootstocks, M5512 and M5489, to phylloxera genotypes G4 and G38. Each vine was infested with eggs belonging to either the G4 or G38 phylloxera. Additionally, Vitis vinifera vines (own rooted vines) were similarly infested with phylloxera and used as a positive control.

Both M5512 and M5489 rootstocks inoculated with G4 or G38 had significantly fewer nodosities, adults and total insects compared to their positive control counterparts. M5512 roots infested with G38 phylloxera eggs showed signs of phylloxera feeding (nodosities), but no individuals of any life stage were observed. This suggests that phylloxera were able to feed and induce damage on this rootstock, but were not able to develop into adults. Nodosities were also present on M5489 roots infested with G38 phylloxera eggs, and a single individual phylloxera was also recorded. Nodosities and individuals of all life stages were observed on both M5512 and M5489 rootstocks infested with G4 phylloxera eggs. Taken together, these results suggest that both M5512 and M5489 rootstocks should be rated tolerant for both the G4 and G38 genotypes. Although no individuals were observed on M5512 rootstocks inoculated with G38 phylloxera eggs, the presence of nodosities suggests that this genotype can feed and induce damage on this rootstock. This information should be provided to growers by updating the Rootstock Selector Tool.

Improved disinfestation procedures: In order to prevent the spread of phylloxera outside of quarantine boundaries, vineyard machinery needs to be disinfested before it can be transported outside of infested or risk zones. The current disinfestation protocol requires machinery to be thoroughly cleaned (including the removal and cleaning of parts that can hide or trap soil), and then placed in a dry heat shed for either 40°C for 2 hours and 15 minutes, 40°C for 3 hours, or 45°C for 2 hours to kill any phylloxera that may have been transferred from the soil or grapevines onto the equipment. These temperatures and time requirements were determined in the laboratory under sterile conditions.

The effectiveness of dry heat disinfestation was assessed under different hygiene conditions, specifically while phylloxera were attached to a piece of root and protected by soil or grapevine material. The current dry heat disinfestation protocols were not 100% effective at killing phylloxera, regardless of the hygiene condition tested.

An improved disinfestation procedure was subsequently tested, exposing phylloxera that were attached to a piece of root to 50°C for 2 hours. This resulted in 100% mortality. The new protocol was then tested in a commercial dry heat shed using phylloxera that were either: i) attached to a root, ii) attached to a root and protected by grapevine material, iii) attached to a root and protected by soil or iv) attached to a root and protected by soil and grapevine material across 6 phylloxera genotypes (G1, G4, G19, G20. G30 and G38). 100% mortality was observed in all genotypes tested when phylloxera were attached to a root in the absence of soil and plant material, except for G30. Four percent of G30 phylloxera initially survived the dry heat exposure however, these insects died before they were able to complete development, suggesting a delayed effect of the treatment. When phylloxera were attached to the root and protected by just grapevine material, 100% mortality was achieved across all genotypes. Unfortunately, 100% mortality was not achieved when phylloxera were attached to a root and protected by soil or by both soil and grapevine material. Forty-two percent of phylloxera protected by soil survived the initial dry heat exposure, with 30% subsequently developing into adults. Similarly, when phylloxera were protected by both soil and grapevine material 50% survived both the initial exposure and the following 30 days, developing into adults. This suggests that soil but not grapevine material offers additional protection from the dry heat treatment.

These findings suggest that changing the disinfestation protocol to 50°C for 2 hours may increase the effectiveness of the dry heat disinfestation treatment. These results also highlight the importance of cleaning machinery and removing parts that can hide or trap dirt before placing them in the dry heat shed to remove any soil that may protect phylloxera from the heat.

Overwintering population dynamics: Phylloxera are slow-moving insects and dispersal is predominantly through human activity. This project investigated the risk of phylloxera dispersal by human activity during winter (outside of peak phylloxera activity), in the North-East Phylloxera Infested Zone (PIZ) by studying phylloxera development in the field during spring, winter and autumn, and assessing phylloxera development and survival at winter temperatures for three phylloxera genotypes (G1, G4, and G19) in the laboratory.

Field surveys found that the number of crawlers on vine roots was highest in winter. However, no nodosities were observed on roots in winter suggesting that phylloxera may not be feeding and may have reduced activity during this season. Laboratory studies suggest that at 4 or 9°C, phylloxera are unlikely to survive or develop into adults, but at 14°C (which is within the maximum winter soil temperatures recorded for the North-East PIZ) a small number of individuals survive and can develop from eggs into crawlers. Taken together this suggests that at least at mild winter temperatures, human-assisted dispersal of phylloxera is possible and effective disinfestation and quarantine protocols should continue to be implemented in winter.

Additionally, laboratory studies demonstrated genotypic variation in phylloxera sensitivity to winter temperatures. G1 and G4 phylloxera eggs maintained at 14°C were able to develop into crawlers and continued developing into adults once returned to control temperatures (25°C). A small number of G19 eggs maintained at 14°C were able to develop into crawlers, but these individuals died following their transfer to control temperatures (25°C). Understanding how different phylloxera genotypes develop and survive in different climates is critical in predicting how the changing climate will influence the prevalence and distribution of different genotypes in Australia. This may have significant implications for growers replanting on resistant rootstocks, as their choice of rootstock is highly dependent on the risk of infestation from specific phylloxera genotypes.

The project also investigated soil characteristics that may contribute to variation in the population dynamics of phylloxera in the field. Field surveys revealed significant negative relationships between soil moisture and the number of root galls and adults observed on roots, and soil pH and the total number of insects observed on roots. Findings from previous research on the relationships between phylloxera populations and soil characteristics have been largely inconsistent. Empirical laboratory studies are likely needed to determine which soil characteristics influence phylloxera population dynamics and whether they can be manipulated to limit their ability to establish, reproduce, or disperse.

Prevalence of leaf galling phylloxera in the King Valley: Recently, occurrences of leaf galling phylloxera in commercial vineyards have increased in many parts of the world including the North-East Phylloxera Infested Zone (PIZ) in Victoria, Australia. The project investigated whether the abundance of leaf galling phylloxera in infested vineyards is influenced by vineyard practices (such as row spacing), grape variety, or rootstock.

Field studies found no significant difference in the abundance of galls between grape varieties or rootstocks, however, several unaccounted-for variables may be masking differences among grape varieties or rootstocks (such as the number of founding phylloxera, time since initial infestation, and the age of the vines) and controlled studies in a greenhouse setting may provide a more definitive answer. There was insufficient variation in vineyard practices to statistically determine if these contribute to the abundance of leaf galling phylloxera. Similarly, controlled laboratory studies where vineyard practices can be manipulated may be beneficial.

Following field surveys, leaf galls were dissected in the laboratory to determine if there was any relationship between the number of galls on leaves and the level of infestation within the gall. There was a significant correlation between the number of phylloxera per gall and the number of galls per leaf. Whether this is due to the length of time the leaves had been infested, or whether specific leaf characteristics are better able to support or attract leaf galling phylloxera requires further study. Interestingly, approximately 17% of the leaf galls dissected did not contain any individuals. Future research could be directed at determining what is driving the absence of individuals inside some galls as this information could be used to develop management strategies that help growers reduce populations of leaf galling phylloxera.

Genetic diversity of leaf galling phylloxera in the King Valley: This study aimed at characterising the genetic diversity of leaf gall phylloxera from the King Valley. The genetic diversity of root galling phylloxera have previously be characterised in this region, this is the first study to characterise leaf galling phylloxera in the North-East Phylloxera Infested Zone.

A total of 513 samples were examined for microsatellite variation from leaf galls (497) and roots (16) collected from six vineyards, including some ornamental vines. A total of 76 genotypes were characterised. Twenty-seven of these genotypes had previously been characterised, but 42 were new. Most genotypes detected were unique to a single vineyard, whilst 12 genotypes were found at multiple sites and in leaves and roots. Almost 50% of leaves had more than one genotype (between 1 and 12), whilst galls contained up to three genotypes.

These results indicate a higher-than-expected genetic diversity of phylloxera in Victoria and future research should consider if sexual reproduction is occurring. Furthermore, this work shows the potential for genotypes to move between vineyards, regardless of the type of vines i.e., ornamental or commercial or, the part of the plant (same genotypes were found in the roots and the leaves). In-depth studies of the genetic diversity of phylloxera such as this enable appropriate selections of rootstock, which should be based on the most abundant local genotype present.

A conservation biological control approach: This study aimed to conduct field surveys to identify potential phylloxera predators in Victorian vineyards and assess the efficacy of commercial aphid predators in laboratory settings for potential use in “inundation biological control”. Field surveys were conducted in Victorian vineyards, focusing on phylloxera-infested roots and leaves to identify resident predatory arthropods. Laboratory trials tested the predation capabilities of ladybirds (Harmonia octomaculata) and green lacewing larvae (Mallada signatus) on phylloxera eggs and crawlers.

Field surveys identified 56 arthropod specimens from 24 families, with predators comprising 38% of the total taxa. Predators included spiders, ladybeetles, lacewings, ants, and predatory mites, primarily found on grapevine leaves. Laboratory feeding trials showed ladybird larvae as effective predators, consuming all phylloxera crawlers within an hour. Ladybird adults and lacewing larvae also predated phylloxera eggs, with ladybirds showing higher predation rates.

The study highlighted the potential of both conservation biological control and inundation biological control strategies. Field surveys cataloged a modest diversity of phylloxera predators, suggesting opportunities for enhancing these populations. Laboratory trials confirmed the predation capacity of commercially available aphid predators, though their practical deployment requires further investigation. Future research should focus on validating predator-prey associations through direct observation or molecular analysis and exploring large-scale application methods for these biocontrol agents.

Biological control presents a promising alternative for sustainable phylloxera management. Enhancing resident predator populations and utilizing commercial aphid predators could assist in reducing the prevalence of dispersing stages of phylloxera. Further research is essential to optimize these strategies and ensure their effectiveness in diverse vineyard conditions.

Objectives

This project is a next-phase research program that will procure knowledge to improve phylloxera management practices and help protect Australian vineyards. It aims to address five key research components:

1. more effective surveillance and diagnostics
2. improved selection of resilient rootstocks
3. improved disinfestation procedures
4. improved knowledge of Australian phylloxera ecology, and
5. improved knowledge of functional biodiversity and conservation biological control.

Approach

The project will employ an integrated pest management approach to:

• Provide more effective in-field surveillance and diagnostics for phylloxera, including new statistical models for improved zoning and rezoning and to establish a level of confidence in detecting an infestation at different levels of prevalence. The newly developed in-field LAMP assay will be used for large-scale phylloxera surveys. Research will determine the detection benefits of using root inspection/bucket traps and LAMP in field surveys and the ability of the assay to diagnose phylloxera outside peak activity e.g. winter.
• Assess selected commercial rootstocks for their susceptibility and resistance status to phylloxera. Commercial rootstocks developed by CSIRO (M5489, M5512 and the newer ‘C’ rootstocks) will be screened for resistance against at least five genotypes of phylloxera selected from diverse clades. The project will add to previous rootstock screening and the ongoing collaborative efforts with CSIRO in rootstock evaluation.
• Determine the risk posed to vineyards by leaf galling forms of the insect. Recent field surveys showed a considerable number of previously unknown gallicoles (leaf galling forms of phylloxera). Further research will conduct targeted surveys to establish the prevalence of gallicoles, characterise the genetic diversity and determine if some exist as both gallicoles and radicoles. The surveys will investigate whether particular rootstocks and vine varieties are more susceptible to gallicoles. The project will also revisit collected data and investigate interactions between phylloxera genotypes, rootstocks and environmental factors (such as soil type, aspect, temperature) and expand on this with further field-collected data. This could help with further rootstock breeding and selection.
• Update machinery disinfestation protocols. Lab-based studies will assess the effectiveness of disinfestation procedures under different hygiene situations that relate to cleaning of harvesters and will included phylloxera survival in soil clods and grapevine material following dry heat treatment. The propensity of insects to survive, develop and reproduce post-treatment will be assessed. The impact of overwintering temperatures on phylloxera survival and factors that induce and break dormancy will be determined.
• Determine the alcohol content for both red and white fermented products at which 100 per cent of a range of phylloxera genotypes are killed. This will add robustness to the disinfestation protocol for naturally fermented products, which currently specifies a number of hours of fermentation before grape products can be moved out of a Phylloxera Infested Zone (PIZ).
• Employ a conservation biocontrol approach to phylloxera by producing new knowledge of phylloxera predators. In conjunction with leaf galling surveys, vines will be inspected for potential phylloxera predators. Laboratory trials will be conducted using commercially available aphid predators to assess their impact on phylloxera.

In addition, the project will consolidate research outputs from this and previous projects into effective extension products and provide a pathway for sector uptake and adoption. Any regulatory implications for the sector (arising from findings in both new and previous work) will also be addressed.

Sector benefits

This project will capitalise on previous projects to enable the sector to be better prepared to manage phylloxera incursions into pest-free states and to protect pest-free vineyards in infested zones.