Grapevine powdery mildew, caused by Erysiphe necator formerly Oidium or Uncinula necator, is the most economically important fungal disease of grapevine worldwide, inflicting losses exceeding AUD$30 million per year in costs associated with control and yield loss (Wicks et al 1997). This widespread and persistent disease affects vine leaves and grape bunches and leads to a decrease in wine quality (Ough and Berg 1979). Furthermore, powdery mildew increases the vines susceptibility to other diseases (Dry et al 2010). This pathogen was first introduced into French vineyards from North America in the 1800s before spreading to all major grape producing regions of the world. Modern grapevine cultivation relies heavily upon the use of fungicides, including sulphur and synthetic fungicides and most grape growers apply between 6-10 fungicide sprays per season (Dry et al 2010). Heavy reliance on use of synthetic fungicides has led to the development of fungicide resistance (Wicks et al 2010). Also, sulphur has undesirable side effects, such as being detrimental to beneficial insects, including natural antagonists of E. necator and can also be toxic to agricultural workers despite of being considered as a natural compound (Calvert and Huffaker 1974). Market and regulatory pressure is building regarding residues and concerns of environmental and human health issues associated with the use of chemical fungicides (Jacometti et al 2010).
Research on alternatives to sulphur and synthetic fungicides to control powdery mildew includes canola based oils, biological oils, inorganic salts, foliar nutrients, surfactants, whey, milk and microorganisms (Crisp et al 2006a). Milk was found to be as effective as sulphur in controlling powdery mildew on zucchini (Bettiol 1999) and wheat (Drury et al 2003) in greenhouse trials. Crisp et al 2006a found that bovine milk has the potential to reduce the incidence of powdery mildew in grapevines in greenhouse and vineyard trials (Crisp et al 2006a&b). Milk when applied once a week at concentrations of up to 50% has been shown as an effective means of control against the powdery mildew fungus in Zucchini and proved to be more reliable than weekly applications of fungicides fenarimol and benomyl (Bettiol 1999). The presence of fatty acids, antimicrobial proteins and production of free radicals in milk have been proposed to damage the fungal structures thereby making milk as an effective means of controlling powdery mildew (Bettiol 1999, Crisp et al 2006). The concept of using milk to control powdery mildew is not new anymore; however, the antifungal activity of milk and the specific compounds that appear to control the disease are not well understood. Milk has not been reported to show any negative impacts on grape and wine quality (Godfrey et al 2010). Research is ongoing to identify and assess active milk components and their mode of action in controlling powdery mildew of grapevines and various vegetable crops (Godfrey et al 2010). Interestingly, a dairy waste stream provided by Murray Goulburn has been found to have antifungal activity against grapevine powdery mildew using in vitro detached leaf studies.
In particular, this research project aims to compare the efficacy of milk with different fat contents (full cream and skim milk), and to evaluate the dairy waste streams identifies to show antifungal activity in vitros. In addition, In vitro detached leaf assays will be undertaken to assess the antifungal properties of specific milk fatty acids. These assays provide an opportunity to test milk components of interest in relatively rapid small scale experiments prior to larger greenhouse trials, providing a more effective way of evaluating and assessing the antifungal activity of milk components. This research project aims to contribute further information towards the identification and assessment of milk and milk components in control of powdery mildew disease of grapevine, with the ultimate aim to contribute to sustainable management practice in controlling this fungal disease.
Aims of Research
The overall aim of this research is to investigate the potential of milk and milk components to control grapevine powdery mildew. The research aims to -
To undertake greenhouse examine whether milk fat content influences the efficacy of milk in controlling grapevine powdery mildew.
To undertake greenhouse trials to evaluate a dairy waste stream which has been shown to have antifungal activity against grapevine powdery mildew using in vitro studies.
To conduct in vitro assays with detached Cabernet Sauvignon leaves to test milk fatty acids for antifungal activity against powdery mildew in grapevine.
Literature Review
Introduction
There is a little or no genetic resistance of the Eurasian wine grape Vitis vinifera to the major fungal pathogen powdery mildew (E. necator) that affects nearly all vineyards and can limit crops loads (Bazar 2009). The fungus is an obligate biotroph that infects all aerial of the grapevine parts including both leaves and berries (Seem 2010). Powdery mildew infection results in severe reduction in vine health, grape quality and yield, resulting in significant economic losses (English-Loeb 2007). Even at low levels, powdery mildew disease can negatively impact wine and juice quality (Gadoury 2001). Economically, grapevine constitutes the most important fruit species worldwide with in excess of 8 million hectares of vineyards across the globe (Vivier 2002). Quality wines are being produced on all arable continents. Grape production in Australia covers 157 290 ha (DFAT 2010). A highly virulent and fungicide resistant powdery mildew has the potential to impose substantial financial damage to the Australian wine and grape industry. Effective protection of the fruit from fungal infection is important because there are many short and long term effects of powdery mildew that include reduction in yields, stunted vine growth, and unpalatable wine. Significant work has been undertaken in this regard and ongoing research focus on reducing the disease incidence and consequent loss in quality and production.
3.2 Grapevine Powdery Mildew
As mentioned earlier, grapevine powdery mildew is major fungal disease worldwide. The powdery mildew fungus can infect all green tissues of the vine, predominantly infecting young tissue. The life cycle of powdery mildew is quite short. The fungus may overwinter as hyphae inside the dormant buds of vines, resulting in diseased flag shoots in spring. Alternatively, the fungus can persist as small black bodies known as chasmothecia (spore structures) on the exfoliating bark of the vine. These overwintering structures are said to be the primary source of infection (Emmett 2008). Immediately following rain, chasmothecia release ascospores that lead to sexual variation in the fungus that has the potential to give rise to the development of virulent strains (Emmett 2008). The infection process from germination to sporulation takes from 5 to 12 days and when conditions are optimum for the disease, the individual organism undergoes multiple reproductive bouts and therefore, for this reason powdery mildew is considered to be polycarpic (Norris et al 2003). The disease spreads steadily during the first 40 days from bud burst and then rapidly until by 80 days most vines in proximity will be diseased (Nicholas, Magarey and Wachtel 1998). The spores are spread through air and the disease does not require free moisture for infection. The optimum conditions for powdery mildew are - temperature of 22 -28°C, excess humidity, filtered light, and a minimum of 2.5mm rainfall. The growth of the fungus is reduced when the temperature is above 35°C (Nicholas et al 1998).
The infection first occurs on underside of the leaves. The symptoms of powdery mildew includes ash grey to white growth on the upper and lower surfaces of leaves and bunches that appears to be oil like patches on leaves, leaf deformation and finally leaf abscission. The incidence of infection is evident on leaves from budburst onwards and the spores of powdery mildew are visible as the leaf spots enlarge. All the succulent tissues on grapevine, the stem, fruit, and leaves can be affected by powdery mildew and shows characteristic symptoms of chlorosis in the area of infection (Lybbert et al 2010). Young shoots shows the symptoms of white powdery spores resulting in stunted growth and occasional death of the shoots (Nicholas et al 1998). Varieties susceptible to powdery mildew include Carignane, Thompson Seedless, Ruby Seedless, Chardonnay, Cabernet Sauvignon, and Chenin Blanc, Semillon, Riesling, Muscadelle. Grenache and Shiraz are much less susceptible. Hybrids with other Vitis species may be resistant (Dry & Coombe 1992). The disease also causes the epidermis to split, reduces the shelf life of table grapes and affects the rate of photosynthesis, thus reducing the sugar content of berry. In severe cases, the infected berries develop a web like growth pattern of spores covering the bunch. Bunches with as little as 5% contamination can be rejected due to the development of off flavours in wine (Nicholas et al 1998).
Current Control Methods
Current control measures for grapevine powdery mildew rely on frequent use of synthetic fungicides like demethylation inhibitor (DMI) fungicides and elemental sulphur sprays in conventional vineyards, and by sulphur and vegetable oils in organic vineyards (Dry & Coombe 1992). However, constant use of fungicides and sulphur contributes to environmental contamination (Hofstein et al 1996). Also, use of DMI fungicides results in decreased sensitivity of the powdery mildew spores and so it makes it hard to eradicate the disease in vineyards (Dell et al 1998). The preventive treatments fail to control powdery mildew effectively once the disease is well established. The predominant use of such control treatments has led to the development of fungicide resistance. For this reason, manufacturers recommend growers to use these fungicides no more than 3times in a season and more importantly no more than 2 consecutive applications. The protectant, quintec is best before the visual presence of disease has been registered. Conventional control is common practice due to commercial availability and low expense. But the use of sulphur may result in phytotoxicity, botrytis infection, vine damage, or taint wine with undesirable residues (Spencer 1978). For these reasons, alternative control measures are warranted. Some agricultural strategies are practiced, including drip irrigation during the most susceptible stage of berry development (Gadoury 2001). Other practices include use of cover crops and canopy management etc.
Alternative Control Methods
Due to the disadvantages of use of sulfur and development of resistance to DMl's has led to need for effective alternatives (Bazar 2009). The alternatives identified as potential replacements to control the fungus include milk, whey, bicarbonates, inorganic salts and canola oil-based sprays (Crisp et al 2006). Another alternative control method is use of biological control measures wherein several species of parasitic or antagonistic fungi, such as Verticillium lecanii, Ampelomyces quisqualis, Sporothrix flocculosa and Tilletiopsis pallescens and Orthotydeus lambi, and a tydeid mite have been evaluated for their ability to suppress powdery mildews of grapevine (English-Loeb, 2007). In addition, B. subtilis, a biological agent was found to be effective in greenhouse experiments but did not provide to be an acceptable control of powdery mildew in the vineyard (Crisp 2004).
Milk as an alternative to control Powdery Mildew
Research on alternatives to sulfur and synthetic fungicides, milk and milk components have been identified to have potential to treat powdery mildew in grapevine. Milk was first used by Wagner Bettiol in 1999 to control powdery mildew on zucchini in greenhouse. Bettiol 1999 found out that milk was successful in controlling the disease and a number of explanations were made for the milk action on powdery mildew including the antifungal activity of milk, presence of fatty acids and the free radical production when exposed to UV light. It was suggested that there is a direct effect of milk on fungus which can be explained on the fact that when milk is exposed to UV rays in sunlight, superoxide anions and oxygen radicals are photogenerated (Korycha-Dahl and Richardson 1978). Milk has also been used to control powdery mildew in wheat and cucurbit in greenhouse experiments (Drury et al 2003).
In general, milk is a heterogeneous suspension of oil, protein (cassein), sugar and a multitude of bioactive trace ingredients including minerals, enzymes and vitamins. The possible modes of action of milk and milk components assessed includes rise in pH of leaf surface, establishment of a protective barrier and antagonistic and systemic resistance induced due to the presence of sodium bicarbonate, oxalate, dibasic or tribasic potassium phosphate, and other salts and amino-acids (Reuveni et al 1993). These actions are highly environment dependent and the timing of the epidemic in regard to the phenology of the crop is an important factor to be considered (Bettiol 1999).
Milk contains many fatty acids and monoglycerides showing antimicrobial activity against fungi (Isaacs 2001). Also, there are significant amounts of polyunsaturated fatty acids present in milk which are biologically active, even at low concentrations. Among free fatty acids, capric acid (C10) and lauric acid (C12) were found to be the most active fatty acids (Jensen 2002). In addition, it has been shown that free radical production in milk contributes to reduction of disease. This is because when methionine, riboflavin and sulfur-rich amino acids are exposed to light, it results in the production of free radicals that tends to lower the risk of disease (Tzeng and DeVay 1989). Furthermore components such as lactoferrin and lactoperoxidase in milk have been extensively researched to show antimicrobial action (Batish et al 1988). Lactoferrin is a glycoprotein that binds to the membranes of fungi resulting in damaging the membranes and causing loss of cytoplasmic fluids (Batish et al 1988). The current research aims to evaluate the components of milk responsible to show antimicrobial activity against powdery mildew and to assess the mode of action. In vitro detached leaf assays, germination assays, greenhouse experiments and small vineyard trials are ongoing to assess milk and milk components efficacy to control powdery mildew.
Research Proposal
Proposed Research Masters: To study the effect of milk on powdery mildew.
Research Questions
In order to understand the antifungal activity of milk against powdery mildew, the research project aims to answer the following questions -
Does fat content present in milk influence the efficacy of milk in controlling powdery mildew in the greenhouse?
Does the dairy waste stream treatment provide effective control of grapevine powdery mildew in greenhouse?
Do specific milk fatty acids have antifungal activity against powdery mildew in detached leaf assays?
Methods
The research project consists of 2 experimental approaches -
Greenhouse trials -Greenhouse experiments with grapevines cv. Grenache and Semillon. The trials involve weekly application of treatments and powdery mildew disease assessment of vines to assess the antifungal activity of skim milk, full cream milk and dairy waste stream.
In vitro bioassay - In vitro detached leaf assay using grapevine cv. Cabernet Sauvignon is used to examine the antifungal properties of specific fatty acids against powdery mildew.
Greenhouse trials
Plant growth
Greenhouse experiments will be carried out with grapevines cv. Grenache and Semillon (DIOVI2 R349A grafted on to Ramsey (R69A) sourced from Temple Bruer wines. Grapevines will be grown in pots of 15-25cm diameter filled with UC potting mix. Temperature in the greenhouse will be maintained at 20-250C. The grapevines will be watered as per required. Recommended fertilizers will be supplied to the vines to meet the nutritional requirements and growth of the vines. The grapevines will be exposed to 12hours of light per day.
4.2.1.2 Trial design and treatments
The grapevine trials were initiated prior to the commencement of this research project. The trials were arranged with five treatments for Grenache and four treatments for the Semillon vines. Each treatment is replicated five and six times in Grenache and Semillon, respectively. A randomized block design was used for both trials. 500ml of treatments are freshly prepared every week and applied to the vines in greenhouse. The treatments used in greenhouse trials are described in the table below -
Table1 - Treatments in Greenhouse trials
Label
Treatment
Grenache
0
Untreated
1
*Water - 500ml
2
Skim milk - 1:10 dilution - 4.75g of powdered milk in 450ml of water* - equivalent for 1:10 dilution
3
Full cream milk - 1:10 dilution - 6.25g of powdered milk in 450ml of water* - equivalent for 1:10 dilution
4
Waste stream - 50ml in 450ml of water* sourced from "Murray Goulburn co-op. Co. Ltd"
*Nano pure water
The treatments were started on 24th June 2010 (Grenache) and 29th July 2010 (Semillon). From 12th August 2010 the treatments will be applied as part of this research project. Treatment will be applied to both surface of the leaves using a hand held sprayer and vines sprayed to runoff. All the treatments will be applied as aqueous suspension using nano pure water/ Millipore water. The treatments will be applied following the initial disease assessment. The growth of vines will also be monitored and the new emerged leaves noted at the time of scoring. The vines were separated so as to avoid cross contamination. The leaves are assessed prior to each application of the treatment.
Disease assessment
Scoring for disease incidence and level is assessed with the help of disease assessment key, where the level of disease is scored from 1 to 10 according to the percentage of the leaf infected with powdery mildew (Appendix 1). The numbers are assigned with respect to the percentage of adaxial (upper) leaf surface infected with powdery mildew. Leaves will be scored for disease prior to treatment. Trials will be run for 8 to 12 weeks depending on greenhouse availability. At completion of the trials, the leaves will be cut off and assessed for the disease on both adaxial and abaxial leaf surface in the laboratory under the microscope.
Statistical Analysis
Statistical analysis will be undertaken using SPSS. Disease assessment data will be subjected to analysis of variance (ANOVA) to test the hypothesis that there is no significant difference in mean disease score among test materials and the control treatments. Least Significance Difference (LSD) will used to assess differences between means. The 5% level of significance (P = 0.05) will be used for all experiments.
4.2.2 Bioassay - in vitro detached leaf assay
The In vitro bioassay will be undertaken using detached leaves of grapevine cv. Cabernet Sauvignon. The detached leaf bioassay is a convenient and effective measure of assessing the antifungal activity of milk and milk components on a small scale in contrast to greenhouse trials.
Glasshouse-grown young glossy grapevine leaves will be collect on ice and sterilized in the laminar flow in sterilizing solution (600ml Milton solution: nanopure water (1:1) with 60 µl Tween 20) for 3minutes, swirling every 30 seconds. The leaves will be rinsed 4times with 500ml of sterile water. Subsequently, the petiole of the leaves is cut to 1cm with a scalpel and the leaves will be placed onto petri dishes containing 1% agar (with 80 µl per 200mL pimaracin). The petri dishes will be kept open in the laminar flow until the leaves are completely dry (approximately 45minutes).
Following sterilization, inoculation is performed. The detached leaf method assesses the protective and curative activity of the material to be tested. In protective assay, Cabernet Sauvignon leaves are pretreated prior to inoculation by suspending the leaf in test material for 5 seconds and air dried for 90 minutes. While for the curative assay, the leaves are treated 3 days before the inoculation by suspending the leaves and air drying as above. The leaves are then allowed to dry in laminar flow. 3 replicate leaves per treatment will be used. For both protective and curative assays, the leaves will incubated in growth room at 250C for 12-16 hour day length and 8 hour dark cycle. The leaves will then be scored for infection 7 to 10 days post inoculation under a stereo microscope. The treatments used in the assays are -
1) Water control
2) Fatty acids at various concentrations, the disease is scored as percent coverage of the leaf.
At last, statistical analysis will be done as for the greenhouse trials.
4.3 Resources required
The resources required in experiments of the project are provided by the research group of Dr Eileen S. Scott (Co- supervisor). The resources used in experiments are as follows -
Greenhouse trial plants - Grapevines cv. Grenache and Semillon sourced from Temple Bruer wines.
Grapevine leaves cv. Cabernet Sauvignon for performing bioassay - detached leaf assay.
Treatments - Milk - Powdered Full cream and Skim milk, Millipore water, Waste stream sourced from Murray Goulburn.
Access to greenhouse for trials and laboratory facilities for preparing treatments.
4.4 Timeline
Core component
Jun
Jul
Aug
Sep
Oct
Research proposal
Literature review
Research component
Greenhouse Trials
Grenache
Semillon
In vitro detached leaf assay
Final Research report
Oral presentation
