«FINAL REPORT to GRAPE AND WINE RESEARCH & DEVELOPMENT CORPORATION Project Number: DNR 02/05 Principal Investigator: David Riches Research ...»
Control of downy mildew of grapevines by
boosting their natural defence system
FINAL REPORT to
GRAPE AND WINE RESEARCH & DEVELOPMENT CORPORATION
Project Number: DNR 02/05
Principal Investigator: David Riches
Research Organisation: Department of Primary Industries,
Control of downy mildew of grapevines by boosting their natural defence system
CONTROL OF DOWNY MILDEW OF GRAPEVINES BY
BOOSTING THEIR NATURAL DEFENCE SYSTEMA final report to the Grape and Wine Research and Development Corporation
David Riches and Robert Holmes Department of Primary Industries Private Bag 15 Ferntree Gully Delivery Centre Vic 3156
This publication may be of assistance to you but the State of Victoria and its officers do not guarantee that the publication is without flaw of any kind or is wholly appropriate for your particular purposes and therefore disclaims all liability for any error, loss or other consequence which may arise from you relying on any information in this publication i Control of downy mildew of grapevines by boosting their natural defence system 1
2 Executive summary
4 Screening experiments
4.2 Materials and methods:
4.2.1 Plant material
4.2.2 Screening trials 1 – 6
4.2.3 Screening Trial 7, Trichoderma
4.2.4 Data analysis
4.3 Results and discussion:
4.3.1 Initial screening
4.3.2 The effect of inoculum concentration
4.3.3 Trichoderma as an inducer of resistance against downy mildew................. 12
5 Characterising elicitor activity
5.2 Materials and Methods
5.2.1 Brotomax dose-response
5.2.2 Chitosan dose-response
5.2.3 Post-inoculation activity against downy mildew
5.2.4 Synergy of inducer treatments with fungicides
5.2.5 Activity on flower tissue against downy mildew
5.3 Results and discussion
5.3.1 Brotomax dose-response relationship
5.3.2 Chitosan dose-response
5.3.3 Timing of elicitor application
5.3.4 Synergy between inducers and copper oxychloride
5.3.5 Antifungal activity on flower tissue
6 Mode of action of elicitors
6.2 Materials and methods
6.2.1 Activity against zoospores
6.2.2 Systemic and translaminar activity
6.2.3 PR-protein hydrolytic enzyme activities
6.3 Results and discussion
6.3.1 Activity against zoospores
6.3.2 Systemic and translaminar activity
6.3.3 Pathogenesis related protein hydrolytic enzyme activity assays.................. 25
7 Performance of elicitors in the field
ii Control of downy mildew of grapevines by boosting their natural defence system
7.2 Materials and methods
7.2.1 Trial design
7.2.3 Disease assessment
7.3 Results and discussion
7.3.1 Downy mildew on inflorescences
7.3.2 Downy mildew control on leaves
7.3.3 Botrytis on bunches
8 Conclusions and Recommendations
Appendix 1. References
Appendix 2: Comparison of approximate fungicide costs
Appendix 3: Staff
iii Control of downy mildew of grapevines by boosting their natural defence system 1 Abstract Fourteen potential elicitors of natural disease resistance were screened for activity against downy mildew on grapevines. Three of these products, Brotomax, AlCl3 and chitosan had high levels of efficacy against downy mildew. It was not proven conclusively that these three products activate host defence responses. In contrast to most inducers of plant defence, Brotomax, AlCl3 and chitosan all had direct antifungal activity against zoospores of the pathogen. Brotomax and AlCl3 also appeared to have some systemic activity while chitosan did not. Contact fungicide activity appeared to be responsible for most of the disease control observed. Both Brotomax and chitosan treatments were trialed in the field where they provided equivalent protection to the standard protectant fungicide (copper oxychloride) on leaves but had lower efficacy than the standard treatments on developing bunches. All products tested showed no post-infection activity and must therefore be used before infections become established.
Brotomax is currently marketed in Australia as a plant nutrient for grapevines.
However, at the dose rates required for downy mildew control, it would be approximately six times more expensive than current synthetic fungicides. This high cost would be a major barrier to the adoption of Brotomax as a fungicide product. Chitosan is a nontoxic and biodegradable product produced from crustacean waste. While chitosancontaining crop protection products are available in Australia, they are not registered as fungicides and at current prices, would not be economically viable for downy mildew control. An Australian potassium bicarbonate product (Ecocarb) was found to have high levels of activity against downy mildew in the glasshouse in a trial conducted late in the project but further trials were not conducted due to time constraints.
1Control of downy mildew of grapevines by boosting their natural defence system
2 Executive summary Stimulating a plant’s own defence mechanisms to protect against disease is a relatively new concept in plant protection. If this mechanism can be exploited effectively we may be able to greatly reduce our reliance on chemical pesticides in future. The first effective plant defence activators or ‘elicitors’ are now commercially available overseas for use in some crops. The aim of this research was to evaluate the potential of elicitors of plant defence for the control of downy mildew on grapevines in Australia.
Potential elicitors of plant defence, identified through literature searches and products currently available in the marketplace claimed to boost plant defence, were screened for efficacy against grapevine downy mildew. Only three of the products screened, Brotomax(a nutrient formulation with claimed plant defence activator activity, currently marketed for viticultural use in Australia), chitosan and AlCl3, had high levels of efficacy against downy mildew. AlCl3 may be unsuitable for use as a fungicide as it was phytotoxic to the grapevines at efficacious doses and likely to pose environmental problems similar to copper. Several compounds claimed to be active against grapevine downy mildew in the scientific literature did not give high levels of disease control in this research.
All of the effective products identified in this study were found to have contact fungicide activity. They were able to prevent zoospore release from sporangia and were toxic to motile downy mildew zoospores. Some systemic protection on unsprayed leaves on treated plants was observed for Brotomax but not chitosan. Antifungal activity was shown not to be cultivar specific with high levels of activity on both Chardonnay and Cabernet Sauvignon varieties. No post-inoculation activity was observed for Brotomax or chitosan which is consistent with protectant fungicide activity or induction of host plant defences. No synergistic effect was observed when combining low doses of either Brotomax or chitosan with a low rate of copper oxychloride. Both chitosan and Brotomax were shown to work on both foliage and flower tissue and had similar efficacy against downy mildew under field conditions as they had in glasshouse experiments. Chitosan and Brotomax were also tested for anti-fungal activity against Botrytis cinerea, the cause of Botrytis bunch rot, but no disease control was observed in the field trial.
The systemic fungicide options available to growers for downy mildew control are limited by the restriction of phosphorous acid to one spray per season, due to phosphite residues, and the resistance risk of phenylamide fungicides (a major problem in Europe and recently detected in Australia). With growers becoming increasing reliant on protectant fungicides, it is important to have environmentally friendlier options than the widely used copper formulations which can accumulate to toxic levels in soil with long term use and dithiocarbamates which are toxic to populations of beneficial mites (Beranard et al. 2001). Other more environmentally acceptable protectant fungicides such as the strobilurin group are also at risk of resistance developing. Clearly there is a need to develop more sustainable control practices for grapevine downy mildew.
2Control of downy mildew of grapevines by boosting their natural defence system
While both Brotomax and chitosan had good efficacy against downy mildew under field conditions, the use of either product in a commercial situation (assuming registration as plant protection chemicals) will depend on the pricing of the products. At current pricing, Brotomax would not be economically viable at the dose rates required to give similar levels of efficacy as current fungicides such as copper compounds. However, Brotomax is currently marketed as a nutrient formulation and application for this purpose at a 1% concentration could be expected to provide effective control of downy mildew and may eliminate the need for a downy mildew fungicide application. While there are unregistered chitosan containing products available in Australia, they are marketed for high value hydroponic applications and would be too expensive to be routinely used for downy mildew control in viticulture.
Chitosan is currently only commercially produced from crustacean waste and squid bone.
Research into producing chitosan from alternative sources such as fungal cell walls may lead to cheaper chitosan in future. Recent Australian research to evaluate the potential of chitosan to enhance plant defence in agriculture has shown promising results for powdery mildew control in tomatoes (Walker et al. 2004). This report recommended the further development of chitosan as a crop protection product, especially in Solanaceae crops.
Thus, there is potentially a large market and economies of scale if an agricultural chitosan were to be registered and marketed in Australia.
Another way the cost of treatment could be reduced is through different application technology such as recirculating sprayers. If the efficiency of the spray deposition process could be sufficiently increased, more expensive but environmentally friendly crop protection products such as chitosan could be routinely used.
A potassium bicarbonate formulation (Ecocarb) was tested against downy mildew after learning of anecdotal evidence of efficacy against downy mildew (D. Madge pers com).
This product showed promising results in a glasshouse trial. Because this experiment was conducted just before the end of the research project, no further studies were conducted with this compound. However, as this compound product is non-toxic, comparable in price to other fungicides, IPM compatible and already has registration for grapes for control of powdery mildew, further developmental work with this product is recommended to determine if commercially acceptable disease control can be achieved in the field.
3Control of downy mildew of grapevines by boosting their natural defence system
3 Introduction For over 100 years, researchers have known that plants can be pre-conditioned to resist diseases. The basis for this ‘acquired physiological immunity’ was unknown at the time and it wasn’t until the 1960’s when the concepts of local acquired resistance (LAR) and systemic acquired resistance (SAR) were demonstrated by showing Tobacco mosaic virus infected tobacco was protected from subsequent challenge inoculations with the virus.
Since these early experiments, SAR has been shown to operate in numerous plant species against not only viral pathogens, but also fungi, bacteria and even herbivores. The discovery that certain chemical compounds could mimic the effect of pathogens in activating the SAR pathway opened the way for the development of activators or ‘elicitors’ of SAR as plant protection products. This led to the concept of induced resistance as a disease control strategy, utilising the plant’s own defence mechanisms instead of applying chemicals to kill or slow the growth of pathogenic organisms. With increasing concern about pesticide use worldwide, due to environmental problems, potential effects on human health and pathogens developing resistance to chemicals, plant protection strategies less reliant on pesticide application are becoming increasingly attractive.
Salicylic acid (SA) is naturally produced by plants and is known to be involved in the SAR biochemical pathway. It has been demonstrated that exogenous application of SA can also activate the resistance pathway in plants (Ryals et al. 1996). The first plant defence activators or ‘elicitors’ developed commercially were structural analogues of SA.
These compounds were able to provide long lasting protection powdery mildew on wheat (Görlach et al. 1996) but were later shown to have potential in many other plant pathogen systems.
Downy mildew of grapevines is a disease that may require frequent fungicide applications to prevent significant crop losses when conditions favour infection (warm wet weather) (Schwinn, 1981). Some of the most commonly used fungicides for downy mildew control have detrimental effects in the environment, are prone to pathogen resistance developing or their use is restricted by wineries due to residue concerns. For these reasons, investigating downy mildew control strategies using induced resistance is an attractive prospect.