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Sustainable control of grapevine powdery mildew ((I n cin ul a n e c at o r S chwe i nitz Burrill) in vineyards in South Australia

Peter Crisp B. Ag. Sc. (Hons), The University of Adelaide

Thesis submitted for the degree of Doctor oflhilosophy ln The University of Adelaide February 2004

Plant and Pest Science School of Agriculture and Wine Faculty of Sciences II 111

We're finally having to acknowledge that if a substance can kill a creature as simple and resilient as a mosquito or a weed, it's probably poisonous enough to destroy more complex creatures like us, simply because all life forms are very much alike.

David Suzuki 2002 AI v

Table of Contents .IX .XI ACKNOWLEDGEMENTS ...... XII CHAPTER 1 - LITERATURE REVIEW.. ... I 1 1.2 Organic agriculture... 2 1.3 Powdery mildew (Uncinula necator) 4 1.3.1 History of the disease 4 1 .3.2 Disease cycle and epidemiology 5 1.4 Management of powdery mildew 8 1.4.1 Cultwal controls 8 1.4. l.l Canopy structure 8 1.4.1.2 Other cultural conhol methods...... 8 1.4. 1.3 Natural enemies 9 1,4.2 Resistance 9 I .4.3 Chemical control...... t2 1.4.3. I Cunent conþol methods...... t2 1.4.3.2 Spray timing t4 1.4.4 Biological conhols 15 1.4.4.I Ampelomyces quisqualis t7 1.4.4.2 Bacillus subtilis 19 1.4.4.3 Pseudomonas spp...... 20 1.4.4.4 Trichoderma spp 2t 1.4.4.5 Other microbial agents 22 1.4.4.6 Mites 23 1.4.5 Novel fungicides,... 25 1.4.5. I Inorganic salts 25 L4.5.2 Surfactants... 27 1.4.5.3 Milk/whey 27 1.4.5.4 Compost extracts 28 1.4.5.6 Chitosan/chitin . 29 1.4.5.7 MR formulation. 30 30 32 33 1.5.3 Eucalyptus oil 34 1.5.4 Tea tree oil...... 34 1 .5. 5 Other foliar coatings.,...... 34 1.6 Summar ..'...... '.'35 CHAPTER 2 - GENERAL METHODS AND MATERIALS...... 37 2. I Greenhouse experiments. 37 2.1.1 Maintenance of vines...... '...... '37 2.1.2 Inoculation, treatment and assessment of vines...... -.'38 2.1.3 Management of pests 42 2.2Field experiments 42

2.2.1.l Temple Bruer Wines vheyards 42 2.2.1.2 Glenara Wines vineyard 44 2.2.1.3 Mountadam Vineyard.,...... 44 2.2.1.4 Warriparinga vineyard.. 45 2.2.3 Methods for field experiments ...... 46 2.2.4Management of pests and other diseases...... '."...... 50 CHAPTER 3 - GREENHOUSE E)GERIMENTS 2000...... 52

3.2 Materials and methods 54 3.2. I Experiment l/2000 54 VI

3.2.2 Experiment 2 I 2000 55 3.2.3 Experiment 3/2000 56 3.3 Results .58 3.3. I Experimerfi I /2000 58 3.3.2 Experimefi212000 60 3.3.3 Experiment 3/2000.... 63 .65 CHAPTER 4 - FIELD EXPERIMENTS 2OOO|2OOT 70 4.l lntroduction .70 4.2 Methods and materials .71 4.2.1 Temple Bmer Wines vineyarcl 72 4.2.2 Glenara Wines vineyard...... 72 4.2.3 Mountadam Vineyard 73 4.2.4 W aniparinga ...... l5 4.3 Results 75 4.3.1 Temple Bruer'Wines vineyard 75 4.3.2 Glenata Wines vineyard 78 4.3.3 Mountadam Vineyard ...... 79 4.3.4 Warriparinga ...... 81 82 CHAPTER 5 - MODE OF ACTION OF SELECTED MATERIALS ...... "...... 87 5.1 lntroduction 81 5.2 Materials ancl methods 89 5.2.1 Detection of free radical activity...... 89 5.2.2 Scanning electron microscopy 90 5.3 Results 93 5.3. I Electron spin spectrometry...... 93 5.3.2 Scaruring electron microscopy 93 5.4 Discussion t0l CHAPTER 6 - GREENHOUSE EXPERIMENTS 2OO1 AND 2OO2 ... I I3 6. 1 Inhoduction...... 113 111 6.2 Metho

CHAPTER 8 - FIELD E)GERIMENTS 2OO2I2OO3 .t44 8.1 Introduction 144 8.2 Methods and materials t44 8.2.1 Temple Bruer Wines vineyard 144 8.2.1.1 Experiment I t45 8.2.1.2 Yield and quality assessment t47 148 8.2.2 Glenara Wines vineyard 148 8.3 Results 149 8.3.1 Temple Bruer Wines vineyard experiment 1...... t49 8.3.2 Yield and quality assessment 155 8.3.3 Temple Bruer Wines vineyard experiment 2...... t57 8.4 Discussion 159 CHAPTER 9 - GENERAI DISCUSSION...... 165 APPENDIX I -FIELD SITES 174 Map I - Glenara Wines and Mountadam Vineyard 174 lllfap 2 - Waniparinga...... ,...... ,t75 Map 3 - Temple Bruer Wines vineyard. ,176 APPENDD( 2 - GENERAI METHODS AND MATERIALS...... 177 A 2.1 Yeast extract medium for Bacillus subtilis ...... 177 A 2.2 Nutri-life 4/20@...... t78 A 2.3 MR Formulation 178 A 2.4 Wheast t78 APPENDIX 3 - V/EATHER AND CLIMATA DATA .... 179 REFERENCES...... 205 IIIA lx

Abstract

Grapevine powdery mildew, caused by the fungus (Jncinula necator Schweinitz

Burrill, is a major disease affecting grape yield and quality worldwide. In conventional vineyards, the disease is controlled mainly by regular applications of sulphur and synthetic fungicides, such as demethylation inhibiting fungicides (DMIs), and in organic agriculture by sulphur and canola-based oils. The impending restrictions on the use of sulphur in organic viticulture, the development of resistance to DMIs in

Australia and elsewhere, and the demand for residue-free grapes create a need for effective altematives to sulphur and synthetic chemicals. This research has identified potential replacements for synthetic fungicides and sulphur in the control of powdery mildew, such as milk, whey, bicarbonates and canola oil-based sprays.

A series of greenhouse experiments was conducted to evaluate 34 potential novel materials and biological agents for efficacy in controlling powdery mildew. The most effective treatments applied were Bacillus subtilis (which reduced disease by

94Yo compared to the untreated control), Synertrol Horti-Oil@ (a canola oil-based product, g2o/o), milk (70%), whey (64%) and Ecocarb@ þotassium bicarbonate, 58%).

Milk and whey provided increased control of powdery mildew as the concentration increased. The efficacy of milk tended to decrease as the fat content of the milk was reduced.

The materials that were most promising in the greenhouse were then assessed in field trials in commercial vineyards. Applications of milk, whey and mixtures of a canola oil-based product and potassium bicarbonate, applied at rates of 300 Ll}:å to

1000 L/ha depending on canopy development, reduced the severity of powdcry mildew. The severity of powdery mildew on vines sprayed with a 1:10 dilution of x milk, 45 g/L whey powder and mixed programs was not significantly different from that on vines sprayed with sulphur (wettable powder, 3 g/L).However, the relative control of powdery mildew by the test materials in field trials was highly dependent on the degree of coverage of the plant surface achieved. In vineyards where coverage was compromised, the degree of control of powdery mildew was reduced, often to commercially unacceptable levels.

Electron spin resonance (ESR) and scanning electron microscopy (SEM) were used to investigate the possible mode or modes of action of milk and whey in the control of powdery mildew. The ESR experiments showed that production of oxygen radicals by various eomponents of milk in natural light was associated with reduced severity of powdcry mildew. SEM images showed that milk and whey caused the hyphae of U. necator to collapse and damaged conidia within 24 h of treatment.

Hydrogen peroxide, applied as a source of fÌee radicals, also caused collapse of the

rlamage to h.-¡rhee¡¡J lJ¡rev of tl necetor within 24 hbut clicl not conidia, ancl appeared stimulate germination. Lactoferrin (an antimicrobial component of milk) ruptured conidia, but damage to hyphae was not evtdent in iactoferrin-treated sampies untii 48 h after treatment. The results suggested that fats, free radical production aiong with the

^f f ^^+^f^*:- ..^-^:Ll" o.o ooo^^iofoá rr¡ifk fho nf ¡lUuUll- -t -,- uI laiLtulçtIlll, 4llu^..l PlJùùrurJ ^+1^^-rrLrrwr ^-^+oi-oPrurvt¡¡ù, 4¡w 4ùùuv!4rvu vY rerr Lt¡v ^^-frnl vv¡rLrv¡ vr powdery mildew by milk.

such as and oil plus bicarbonate mixtures, were Novel soft fungicides, milk I effective alternatives to sulphur and synthetic fungicides in certain South Australian conditions. Biologica-'l agents (including B. subtilis, which was highly effective in greenhouse experiments) did not provide acceptable control of powdery mildew in the vineyard. x1

DECLARATION

This work contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text.

I give consent to this copy of my thesis, when deposited in the University Library, being available for loan or photocopylng. xl1

Acknowledgements

I would like to thank my supervisors Associate Professor Eileen Scott and Dr

Trevor V/icks. I thank Eileen in particular for editing my manuscript, for her belief in the project and providing motivation when it was needed. Thanks also to Dr David

Gadoury who, as an adviser to the project, contributed useful information from his

experience with powdery mildew and to Dr Kerrie Davies for reading the manuscript

and also her interest in the research.

The research was conducted with support from the Arrstralian Rescareh Council

and industry partners, Temple Bruer'Wines, Glenara Wines and Mountadam Vineyard.

Special thanks to David Bruer from Temple Bruer Wines for his trust, knowledge and 'Wines commitment to the project and Leigh Venall from Glenara for advice and

experience in organic viticulture. Also, thanks to Dr Gordon Troup, Monash University in Melbourne, for his assistance with the Electron Spin Resonance spectrometry, and the staff at Adelaide Microscopy for assistance with Scanning Electron Microscopy.

I also thank the many other people who assisted in the laboratory, gteenhouse and field, particularly Karolina Pniewska and Judy Belatti, and honours students,

Robert Marlow and Lachlan Palmer. I would also like to thank the other students and staff in ia'ooratories Ni05 and l.{iû7 ior iheir support anci patience. Other support stafi who assisted in my research including Terry Feckner, Gary Taylor, Heather Fraser and the staff in the Plant Research Centre, thanks to you all.

To my parents for helping me despite some concems, especially Dad who will never have the opportunity to read this manuscript; thanks. A final and special thanks to Lynda for supporting me through this huge challenge and to Matthew, Jasmine and

Andrew for putting up with me not letting them near my computer while writing up. IIIX ,

^IX 1

Chapter 1 - Literature review

1.1 Introduction Vitis vinifera L. (European grape) cultivars are grown in many countries of the world for wine, dried fruit and table grape production, ffid this species is most commonly used for wine production in Australia and Europe. Powdery mildew of grapevine, caused by the fungus (Jncinula necator Schweinitz Burill, is a major disease in many countries (Magarey et al. 1997) and is estimated to cost the Australian grape

and wine industry at least A$30 million each year (Wicks et al. 1997). The costs are a

combination of the $10 million associated with controlling the disease and losses of $20 million due to unusable berries, reduced bunch weight (Chellemi et al.1992; Reuveni et

al. 1995; Wicks et al. 1997) and a decrease in wine quality (Ough et al. 1979; Olmo

1986; Amati et al. 1996).

(J. necator is associated with a reduction in photosynthesis, transpiration and

water use efficiency by infected leaves (Lasko et al. 1982; Shtienberg 1992 ) attd, in the

long term, causes a reduction in vine size and bud fruitfulness (Poole 1984:, Chellemi et

al. 1992). The disease reduces wine quality by reducing colour intensity in red wines

and increasing levels of caftaric and coutaric acids (Amati et al. 1996).

The probable origin of [J. necator is North America where it is associated with

native vines, which display moderate to high levels of resistance to the pathogen (Li

1993; Staudt 1997). The North American Vitìs species also have some physical

differences that make colonisationby U. necator more difficult and provide protection

for antagonists of the pathogen (English-Loeb et al. 1998).

Control of powdery mildew in the field relies heavily on the application of

synthetic chemicals (Rankovic 1997) and sulphur. However, the development of 2

resistance to DMIs in Australia (Savocchia et al. 1999) and the USA (Gubler et al.

1994; Erikson et al. 1997) and the demand for residue-free grapes provide incentives for

minimizing inputs of synthetic chemicals in chemically assisted viticulture. Alternative

control measures include anti-transpirants, compost "teas", silicon, chitosan, milk, potassium salts, antagonistic fungi, bacteria and nutrient sprays (Ziv et al. 1993). Some

of these alternative controls are available commercially in South Australia.

With the possible withdrawal of sulphur from the schedule of acceptable inputs for organic vineyards (EC regulation 2092191: International Federation of Organic

Agriculture Movements), and the negative health and environmental impacts associated with its use, such as respiratory ailments, worþlace injury (Anon. 1996 b) and air pollution, there is a need to look at alternatives for organic viticulture. Biological and novel controls have the potential to reduce loss due to powdery mildew. The aim of this research project was to assess the efficacy of a range of biological and novel eontrols of f | øôînt^v ll.'ql o¡a ot til oLlo fnr rrca in fha .rifinttlfttrp inrlr rcfn¡ ìn cnrrfhpm urr4r sr v ra¡v ^.-o-ì^v¡ 64¡uv

Australia, and to establish the mode of action of the most promising agents.

1.2 Orgrnic agriculture In this thesis the term "organic agriculture" refers to the registered term as defined in Australian legislation and by the International Federation of Organic

Agriculture Movements (IFOAM). The term "chemically assisted agriculture" includes all agricultural systems that include synthetic pesticides, whether conventional, low input or IPM-based systems are used. There is an international increase in demand for organicaiiy grown iiuit. vegetabies, and iheir secondary products ineiuding wirre. Irr

Europe, the market for organic products was worth approximately A$7.5 billion in 1999 and is increasing by 20 - 25% each year. If current rates of growth are maintained, organic produce will account for 30%o of production by 2010 (Kirurear 2000). J

Production in the US was approximately US$6.5 billion in 1998, growing to US$9 billion in2002 (Moore 2003) and in Japan production is between A$1.5 and A$2 billion and was predicted to reach A$10 billion by 2006 (Kinnear 2000). Global production has grown from US$8 billion in 1990 to an estimated US$25 billion in2002 (Moore 2003).

The organic industry in Australia had 1500 certified members in 2000, including grorwers and processors servicing a market valued at approximately A$140-200 million

(RIRDC 1999; Kinnear 2000), growing to A$250 million in2002 (Baldwin 2003). The industry is growing at a rate similar to that of overseas markets. The export value of organic food produced in Australia was approximately A$40 million ín 1999, and most of this was exported to England and Japan (Kinnear 2000). Although the value of the

Australian organic viticulture industry could not be quantified, in 1999 members of the

Australian Organic Viticulture Association produced 864.5 tonnes of berries that were either certified organic or in transition to organic production (Anon. 1999).

At present control of powdery mildew in organic viticulture is mainly by application of sulphur. However, proposed changes to the regulations controlling the accreditation of organic enterprises may restrict the use of sulphur, which, although considered a natural compound, has undesirable side-effects, and toxicity to beneficial mites and insects (Calvert et al. 1974) which may promote outbreaks of secondary pests. In some circumstances sulphur can be ph¡otoxic and can impart off flavours, which reduce the quality and value of the wine (Martin et al. 1931; Gubler et al. 1996).

In California, sulphur was responsible for the greatest number of pesticide poisonings of agricultural workers (Robinson et al. 1993), and viticulture was one of the six agricultural industries accounting for most worker injuries for the period 1984 - 1990

(Robinson et al. 1993). The majority of the sulphur-related injuries reported in 4

Califomia in 1994 were systemic/respiratory injuries, followed by eye and skin injuries

(Anon. 1996 b).

Some of the fungicides used to control U. necator and other fungal and insect pests, including sulphur, have a negative impact on natural antagonists of U. necator and pollute the environment (Ziv et al. 1993; Hofstein et al. 1996). The loss of the natural controls of powdery mildew, such as antagonistic fungl and mycophagous mites

(Gadoury et al. 1998; Norton et al. 2000), further increases the reliance on applications of fungicides. Since the introduction of sulphur in 1850 there have been a number of attempts to control U. necator, including the use of compost "teas", plant extracts, silicon, salts, baking soda, oils, biological controls and synthetic fungicides (Calpouzos

1996; Bélanger et al.7997). These approaches will be discussed in more detail later in this chapter.

1.3 Powdery mildew (Uncinulø necator)

1.3.1 History of the disease (J. necator was first described by Schweinitz in 1834 on native American grapes. The first record of U. necator in England was in 1845 (Pearson et al. 1992), where it was named Odium tuckeri from 1847 until the discovery of cleistothecia in

1892 (Pearson et al" 1992). Between 1845 and 1850 tl necator spread across Europe where it reduced grape yields by 80% from 1847 to 1854 in France (Bulit et al. 1978).

The yields recovered to "1847 levels" in 1857 after the discovery that regular I application of sulphur could control powdery mildew (Pearson et al. 1992). Records of

(i. necator in Australia date back at ieast 130 years, bui eleistothecia were not found irr

Australia until 1984 (Wicks et al. 1985). 5

1.3.2 Disease cycle and epidemiology .U. necator is an obligate parasite of plants in the family Vitaceae, developing on green tissue during spring, srünmer and into autumn (Pearson et al. 1992). The fungus overwinters as either 1) dormant mycelium in buds (Pearson et al. 1985) or 2) cleistothecia caught in the bark of the vine or on dried berries (Pearson et al. 1985;

Jailloux et al. 1998) (Figure 1.1). Mycelium, which has overwintered within infected dormant buds, emerges and spreads when the buds burst in spring, producing severely diseased "flag shoots" (Figure l.l) (Pearson et al. 1985).

Figure 1.1; Infection cycle of U. necator

From Sticht (Pearson and Goheen 1988)

The density of flag shoots and infections originating from cleistothecia are dependent on the severity of the disease in the previous season, environmental conditions and the fungicide regime (Steinkellner 1998). The overwintering powdery mildew mycelium in closed buds is protected from attempts to control the fungus 6

(Loveless 1991) but, if infected buds are killed by frosts, the mycelium does not survive, delaying disease development (Gadoury et al. 1997 b).

As is common among polycyclic diseases, dispersal of conidia is a critical factor in the incidence and severity of the disease (V/illocquet et al. 1998). Conidia are dispersed by wind, rain splash and mechanical means, and have the potential to cause rapid spread of the disease through a vineyard (Pearson et al. 1992; Willocquet et al.

1998). Dispersal of conidia is mainly diurnal in dry periods and airborne density of conidia is directly related to wind strength (Willocquet et al. 1998), with a minimum wind speed of 2.3 ms-r required (Willocquet et al. 1998). The wind speed required for dispersal af 9Ao/" of eonidia within a vineyard is 25 ms-r or 90 kph (Willocquet et al.

j- r rrñ 'l i f f lL1 1 - L1- L---1---1^-^^ ^^---^-1-. Iyyó).^^ñ\ wnIIc rarcty fgacllgu wltllllr ùllç ualrupy, Suulr---^1- [uruulrrrlus ls uult[llullry Ëtrrlçr¿lrçu-^-^-^+^) by high-pressure spray equipment (Smart 1985).

Conidia are shaken off the leaves by the impacts of raindrops after light rainfall, rr* t¡ t //\l/;ll^^^"-f af ol l OOa\ l"n.t¡arrar oi.l-^-o nnnìrlia qrc mncf ohrrnrlqnf UP rV L -- tttttr \ rY l¡¡vwYuw! vl 4rr L t tvr) vr, s^¡ during dry periods, suggesting that periods of extended rainfall may have a detrimental effect on the spread of U. necator (Willocquet et al. 1998).

While in many areas U. necator overwinters predominantly as mycelium in buds

1 ' , )1 ' 1 --- -f :,-----l--,--:,- -L1^ //-1^¿^^: (rearson eI al. Iyö)), clelstotneola arc ntaJOr soul'çss u-l luuuulullr lrr uurçr ¿11 ças \LUrrçrr

¡ I r^^ô\ | ) L1 | lll- ¡I-^¿ glal. ly9/ a: JalllouxeLaL Lyyó).urclstotngulail'çI'cslllElltsulVrvaISuuçtulçsulat¿lltt^1 -r - -,--i---f ^-^ formed by the fusion of hyphae from two compatible mating types of the fungus. The cleistothecia of (1. necator are spherical and range in colour from yellow when i-.,-af,rra f^ Llo^L af mofir#fr¡ (l'lq¡7¡ttrtt ct ql IORR\ Thcr¡ nnnfqin fhe qqnncnnreq in rrrurrdlwv Lv ursvr\ s! rrrsrw¡rJ \vsvv4rJ r"^-'^-' asci (Cortesi et al. 1997 b; English-Loeb et al. 1998; Jailloux et al. 1998). The release of ascospores occurs after rainfall when the temperature is >1OoC, typically around bloom, when vines are most susceptible to infection (Gadoury et al. 7997 a). 7

Hyphae of U. necator grow superficially and penetrate individual epidermal cells by means of appressoria, which form penetration pegs (Heintz et al. 1990). The penetration peg swells within the host cell and forms a haustorium through which the fungus absorbs sugars and other nutrients from the plant cell. Conidia form on specialised hy,phae on the surface of the leaf or berry. Cor-ciia form 7 days after infection at 19oC and after 5 days at temperatures up to 30oC (Chellemi et al. 1991).

The number of conidia produced by a colony of U. necator varies with the age of the infected tissue; fewer conidia are produced by colonies on older tissue than on younger growth (Chellemi et al. 1991). There is also variation in the number of conidia formed by colonies between cultivars, particularly at lower temperatures (Chellemi et al. 1991).

If environmental conditions are ideal (ca 25"C, high humidity (>70%) and low light), U. necator can complete its life cycle in approximately 5 days. As temperatures reach 33oC, the life cycle is extended to 15 days (Delp 1954; Gubler et al. 1994) and at

35oC growth stops (Chavan et al. 1995). The minimum germination temperature for spores of U. necator is 6oC, and for infection and growth a minimum of 7oC is required

(Delp 1954). If multiple infection periods occur before bloom or fruit set, the proportion of fruit that is infected increases proportionally (Wilcox et al. 1997; Gadoury et al. 1997 b). Infection periods which occur after fruit set have less influence on final disease levels than infection periods occurring at the critical period from bloom to fruit set

(V/ilcox eI al. 1997).

Ascospores and conidia infect young green tissue, but can not easily infect older tissues (Gadoury et al. 1999). The percentage of conidia that germinate and successfully infect leaves is inversely proportional to the age of the leaves and berries (Delp 1954;

Doster et al. 1985 b; Gadoury et al. 7999). Also, few hlphae on mature leaves are able to form penetration pegs (Doster et al. 1985 b; Gadoury et al.1999). 8

1.4 Management of powdery mildew

1.4.1 Cultural controls l.4.l.l Canopy structure Chemical, biological and novel controls can be supplemented and enhanced through canopy management (Pearson et al. 1992). Increased canopy width provides high humidity, moderate temperatures and protection from ultra-violet light, conditions suited to the establishment of (J. necator (Smart 1974; Smart 1985; Pearson et al'

I9g2). Mycelial growth and spore production by U. necator are restricted by exposure

: - ¿t-^ l---^a:^- ^f ¡rC*^'.+ l Oa<\ I tO Ultfavlolet llgnt (UV,l ,tt, lïTespegtrve UI-t ulç uul¡lllull ur tJÃPUùurç^-,-^^,,-^ \ùurcrrL L7orr. Lvvt^.r' levels of tIV light increase the susceptibility of Chardonnay and Cabernet Sauvignon to powdery mildew through reduced production of phenolic compounds and reduced cuticular wax deposition (Keller et al. 2003).

Narrow, open canopies reduce the incidence and severity of powdery mildew, by providing an unfavourable microclimate (Smart 1985) and increased penetration of anti-fungal treatments. A reduced canopy has very little effect on the total

+L^.,i-- tha o,.rfqne lear¡cc nqrrr¡ nrrf rrrnst nf fhe nhofnswtheSis- Prrutuùyrrtrlçslù-L^+^^--+L^^:^ ur^f Llrv Yruv 4ù^o rrrv ---J ------2 with a "three leaf thick" canopy considered ideal (Smart 1974).

1.4.1.2 Other cultural control methods The removal of basal leaves can reduce the severity of infection of U. necator on berries (Chellemi et al. 1992), but not the incidence. The reduced severity is thought to be a combined effect of reduced humidity and increased light penetration (Chellemi et al. 1992). Removal of many leaves around bunches does not affect berry yield or weight but can affect the pH, aroma and flavour of the resulting wine (Bledsoe et al.

1938). The practice of removal of leaves is labour intensive, time consuming and costly, making it impractical for medium to large commercial vineyards. 9

1.4.1.3 Natural enemies (J. necator, like all living organisms, is not free of competitors, antagonists, predators and parasites. Natural enemies include bacteria, other fungi, insects and mites that vary from highly specialised to generalist antagonists. The efficacy of antagonists varies in relation to the suitability of environmental conditions and their ability to locate, if appropri ate, (J. necator on the vine. The impact of many natural enemies of U. necator, particularly arthropods, has not been extensively studied (English-Loeb et al.

1ee8).

Natural enemies must survive in the same environment as the pathogen and be

able to survive exposure to any pesticides applied to the vines (English-Loeb et al.

1998). Sulphur is toxic to most fungi, including many that are antagonistic to U.

necator, and to many of the mycophagous arthropods (English-Loeb et al. 1998). A

range of natural enemies of (J. necator, including the fungus, Ampelomyces quisqualis,

and the mite, Orthotydeus lambi (Acarina: Tydeidae), will be discussed in more detail

in sections 1.4.4.1 and 1.4.4.6 respectively.

1.4.2 Resistance Agrios (1997) defined resistance as the ability of an organism to exclude or

overcome, completely or in some degree, the effect of a pathogen or other damaging

factor. The response in Vitaceae species and cultivars to U. necator varies from highly

susceptible to moderately resistant (Doster et al. 1985 a;Li 1993; Staudt 1997). Based

on leaf infection rates, V. vinifera is one of the most susceptible species, although there

is some variation between cultivars (Olmo 1986). The least susceptible cultivars are

either American in origin or crosses of American and European vines (Doster et al.

1985 a).

The resistance of V. viniþra cultivars to U. necator shows a continuous

distribution, suggesting that resistance is polygenic and is the cumulative result of a 10 range of physical and chemical processes (Li 1993; Staudt 1997). This increases the

difficulties assooiated with breeding for resistance. There is a strong correlation between the resistance levels displayed by leaves and berries on individual cultivars (Li

1993). Plant defences include physical barriers, enzymes, hypersensitive responses

(Hahlbrock et al. 1987; Heintz et al. 1990; Agrios 1997) and, in some cases, mutualistic interactions with beneficial arthropods (O'Dowd et al. 1989; O'Dowd et al. l99I;

Agrawal 1997).

The physical ban'iers include \ryaxes, cutin, suberin, other cell wall components and leaf hairs. If the pathogen overcomes these barriers, the plant can respond to infection with enzymes, including $-1,3-glucanase, chitinases and peroxidases that atiack iirc irurgai ceil wails ¡ud nrc¡rrbraues (Hcirii¿ ei al. i99û; Vigei-s ef ai. i99i; Dan'r et al. 1996). Host cells can also develop thick papillae composed of layers of carbohydrate, silicon and phenolic compounds that prevent the penetration pegs

L-oo^I".i-- fLo /IJainJo at ¡1 l OOn\ Dlonf ¡lafan¡ao nnof fhc nlonf oncrort qnÁ ulv4vauué nlv ^ollvvll 'r'allvv4r¡ \rawrar!¿ wç sr. rttv), r l4r¡l uvav¡rvvu vvùr nutrients that could otherwise be used to grow and compete against other plants (Harms et al. 1992). Thus. the plant must strike a balance between defence and growth. The variation in susceptibility leads to the possibility of different control programs for different cultivars, with reduced spray appiications requiretÍ Íì¡r iess suscepti'ole cuitivars (Doster et ai. i 9E5 a; 'Wicks ei al. 't 992; Li ï993; Gatioury ei ai. 1998).

The variation in susceptibility to U. necator appears to be partially linked with the osmotic potential of the leaves (Goheen et al. 1963). Leaves with low water content onrl hich ncmnfin nnfanfiql qrê ñ^rê recicfanf fhan leor¡cc r¡¡.ifh hiohcr r¡¡qfcr nnnfenf cnd lower osmotic potential (Goheen et al. 1963). U. necator may not be able to form penetration pegs on leaves with osmotic potential greater than within its own cells

(Goheen et al. 1963). There is also evidence that elevated nitrogen levels in the leaves 11 result in increased susceptibility of grapevines to both U. necator and Plasmopara viticola (downy mildew) (Keller et al. 2003). Also, reduced levels of nitrogen lead to increased production of some phytoalexins, which have been associated with anti- fungal effects (Bavaresco et'al. 1987). Susceptibility of tissues of an individual vine also variesi yomg, green tissues such as new leaves, shoots and buds are more susceptible to infection by U. necator than older tissues (Doster et al. 1985 b; Robinson et al. 1997; Salzman et al. 1998). Excessive nitrogen fertiliser results in large quantities of young, green growth providing an ideal canopy for the establishment of powdery mildew.

The susceptibility of young leaves to infection by U. necator is partially related to the thickness of the cuticle plus the wall of epidermal cells. Leaves with the thinnest cuticle are the most susceptible (Heintz et al. 1939). The susceptibility of fruit decreases as levels of the thaumatin-like protein, VvTLI (Tattersall et al. 1997), chitinases (Robinson et al.1997) and other anti-fungal proteins increase (Salzman et al.

1993). The accumulation of these anti-fungal compounds appears to be a developmentally-induced defence mechanism that reduces the susceptibility of ripening fruit to attack from certain fungal pathogens (Salzman et al. 1998).

Systemic acquired resistance (SAR) is a non-specific defence system activated by salicylic acid produced by cells that are either damaged by disease, arthropods or by mechanical means and is effective against a range of pathogens and pests (Atkinson

1993; Gaffirey et al. 1993; Dempsey et al. 1999). Salicylic acid mediates the production

of proteins, enz)¡mes and free radicals that activate the defence mechanisms in cells remote from the initial damage (Dempsey et al. 1999). The proteins induced by salicylic

acid act by either directly attacking the pathogen, or strengthen the plant cellwall. T2

It is possible to activate SAR in plants by application of chemicals or avirulent races of pathogenic organisms (Dann et aI. 1996). Foliar application of salicylic acid,

2,6-dichloro-isonicotinic acid (DiA), ferric chloride, chitin and other compounds has been associated with the induction of SAR in some plant species @everall 1993; Dann et aI. 1996: Manandhar et al. 1998; Dempsey et al.1999).

While inducing resistance in vines has some potential as a means of controlling

U. necator, most of the benefit of SAR is in preventing disease and the materials have only limited application as curative treatments, therefore, once powdery mildew is established in a vineyard, materials that induce SAR are ineffective.

The variation in resistance of grapevine to U. necator creates an opportunity for plant breeders to develop cultivars with increased resistance to the fungus. Current breeding programs aim to produce vines that have increased resistance to a number of diseases, including powdery mildew, while maintaining or improving grape quality and y'reld (Olmo 1986). Species sr¡ch as Mu,scorlinia rofundiþlia (Small) have heen crossed with V. vinifera cultivars to produce new cultivars that offer resistance to disease (Olmo

198ó). Keller et al. (2003) showed that the ciitTerence in susceptibiiity between the cultivars Chardonnay and Cabernet Sauvignon to U. necator was due at least in part to

+L^ +Li^1.-^-- l^^"^- -^"' --..i.+^-+ uurLr vdrù h-"o rr4vu Lllv LlJlvlurvùù ut^f vLtLtvL¡tdt ^"+ì^"1^* "'^-tta^ ^-vtr tw4ywÐ. nrLrrwuórr^l+L^"-L llwvv ¡vÐrùLdrrf, ^"1+;"^-. ^^fa-fiolyvLv¡¡lr4¡ for use in new plantings, they do not solve the disease problems in existing vineyards, some of which are over 100 years old.

1.4.3 Chemical control

I a I f-¡rrrpnf r.nnfrnl vnpfhn¡ls ^ U. necator is controlled in most Australian vineyards by regular applications of sulphur, either as a wettable powder, dust, or flowable formulation, andlor synthetic fungicides (Pearson et al. 1990). Sulphur and vegetable oil formulations are the main 13 means of control of (J. necator in organic viticulfure but other materials, such as foliar coatings, e.g. clay and silicates, and inorganic salts, are used by some growers.

The choice of application methods for sulphur is dictated by environmental conditions; dusts are preferred in dry areas and other formulations with superior adhesion properties are used in areas with high rainfall (Pearson et al. 1992). Wettable sulphur is the preferred formulation in Australia mainly because rain is likely during the spring and early summer when vines are most susceptible to powdery mildew. The vapour phase of sulphur is the most active phase for control of U. necaror, providing effective control even if spray coverage is poor. One major advantage of sulphur over synthetic fungicides is that, despite repeated and continuous use over the last 150 years, no case of resistance has been reported. Sulphur is a multi-site frrngicide, which inhibits the production or activity of a range of enz¡rmes (Magarey et al. 1997). The development of resistance would, therefore, require multiple simultaneous mutations.

Disadvantages of sulphur are its poor perforrnance at low temperatures (Wicks et al. 1997), the optimal range being between 25o and 30oC (Pearson et al. 1992), detrimental effects on beneficial arthropods (Calvert et al. 1974) and its potential to aggtavate respiratory illness in some people. Sulphur can be phytotoxic, particularly above 30oC, causing leaf damage (Martin et al. 1931; Gubler et al. 1996), and is not recommended for use when temperatures over 35oC are expected. As mentioned previously sulphur can also taint wine, resulting in unpleasant odours and flavours

(Gubler et al. 1996).

While most synthetic fungicide treatments are less phytotoxic, and are active over a wider range of temperatures, than sulphur the risk of fungicide resistance is greater, as many have a single target site (Reh et al. 1995; Wicks et al. 1997).

Resistance of (J. necator to benomyl in the United States and Romania was reported t4 after three to four seasons of regular use (Pearson et al. 1992). Synthetic fungicides may be less harmful to beneficial insects and mites than sulphur but some still have a range of effects on non-target organisms (Ziv et al. 1983).

Resistance to DMI fungicides was first recorded in California in 1985, three years after the introduction of the DMI, triadimefon. Due to high temperatures and low disease pressure, no losses were incurred until 1989 when disease pressure was high

(Gubler et al. 1994). In 1986, growers in some affected areas reverted to sulphur to control powdery mildew; despite this, the level of resistance to triaclimefon dicl not decrease (Gubler et al. 1994). Cross-resistance to other DMI fungicides has also been reported (Pearson et al. 1992; Erikson et al. 1997: Ypema et al.1997).

ln view of the resistance af U. necator to synthetic fungicides and possible restrictions on the use of sulphur in organic vineyards (EC regulation2092191, IFOAM

1998), reliable, effective and inexpensive biological or novel controls need to be

develoned--" -- --'f -

1.4.3.2 Spray timing To control powdery mildew eflèetively, caretul ttmtng ot'application is required to optimize the effectiveness of the control agent and limit the establishment of U.

/\ Ã^-^-^-, 1ôfì-\ Tl-^,,^-+^+:.,a kan4aaa+^ f^- *^,,,,¡^*,, *.:1,{^.,, *,,^+ L^ necut,ul tIvIaB4It y çt^+ ¿r, ^l I a7 t ). L ruvçrrl4lrvç rrv6r,Lrlrvlrrù r\rr yl!/w\!vrJ rrrrr\¡çw rrruùL u! applied before outbreaks become so severe that the control method is no longer effective. If application of control agents or chemicals were timed to coincide with peak infection periods, disease could be maintained below the economic threshold lcnnrn'r¿irnrfelv\*rr.-..^...----_--.'.,4¡la ?-5ol" hnnch infectionl wifh fewer firnsicide annlications. A hiolosical control agent able to persist in a vineyard and provide control of U. necator in both the short and long term would reduce the number of applications required. 15

Reducing the number of spray applications required to control powdery mildew leads to reduced dispersal of conidia by spray equipment, soil compaction caused by tractors and production ofgreenhouse gasses.

1.4.4 Biological controls The definition of biological control varies according to author, but usually involves the use of a living organism to control a pest organism (Thomas et al. 1998). In terms of plant disease, it is often defined as: "the decrease in the volume of inoculum of a pathogen or a decrease in the severity of the disease by one or more organisms, including the host plant" @aker 1937). Biological control is an attractive alternative to the use of synthetic chemicals for controlling pests, as there is usually minimal disruption to the ecosystem of the area (Nechols et al. 1992). The costs of control can be reduced if the control organism persists in the field, and keeps the disease below its economic threshold (Nechols et al. 1992).

Biological control can be divided into three main categories: natural, augmentative and classical control.

Natural biological control is the control of pest species by predators and parasites occurring naturally in the target area (Thomas et al. 1998) without human manipulation of populations. This can be enhanced by managing the environment to conserve populations of natural control agents (Batra 1981). The use of sulphur in vineyards could limit the potential of natural controls.

Augmentative biological control uses naturally occurring organisms that are mass produced and released in large numbers when required to control pests or disease

(Batra 1981). Augmentative biological control of U. necalor using antagonistic fungi, bacteria ancl fungivorous mites would be most effective if conditions were favourable to an epidemic of the biological control agents (Stack et al. 1990). Again, applications of 16

sulphur are likely to compromise the efficacy and persistence of organisms used as

augmentative control agents of powdery mildew.

Classical biological control uses introduced predators and parasites, usually

from the pest's centre of origin, to control the pest organism (Hall et al. 1980; Batra

1981; Murdoch et al. 1985; Howarth 1991). If a classical biological control agent is to

be effective, it must be able to colonise infection sites, suppress the pathogen, disperse

to new tissue and persist on the plant (Schreiber et al. 1988). Before any classical

biological control agent can be recoÍrmended for use in controlling U. necator, various

criteria must be addressed, including the potential of the agent to affect non-target

organisms. These criteria also include reliable formulation, ease of production, storage,

rfailspurt-,r anu-,-t ¿{Jprrualluil -,-,-1:^^a:--- (r-ullrsuçIl/Í --,---)--- sr^¿ ^1dt. 1l.ìOl\tTot).

The first recorded attempts to use biological agents for the control of plant

pathogens was in 192I, when Hartley attempted to control damping off, caused by

D,'lL;"'^ .1-h^^'^-,'- 1-., innnrrlqfi-- nrrrcen¡ cnil rx¡ith 1? onfocnnicfin firnoi (Rql¿er t )/Ot,.Uttta ØvUqt tØr.Øtr.) vJ

19S7). Hartley's attempts were partly successful, reducing the occurrence of the disease

in his nursery by up to 65%o (Baker 1987). Since 1921 there have been many attempts at

biological control of a range of plant pathogens using other microorganisms (Besson et

I r^-^ ñ I l^ôá ñft L 1 1^^A- t l-if l^^/ TT^:l^^--i 1ôn/ an, t>/y; fJilKer työt; ttclalrger gt al, r7>+; rldJtduul----i trr-¡ al.-1 L>>+ 4)^- rri{ri4uur trL^¿ ^ldt. t>>+ b; Falk et al. i995; Asaka et ai. 't996; Dik ei ai. 1998). Approaches used include

antagonists that act as hyperparasites of the pathogen, e.g. Ampelomyces quisqualis, competitors for space and nutrients, and organisms that produce antibiotics that kill the

nqfhncan (Qiecrà lARl'L'vt) Flql¿cr 1QR7' Askarv ef el 'Pnzqnnv¡ lQQO' Korsfen et al 1qq5' l/s!¡¡v¿rv¡¡ '.vt).

Hofstein et al. 1996).

The risks and difficulties associated with the introduction of fungi or bacteria into Australia, and the presence of many suitable organisms in Australia, makes T7

augmentative biological control the most attractive option for this study. Formulations

of organisms antagonistic to U. necator and developed in other countries would be

evaluated if the organism were already present in Australia. Literature relating to the biological control of powdery mildew, particularly to organisms that were evaluated in this project, is reviewed in sections 1.4.4.1 to L4.4.6.

1.4.4.1 Amp elo my c es quìsqu alìs Fungi within the genus Ampelomyces aÍe parasites of a range of species of fungi

in the family Erysiphaceae (Sundheim 1982; Sundheim et al. 1988; Rankovic 1997).

Ampelomyces spp. were found on 75 of 229 plant species, including V. vinifera,

sampled by Rankovic (1997) and were shown to be hyperparasites of 33 species of

fungi. A. quisqualrs penetrates the hlphal cells of (J. necator by producing appressoria

and penetration pegs and then grows from cell to cell through the septal pores

(Sundheim et al. 1988). Ampelomyces spp. form pycnidia within the hlphae and

cleistothecia of U. necator (Sundheim et al. 1988).

The efficacy of A. quisqualis varies between species of plant and pathogen

(Sztejnberg et al. 1988; Rankovic 1997). A disadvantage of A. quisqual¡s, common

among biological control agents, is that its development often lags behind that of the pathogen resulting in damage to the host plant prior to the control of powdery mildew

(Falk et al. 1995). Another disadvantage is its requirement for free water for

germination, which compromises its efficacy in dry weather (Falk et al. 1995). Its

efficacy as a control agent is variable. In glasshouse trials on cucumber, A. quisqualis

did not control powdery mildew (Sphaerotheca fuliginea) on either susceptible or resistant plants (Dik et al. 1998). However, similar trials'have resulted in control equal to that of synthetic fungicides (Sundheim et al. 1988; Ado-Foul et al. 1996; Pasini et al.

1997). A. quisqualzs provided 90% control on U. necator during field trials in eastern 18

Russia when the relative humidity was greater than 70o/obut was less effective (<50%) if the humidity dropped below 70o/o (Puzarrova 1990).

The commercial formulation, AQ10@ @COGEN Inc.), maintained powdery mildew below economic thresholds if applied before the disease became prevalent and, ideally, before peak infection periods (Hofstein et al. 1996). If infection has reached

10Yo onthe foliage of vines, control by AQ10@ is less thar. 5o/o, therefore applications must be before the disease affects l}Yo of the canopy. AQ10@ should be applied just belore bud break, between flowering and bunch closure and just bcforc vcrasion if effective and reliable control of U. necator is to be achieved (Hofstein et al. 1996).

Trials conducted by Falk et al. (1995) and Bosshard þers. com., 2000) showed that A. quisqualis parasitised colonies oi U. necator effectively after 2 days of rain but was unable to provide reliable control in dry conditions. The variation in the level of control of the powdery mildews by A. quisqualis in these trials could be caused by val-lallon ln tne slraln oIf A, / qußquuus) | 1: :--lil llulrlrulry 1---,-^: l]u- allu--- I ¿1--rll9 ¡lliluuilt^--^^---. ut ^f .c.-^^lltrc ---^L^-walçl rrrt^- ¿L^Lllç surface of the leaves. A. quisqualrs overwinters in the cleistothecia of U. necator, reducing inoculum and providing populations of the hyperparasite at the beginning of the next season (Falk et al.1995; Hofstein et al. 1996).

AQ10@ is a stable formulation of A. quisqualis that can be stored fbr up to 12 months without loss of viability, and can be applied easily using existing spray equipment (Hofstein et al. 1996). While early formulations of A. quisqualis failed to protect vines in Australia (T. V/icks, pers. com., 1999), the suitability of A. quisqualis and new formulations of AQ10@ fo.r'.re as a control agent oi (J. necator in southern

Australian vineyards is largely unknown. Here, AQ10@ will be evaluated in both glasshouse and field trials as a means of control of U. necator. 19

1.4.4.2 B acillus subtilis Bacillus subtilis, a soil-borne bacterium with antibiotic and anti-fungal properties, has the potential to control powdery mildew (Asaka et al. 1996; De Lucca et al. L999). B. subtilis produces iturin A group antibiotics, surfactin, bacillomycin

(schreiber et al. 1988; Maget-Dana et al. 1992; De Lucca et al. 1999), mycosubtilin

(Schreiber et al. 1988), plipastatins (Yamada et al. 1990), a range of lytic enz)rmes, antibiotics (Asaka et al. 1996) and siderophores (Berg et aL.1994).

Antibiotic production by Bacillus spp., including ,8. subtilis, is highly dependent on the growth substrate used (Liefert et al. 1995). Liefert et al. (1995) found that the substrate inducing maximum antibiotic production was homogenized cabbage tissue.

Antibiotic production by B. subtilrs appears to be directly related to sporulation by the bacillus, therefore a continuous cycle of growth and sporulation must be maintained on the leaf surface for long-term control of U. necator (Liefert et al. 1995). If suitable conditions cannot be maintained on the leaf surface, B. subtilis sprays must be applied regularly on a program similar to current synthetic fungicides (Liefert et al. 1995).

B. subtilis has been used in various ways to control fungi, including soil inoculation and seed coatings for diseases such as damping-off caused by Pythium spp.

(Becker et al. 1988), foliar sprays and post-harvest sprays for control of stone fruit brown rot. B. subtilis has also been shown to provide protection against Aspergillus níger, Candida albicans and Fusarium oxysporum. Autoclaved supernatant has been used to control bean rust in glasshouses, but the level of control varied between plant species (Baker et al. 1985).

There are two potential drawbacks associated with the use of B. subtilis. Firstly, there is the potential for a negative impact on non-target organisms and, secondly, ,8. subtilis may inhibit Saccharomyces cerevísiae, the yeast used in wine production. Iturin

A, mycosubtilin and bacillomycin L are active against S. cerevisiøe (Besson et al. 20

1979). Mycosubtilìn inhibits S. cerevisiae at concentrations as low as 10 pg/ml (Besson et al. 1979), iturin A at 20 ¡rm/ml (Latoud et al. 1987) and bacillomycin L is inhibitory at 30 pglml (Besson et al. 1979). Itwin A damages the cell wall and membrane of S' cerevisiae causing the leakage of fatty acids and alteration of lipid composition within the cell, leading to cell death (Latoud et al. 1987). Also, bacillomycin L and iturin A have been found to be haemolytic, causing the release of haemoglobin and potassium

(K*) ions from erythrocytes (Latoud et al. 1987) and therefore may have adverse effects on the heaith of vineyard staff.

Serenade@, a formulation of B. subtilis produced by Agraquest, is registered for use against (J. necator in vineyards in the USA. Its suitability in South Australia

.,:.^^,,^-,{^,,,^^vlllçJ4ruù w4ù ñ^+tl\,L vv4lucLvu^,,^1,,^+-.1 Lo*o llw¡v uuw'l,,o t^.liflìnrrlfipcLU urrr¡vsrl in nhfoininovvrsr¡¡rrrÞ crrnnlies Other live cultnres of B. subtili,s were available commercially and these were evaluated along with a strain from South Australia from the collection of Prof. D. Pinnock (University of

Adelaide). The potential of the various fomrulations, and filtered or autoclavcd cultures, for control of grapevine powdery mildew in Australia has not been fully evaluated.

1.4.4.3 Pseudomonøs spp. Several species of Pseudotnonas produce antibiotic and anti-fungal compounds

(Sorensen et al. i996; De Lucca et al. 1999). The antibiotics produced by P. syringae are mainly small cyclic lipodepsinonapeptides called syringomycins, syringotoxins and syringostatins (Sorensen et al. 1996; De Lucca et al. 1999). Syringomycin E forms charge sensitive ion channels in the membrane of plants and yeasts, allowing K*, H4

. r and L-^).+ rons ro lear fiom cells, everrtually leading to cell death (De Lucca et al' 1999).

The compounds have strong anti-fungal properties but are generally more effective against yeasts than filamentous fungi such as (J. necaror (Sorensen et al. 1996). P. syringae also induces SAR within some plants, including cucumber and rice' 2l

Two commercial preparations of P. syringae, Bio-save 10@ and 11@

(Ecoscience), controlled Penicillium expansum and Botrytis cinerea as effectively as the synthetic chemical thiabendazole (Janisiewicz et al. 1997). The Bio-Save products may cause mild eye irritation and have mammalian LD 50 levels of 5000 mg/kg making them a low risk product to users (Anon. 1996 a). The major drawback of the Bio-Save preparations is the need to keep the product frozen at -56oC to avoid deterioration

(Anon. 1996 a), making long term storage on individual properties impractical.

Preparations of P. syringae have not been tested against powdery mildews.

P. syringae shows many of the features required of an augmentative biological control agent, having low mammalian toxicity, being easily produced and applied and potentially cheaply produced. However, its potential as a control of powdery mildew is largely unknown. Here, a strain of P. syringae ftom The University of Adelaide's Waite

Campus undergraduate teaching unit was tested as a biological control of U. necator.

1.4.4.4 Tríchoderm¿ spp. Trichoderma spp. are mycoparasites, normally found in soil, that are antagonistic to a range of plant pathogenic fungi (Cherif et al. 1990; Lorito et al. 1993). and can be used to control some phytoplane and rhizosphere pathogens (Ricard 1981).

Trichoderma spp. rarely penetrate the mycelium of the pathogen, but produce extracellular chitinases and B-1, 3-glucanases that disrupt the hyphal wall of the pathogen allowing the cytoplasm to leak out (Cherif et al. 1990). Trichoderma spp. also produce antibiotic compounds that are effective against the hyphae of plant pathogenic fungi (Lederer et al.1992).

A strain of T. virens was evaluated for suitability as a biological control agent of

U. necator in greenhouse and field trials. 22

1,4.4.5 Other microbial agents Pseudozynta flocculosa, a yeast-like fungus, is antagonistic to a number of species of Erysiphales and appears to act through antibiosis (Hajlaoui et al.1994 b). P. flocculosa causes rapid breakdown of the cytoplasm of U. necator and the retraction of the plasmalemma (Hajlaoui et al. 1994 a). Metabolites from P. flocculosa also reduced the growth and sporulation of B. cinerea (Hajlaoui et al. 1994 a). However, P. flocculosa appears to be most effective when the relative humidity is about 70olo, makirrg it bettur suited to control of powdery mildew in greenhousc crops than in vincs.

Bacillus species, including B. brevis, B. lichenlforrnis (De Lucca et al.1999). B. thuringiens¡s and B. cereus, have anti-fungal properties. B. licheniþrmls produces a range of enzymes and peptides that either bind chitin or inhibit cell growth, and effectively controlled plant pathogenic fuirgi in the glasshouse (De Lucca et al. 1999) but there are no publications reporting its ability to control U. necator. B. cereus and B. licheniformis controlled some post-harvest fungal diseases through antibiosis, causing cell lysis and reduction in spore formation (Korsten et al. 1995). A product containing two Bacilhrs spp. that is marketed for control of fungal disease in hay, Uayrite@ (Bio-

Care Technology Pty Ltd, Australia), was evaluated as a possible alternative to -8. subtiiis in Íield experiments. B. thuringiensis was not evaiuated in these experiments as it has insectici

Pythium oligandrum, a soil borne mycoparasite which may control powdery mildew, is available as the biofungicide Polyversrrmt lBiopreparoty Spol. S R O). A poiyoxin B compounci pro

Pharmaceutical as Polar@ (Reuveni et al. 2000). Polar af 0.025o/o (v/v) provided protection from U. necator and other powdery mildew fungi equivalent to 1% wettable 23 sulphur (Reuveni et al. 2000). Neither Polar@ nor Polyversumt was available to this project.

Azotobacter is a soil bacterium that has been reported to control some plant pathogenic fungi, including U. necator (Becker et al. 1988). Research to date suggests that the siderophores producedby Azotobacter or other bacteria, including fluorescent

Pseudomol,ds spp., inhibit the germination of fungal spores (Becker et al. 1988). Here, a coÍrmercially available product, Bioplex@ (Nutri+ech Solutions Pty Ltd), containing

Azotobacter sp. will be evaluated for biological control of U. necator.

While these and other microorganisms have shown potential as biological control agents of U. necator and some are available in commercial blends as compost boosters, drain cleaners and fungal retardants, only AQ10@ has been subjected to limited evaluation as a biological control agent for U. necator in Australia.

1.4.4.6 Mites Mutualistic associations between plants and arthropods have evolved over millions of years and usually take the form of the provision of food or shelter by the plant and performance of some task by the arthropod. Mites make up 90%o of the arthropod populations on the leaves of plants, and can be phytophagous pests or be beneficial, either as predators or by feeding on pathogenic fungi (Krantz 1978; Bailey

'Weeden 1979; Waite 1988; Croft et al.1992; et al. 1998 a; Weeden et al. 1998 b). Some grapevine species, e.g. V. riparia, have evolved leaf hairs and domatia, small protective structures on the underside of the leaf (Agrawal1997), where tydeid mites that feed on

U. necator can take refuge. Tydeid mites reduce powdery mildew on V. riparia (KrarÍz

1978; Agrawal 1997; English-Loeb et al. 1998; Jones 2000; Norton et al. 2000).

Domatia are less coÍrmon on European grape varieties and the populations of beneficial 24 mites and other arthropods are usually lower than on native American varieties

(English-Loeb et al. 1998).

Orthotydeus lambi is common on V. riparia in North America and has a significant effect on powdery mildew, reducing disease by 85% compared to plants without mites (English-Loeb et al. 1998; Jones 2000). O. lambi populations also reduce the number of cleistothecia (Jones 2000; Norton et al. 2000). Control of U. necator on commercial cultivars, such as Chardonnay, Riesling and Sauvignon Blanc, was less on leaves, 25 - 50yo, than on V. ripariø but similar on bunches at around 85% (English-

Loeb et al. 1998; Jones 2000). The reduced control of U. necator on the leaves of the commercial varieties in the USA is though to be related to the differences in leaf structure between commercial and native vadeties (Jones 200A; Norton et al. 2000).

Other tydeid mites, including Homeopronematus anconai Baker (Knop et al.

1983 a), found on the phytoplane of grapevine feed on powdery mildew but appear to

L^ I^-- +h^^ /-l l^^A; lWnnn al ql IOQ? o\ Dnnrrlofinnc nf Éf nø¡nmni nn rrine uv rvùù wllvvll^ff^^+;,,^ v w !ll4lr v. aurt,ua \¡ulvP vL vL. L / e¿ sr. L vP4L¿L leaves increase after the addition of pollen to the leaves; the larger populations are then able to control U. necator more effectively (Knop et al. 1983 a).

There appears to be a diverse community of tydeid mites in Australia but they are largely unidentified and feeding preferences are largely unknown. However, it is likely that some would feed on fungi suctr as [J. necator and could contribute to the control of powdery mildew (Childers, pers. com., 2003). Tydeid mites are highly susceptible to sulphur (Knop et al. 1983 a) and other pesticides which are commonly

,rooáqùvg i-¡rr .rina.rorr{cv rrrvi4uú fn ru vv¡¡rrv¡ ¡nnfrnl f I mo¡ntnv lTnnec tnOO\ O lnwhi ic fhe tnnst^.^""- nrnrnisinqr- - _'_^'^__Ð arthropod biological control agent of U. necator but it has not been recorded in

Australia. 25

1.4.5 Novel fungicides

1.4.5.1 Inorganic salts lnorganic salts, including dipotassium hydrogen phosphate 1çHPO+) and potassium di-hydrogen phosphate (KH2POa), have been used in the control of a range of powdery mildews (Reuveni et al. \995; Manandhar et al. 1998). The phosphates, applied with the surfactant Tween 20, proved as effective in controlling U. necator as the fungicides benomyl, penconazole EC and Dorado@ 1p¡fenox 200 mil)(Reuveni et al. 1995). Given that these experiments did not include a treatment of Tween 20 alone, and that surfactants including Tween 20 have been shown to provide partial control of powdery mildew (Schneider et al. 1990), the results of Reuveni and Reuveni (1995) may not be indicative of the efficacy of the salts alone.

Bowen et al. (1992) compared the efficacy of 17 mM soluble silicon sprays with

KzHPO¿ (3.7 and 11 mM) and water. The silicon sprays significantly reduced the number of colonies of U. necator after 16 days, whereas the number of colonies on

leaves treated with KzIIPO+ and water did not differ significantly.

Control of powdery mildews by phosphates is both local and systemic (Reuveni

et al. 1995). The treatment of grapevines with phosphate results in a significant increase

in peroxidase activity, which might damage the cell membranes and the metabolic processes of the pathogen. ln cucumbers, foliar application of potassium phosphate salts

induced elevated peroxidase activity (kving et al. 1990). kving and Kuc (1990) recorded increased activity of chitinase and B-1,3 -glucanase in cucumber plants treated with phosphates. As both enzymes are involved in SAR, this may have contributed to

the control of powdery mildew.

Bicarbonate solutions have also been used to control powdery mildew.

NatICO¡, mixed with oil (Sun spray ultra fine@), was more effective than Bayleton 50 26

DF@ and Benlate 50 WF@ at controlling powdery mildew on grape clusters (Pearson et al. 1990).NaHCO3andKHzPOa,atTo/oand0.5o/orespectively,inhibited S.pannosavar. rosae on rose in glasshouse trials when sprayed either weekly or forfnightly (Horst et al.

1992; Pasini et al. 1997). Pasini et al. (1997) found that NaHCO¡ caused leaf scalding on treated rose leaves. NaHCO3 provided good control of U. necator on V. vinifera cv.

Bacchus at rates of 0.5Yo and lo/o without any negative effects on wine quality (Reh et al. 1995). Potassium bicarbonate has caused severe leaf scalding and leaf drop on vines sprayed with a 2olo solution in conditions of high temperatures l> 30o C) in association with hot "dt\Ì'winds (L. Venall, pers. com. 2000).

Potassir-rm silicate gave the same level of proteetion from LI. necøtor an V. vinifera (cv. Bacchus) as Kumuius S@ (granuiar suiphur) in iow risk yeals (Reynoicis et al. 1996), but was less effective in years of high disease pressure. High concentrations of silicon have been observed in cell walls of infected leaves and around areas of

I I Ll f l------pilystoar', uarltagc,-r- - - apPrrc,atruüs-11 ur puldssrulrr--L- --: ---- slrru¿lrç-'-1:--r- rrlay supplgruErrt-----1---- çrruuBËlluus slllL]ul-:1:---- thus assisting the plant to restrict the infection (Bowen et aL 1992). Hyphae do not develop on areas of the leaf surface with thiek silicon cleposits, ancl plants are able to translocate the silicon to other areas of the leaf and surround the appressoria of U necator (tsowen et al. 1992). Srlrcon applied to roots is taken up by the piant anci translocated to the ieaves then depositeei arounri the appressoria of U. necator in a similar manner to silicon applied as a foliar spray, but no inhibition of the colonies of

U. necator occurs (Bowen et al. 1992). This suggests that the silicon on the surface of

¿r-^ f i-^1^i1^.:J^ rL^ J^--^f TT L,.+ ¿L^ LIIE lg¿1l^^f ltuiluttS utç ut vt tuplll\illt^--^-+ ul^f u, ilecutut--^^^¿^-. uuL ulç auuuillul¿1lluil^^^,..-,,1^+i^- ul ^f slilurjll^:l:^^.^ aruulru^-^.,-J appressoria does not.

Ferric chloride provided control of rice blast (Pyricularia oryzae), proving equal to the synthetic fungicide Hinosan 50 EC@ (edifenphos, Bayer, lndia) and better than 27 salicylic acid or þFIPO+ At rates greater than 50 mM, both ferric chloride and salicylic acid caused phytotoxicity and damaged the rice leaves, whereas þHPO+ did not cause damage, even at higher rates (Manandhar et al. 1998).

While there has been extensive research into the use of salts for the control of powdery mildew, there are still concerns among growers regarding phytotoxicity, particularly when mixed with oils, and delays in maturation of berries. Salts were included in this project to assess application rates for Australian conditions when applied alone or mixed with vegetable oils. Salts such as NaHCO¡ and KHzPO¿ are readily available, inexpensive, easily stored and can be applied using existing equipment, making them potential treatments for U. necator.

1.4.5.2 Surfactants Applications of some surfactants in association with pesticides have fungistatic and fungicidal actions (Schneider et al. 1990). Schneider (1998) found that regular application of Agral 90@ (nonylphenoxy polyethyoxyethanol, Norac Concepts Inc.) reduced the severity and rate of spread of S. íuliginea on cucumber. Although surfactants show potential to control U. necator, their efficacy has not been extensively evaluated in Australia. In this project, surfactants were evaluated in greenhouse and field experiments, alone and in conjunction with other products. The interaction between surfactants suitable for use in organic agriculture and novel products was also investigated.

1.4.5.3 Mitk/whey Milk was effective against powdery mildew on zucchini in glasshouse trials, in which weekly applications of milk at concentrations of up to 50o/o proved more effective than weekly applications of the synthetic fungicides fenarimol (0.1 mW 2.5%o emulsifiable concentrate) and benomyl (0.4 mUL S}o/oberuimidazole) (Bettiol 1999). 28

Whey, which is a byproduct of cheese production, mây retain the fungicidal

properties of whole milk. Each kilo of cheese produced results in at least 7 kg of whey.

Worldwide production of whey in 1990 was over 110 million tonnes and has been

increasing by approximately 3%o per year (Sienkiewicz et al. 1990). Whey is used in a

range of products, including ice cream, infant formula and beverage and yeast

production but much is still disposed of as waste (Sienkiewicz et al. 1990). The

Australian dairy industry produced 60,000 tonnes of whey in 1999 (Australian Dairy

Corporation 1999 a); of this 45,000 tonnes was processed and the remaining 15,000

tonnes disposed of as waste (Australian Dairy Corporation 1999 a).

If whey has the same fungicidal properties as milk, its use in agriculture would

reduce the volume of waste. Based on the report by Bettiol (1999), milk ¿nd whey were

assessed as novel control agents of U. necator.

1.4.5.4 Compost extracts D-,¿-^^¿^ 7^^-- /11 ¿^^-ll\ l----^ l- L^ r_i^uaurs ul^f uurrrPuùr ^^--^-L arru^-l uulsu lllalluls ( uulllpust-^-^--- --¿ Lvidö ) il¡1v9 uvglr---- siluwrr-1^^---- tu 'Weltzien control U. necator and other mildew fungi (Ketterer et al. 1987; 1989; Elad et

al. 1994; Sackenheim et al. 1994). The mechanisms of control may involve a combination of factors such as competition, parasitism and induced plant defences

(Béianger et al. 1998). A possible advantage of compost teas is that multipie microorganisms are more likely to survive than a single organism in the vine canopy, as conditions optimal for a single organism in the field may be rare (Sackenheim et al.

tee4).

'T.L^r rlv v^rr4vLÐ^.,+-^^+^ (uç^-^ +L^Lllt; llILçIçL.tf:l+^-^l l.lul\.Ifl.,:l tl\IltÂ.^* d,^ tçrtllçllL¿Lrutlf^*^-+^+:^- uI^f ¡altltltd'l^-:*^1 .-^..,.-^ llt4llrilç (ulu^-l water, with additives, such as sugar, molasses, yeast or other nutrients, to promote microbial growth (Ketterer et al. 1987; Weltzien 1989; Sackenheim et aL 1994). The microbial communities present in compost teas vary with starting material, 29 environmental conditions, length of fermentation and additives (V/eltzien 1989;

Sackenheim et al. 1994). Grape marc has also been used as a base for a compost extract, which was effective against B. cinerea on grapevines after 10 days of fermentation even when diluted 25 times (Elad et al. 1994).

The variable nature of compost teas, the difficulties involved in brewing and filtering and the concerns of some growers that human pathogens may be applied to grape berries, makes them unsuitable for this project. However, the commercial microbial complex Nutri-life@ 4D0@ (which contains four fungi and 20 bacteria, from

Nutri-life Products Pty Ltd), was included in the greenhouse and field experiments.

1.4.5.6 Chitosan/chitin Chitosan (B-1,4-D-glucosamine), a component of crab shell, has been found to inhibit some fungal diseases on vegetable crops (Benhamou et al. 1992; Sathiyabama et al. 1998). Chitosan appears to induce resistance, inhibit fungal growth and increase plant growth rate (Sathiyabama et al. 1998). Chitin and chitosan are stable compounds that are easily stored and handled but chitosan is soluble only at a pH of less than 5.5 and chitin is almost insoluble. Applyng sprays with a pH of 5.5 may result in leaf damage so suspensions of chitin or chitosan powder may be the only effective and safe method of application. Despite these difficulties, chitin and chitosan were tested in the greenhouse.

A number of foliar nutrient formulations contain chitosan and it is claimed that

they reduce the severity of powdery mildew. Such products have not been extensively

tested in the field in Australia. Aminog¡o@, produced by Organic Crop Protectants Pty

Ltd, Vita Kelp@ and chitosan (Sigma Chemical Company) were evaluated for the

control of U. necator. 30

1.4.5.7 MR formulation Mixtures containing methionine and riboflavin (MR formulation, see Appendix

2 section A 2.3) reduced the severity of powdery mildew on grapevines in glasshouse

trials (Tzeng et al. 1989; Wang et al. 1998). Methionine and riboflavin are sulphur-rich

compounds that, exposed to UV light, produce free radicals, including hydroxyl radicals

and singlet oxygen, which are biocidal to a range of microorganisms (Wang et al.

1998). MR formulations have also been shown to inhibit Verticillium dahliae, Fusarium

oxysporum, Colletotrichum clematium anú Phytophthora infestans (Tzeng et al. 1984;

forrlan ef-'--- al 199?l"'-/-

While MR formulation has been successful in inhibiting microorganisms in

laboratory situations, its efficacy in controlling powdery mildew in the field is largely

unknown. Concentrations of methionine and riboflavin in MR formulations are low (see

Appendix 2 section A2.3) but the cost of the material at rates required in the field may

make it uneconomic. Nevertheless MR formulation was evaluated in this project.

1.5 OiIs Oils have been used as an alternative or supplennent to sulphur and synthetic

fungicides as control agents of powdery mildew and are often applied mixed with

inorganic salts. Calpouzos (1959) discussed the use of petroleum oils to control a fungal

disease in bananas. The first recorded trials of oils were in l9I4 by Barker and Lees

(Martin et al. 1931). [n a comparison of three mineral and two plant oils, canola

(Brassica napus) and soy (Glycine max), Northover (1996) found that the mineral oils displayed moderate protective and curative properties but the plant oils reduced disease only marginally. Likewise, Wicks and Hitch (1998) reporte<Í minerai oils to be more effective than vegetable oils for the control of powdery mildew but less effective than synthetic chemicals. Other researchers found vegetable oils to be effective against 31 powdery mildew on grapevine, particularly if mixed with either sodium or potassium bicarbonate (Ziv et al. 1993).

Oils may cause leaf burning if applied at rates greater thartl/o and over 1000

Llha (Calpouzos 1996) and are only effective if almost complete coverage of the leaf and fruit surface is achieved (Wicks et al. 1998). Several vegetable oils, including canola oil, grape-seed oil, garlic oil, soy-bean oil and neem oil, have been evaluated for control of powdery mildews (Rovesti et al. 1992; Ziv et al. 1993; Wicks et al. 1998).

Tests of a range of vegetable oils against hop powdery mildew (Sphaerotheca humuli) showed that cotton seed and sesame oils were effective if applied at rates of 0.25%o

(Northover et aL 1996). Olive oil and peach kernel oil were effective at rates of 0.5%o, canola oil at 2o/o and castor oil at 4% (Martin et al. 1931). úr all cases, l00o/o leaf coverage was required to obtain control of S. humuli, and only canola oil caused leaf damage (Martin et al. 1931).

Essential oils such as tea tree oil, neem oil and eucallptus oil have been used extensively as traditional treatments for a range of bacterial and fungal human pathogens (Hammer et al. 1999), however, their effects on powdery mildew fungi have not been widely evaluated. They may impart unwanted flavours to wines produced from berries sprayed just prior to harvest, whereas the application of mineral oils had no effect on the flavour of wine (Wicks and Hitch 1998).

The mode of action of oils is poorly understood. Mineral oils do not prevent production or germination of spores and do not appear to have a significant effect on the growth of mycelium (Calpouzos et al. 1959). Mineral oils are not permitted in organic viticulture, therefore, only vegetable oils are considered further in this review. 32

1.5.1 Canola oil Canola oil has been evaluated as a control agent of powdery mildew fungi including (J. necator (Schneider et al. 1990). Mixtures of canola oil and Agral 90@ controlled powdery mildew of cucumbers and reduced the spread of the pathogen to new growth (Schneider et al. 1990). Application of such mixtures provided more effective control of S. fuliginea than Agral 90@ alone, however, repeated used caused some pitting and leatheriness of leaves (Schneider et al. 1990).

The efficacy of canola oil against powdery and downy mildews of grapevines

L^- L-^- :- .,^*:^+., It¿ts uçtrrl E;v¿1'luatgu^-.^l-,^+^-¡ ?IL ^4 a7 ^ -^--^tiTJlëç ul^f uullt^^.^^^..+-^+i^.^^ çlrll 4Lrulrù 4lr(l^..l llr 4^ v(lrrsùy \Jr^f rJ\JrlL¡lLrLrls. ^^-l:+.:^-^ 'T'L^r lrE poorest performance suggested that l%o canola oil mixed with Agral 90@ was no better than Agral 90@ alone and provided only minimal protection when compared to a water control (Northover et al. 1996). Canola oil controls U. necator at rates of 2o/o or above

(Martin et al. 1931). The use of canola oil can lead to leaf burn if applied in full sun when the temperature exceeds 30oC (schneider et al. 1990; Pasini et al. 1997).

Synertrol Horti-Oil@ (Organic Crop Protectants Pty Ltd), canola oil with 0.5% eucaiyptus oii, prociuceci in Austraiia, controis severai species of pow

Synertrol Horti-Oil@ is that, as with other canola-based oils, it may damage leaves if applied during hot weather. Synertrol Horti-Oil@ has also displayed phytotoxic effects

, 1 ' r 1 t' 1 a /ñ--:-: i0 rose isavss tn giassilousc inais Anq Cail--,, CaUSe- Soiiie gieasiiiess-----:,---- Ol-c1-- ieAves-- (-i--Asljli et-1 ai.-1 reeT).

In this study, Synertrol Horti-Oil@ was evaluated in greenhouse and field trials, either alone or in mixed with salts and other materials. aa JJ

1.5.2 Neem oil Oils extracted from neem trees, Azadirachta indica, have been used in a number of countries to control insect and nematode pests (Rovesti et al. 1992) and are known to have antimicrobial properties (Schmutterer 1995; Pasini et al. 1997). The active component of the neem extracts is azadirachtin (Rovesti et al. 1992). Neem kernel extracts inhibit some plant pathogenic fungi, including S. fuliginea and Erysiphe graminis, but have little effect on others, such as Phytophthora infestans and

Cercospora beticola (Rovesti et al. 1992). Control of pathogenic fungr in soil by neem kernel extracts were originally thought to be due to an increase in populations of beneficial organisms but Rovesti et al. (1992) showed that the extract has a direct

antimicrobial effect on phytoplane fungi.

Neem extracts were effective against S. pannosa vaÍ. rosae when applied at a

rate of 0.5Yo vlv, providing control similar to Synertrol Horti-Oil@ and slightly better

than AQ10@ (Pasini et al. 1997). However, neem oil applications at rates greater than

l% (wlw) can be phytotoxic and may reduce yield. Toxicity is due to the solvent used

for application of the oil (Ermel et al. 1995).

As neem is a broad-spectrum pesticide (Ermel et al. 1995) there is a risk of

negative effects on non-target and beneficial organisms within the vine canopy.

However, a review by Schmutterer (1995) found that the effect on most non-target

organisms was small and, in most cases, acceptable in an IPM program.

Here, a potassium soap of neem oil, Neemtech@ (Moeco Pty Ltd), available in

Australia as a surfactant and nutrient supplement, and Azamax@ (Organic Crop

Protectants Pty Ltd), an azadirachtin sesame oil formulation, were evaluated for their

ability to control U. necator. 34

1.5.3 Eucalyptus oil The essential oil obtained fuom Eucalyptus polybracteahas been tested, in vitro,

on a range of Gram negative and Gram positive bacteria and yeasts and was inhibitory

at 0.5 - 2.0 % v/v (Hammer et al. 1999). Due to the high cost of eucalyptus oil it was

evaluated only as a component of Synertrol Horti-Oil@ in this study.

1.5.4 Tea tree oil Melaleuca alternifolia, tea tree, is endemic to Australia and has been used by

Australia's Aboriginal people for treatment of ailments, including dermal fungal

:-^C^^L:^--- f^-- ¿I-^-----^)- ^1 ----,-- / ô^^^ - l \ mt 'l ¡ ' rrrrsuuurrs, rur rflur.rs¡lilus ur yËars (.É\fluil,^,--,- zvvv a; .Él_Iton.^ zvvv^^^^ D). tne oll conlatns

terpines, cineol, terpinol and a range of other compounds with broad spectrum

antimicrobial properties (Altman 1 989).

An Australian product that contains 3o/o tea tree oil (TTO), provided little control of fruit rot in strawberries, caused by B. cinerea (31oá control), but controlled some other fungi (V/ashington et al. 1999). However, due to the cost of TTO and the potential for leaf damage it was not evaluated in this project.

1.5.5 Other foliar coatings Foliar coatings assessed for their ability to protect plants from disease include rvaxes, silicon. polymer coatings (Ziv et al. 1983) and liquid paraffin (Martin et al.

1931). These provide physical or chemical barriers to infection and act as anti- transpirants to "suffocate" the pathogen (Ziv et al. 1983). Nu-film 17@ (poly-l-p-

Methene) and Vapor Gard@ (pinolene) provided moderate control of U. necator oî grapes, but were less effeetive than Bayleton 50 DF@ (Pearson et a-1. 1990,ì. Wilt pt'-tf'

(di-1-p-menthene, Nursery Specialty Products Pty Ltd), an anti-transpirant, and Sta- fresh 460@ (thiabendazole, FMC Co., Lakeland, FL.), a fruit storage spray, provided control of powdery mildew on wheat, similar to that due to Benomyl (Ziv et al. 1983). 35

However, similar products have been ineffective, for example, BIO-S@ (sulphur-rich bioremediation product) was effective against U. necator only if disease pressure was low and Algifert@, a seaweed extract (Algea AS, Norway), provide no protection against U. necator, B. cinerea or Plasmopara viticola (Holz 1983).

Other foliar sprays that have been used to control U. necator include paraffin and whitewash (fine clay). Liquid and medicinal paraffin were effective at 2Yo and 3o/o respectively when mixed with surfactants. However, 100% coverage is required and leaf scald may occur (Martin et al. 1931). Application of whitewashes (10% w/v) and clays has been shown to control powdery mildew of squash caused by S. fuliginea

(Marco et al. 1994).

Giberellic acid (GA3), applied to leaves of grapevines after budbreak, during flowering and before bunch closure in two seasons, reduced infection by B. cinerea but had no effect on infectionby U. necator (Amati et al. 1996). Other foliar treatments that had no detrimental effects on must or wine quality include a range fatty acids, the best of which provided control of (J. necator apptoximately equal to sulphur (Kumulus

80 WPl (Lattore et al. 1996).

Foliar coatings usually provide inconsistent control of U. necator and are rarely effective at high disease pressure. They were not individually evaluated in this study.

However, the effectiveness of fatty acids may explain the success of milk as a control agent for powdery mildew on cucurbits demonstrated by Bettiol (1999).

1.6 Summary Despite the considerable research on the control of various powdery mildews, there is no program to control powdery mildew in organic vineyards in Australia which does not involve sulphur. This project was established to develop such a program by evaluating a range of materials selected from the literature and elucidating the mode of 36 action of the most promising materials. Some materials, such as Serenade@, were considered worthy of evaluation but were not available for testing.

The materials were chosen to represent the groups discussed in this chapter and included bacteria, fungi, oils, salts and novel fungicides. While the main criteria for selection in the experiments and final programs was efficacy in the control of powdery mildew, cost effectiveness, ease of handling, storage and application, impact on the environment and human health, and suitability for the organic industry, were also considered. 37

Chapter 2 - General methods and materials

2.1 Greenhouse experiments

2.1.1 Maintenance of vines The greenhouse experiments were conducted at the Plant Research Centre

(PRC) at the V/aite Campus of The University of Adelaide. The vines used were own- rooted V. vinifera cv. Viognier or cv. Cabernet Sauvignon, supplied by Temple Bruer

Wines Pty Ltd. They were grown in 15 or 25 cm diameter pots filled with UC potting mix (V/icks et al. 1998). The plants were maintained at 20 - 25oC and watered as required, using a drip irrigation system to prevent splash of water onto the leaves.

Thrive@ (Arthur Yates and Co.) liquid fertiliser was applied to the soil monthly (100 ml of 8 g/ 4.5 L water per pot), to maintain plant health and promote growth of new, powdery mildew susceptible leaves. Growth was maintained throughout the year by exposing the vines to a minimum of 12 h of light per day, using natural light supplemented from April to October with two 1000-watt halide lights.

Cv. Viognier was selected for greenhouse trials as it is susceptible to U. necator and was readily available from Temple Bruer Wines at the time, whereas cv. Cabernet

Sauvignon was used when Viognier vines were not available. Cabernet Sauvignon had been used in greenhouse experiments on previous occasions (Stummer et al. 2000);

(Savocchia et al. 1999).

The plants were protected from infection by U. necator by suspending wicks impregnated with the fungicide, Topas@, between the plants until 4 weeks prior to the first experiment (Evans et al. 1996). After that time, no action was taken to prevent incidental infection by U. necator. 38

2.1.2 Inoculation, treatment and assessment of vines After the removal of the Topas@ wicks, the vines used in the first experiment were inoculated by brushing conidia onto leaves. The U. necator used for the first inoculation was grown on cv. Viognier in a separate growth chamber with 14 hour /10 hour day / night periods, at 25oC and approximately 50%o relative humidity. The cultures used to inoculate these vines comprised a mixture of eight clonal lines isolated by B. Stummer and S. Savocchia in 1997-99 and maintained on micropropagated plantlets of cv. Cabernet Sauvignon (Savocchia et al. 1999; Stumrner et al. 2000); fF ¿ 1 l^^/\ : -- I - ':,^-- lfct ---^'¡:--- /ì,f..-^^L:-^ (.úvans eI al. tyYo). -iI nemlcropropagatcu vlrrss wtrrç gruwrr-:-- lrlrvlù rilsululrl uvrur.lùruËç et al. 7962) with nutrients at half strength, and 15 g/L sucrose in 0.7 o/o agar (Bitek,

Difco Laboratories, Michigan) in 500 ml culture tubes with breather lids (Evans et al.

1996). To maintain a consistent supply of inoculum for the scanning electron microscope experiments, every 8 weeks conidia from plantlets were transferred to newly micropropagated plantlets using a sterile artist's brush and the dual cultures were maintained at 25oC with a 16 h photoperiod. Leaves with sporulating colonies of U.

øanntnv oa¡7 n^nirlìo .¡¡aro lrnrchcã nnfn fhc lcqr¡pc nn cqnh r¡ine rrcing :¡ rù9vøLv, "ro-ovY vrv vvr¡vvlvu^^llo¡Iaà 4uu vvruurs sterile artist's brush; one infected leaf was used to inoculate each experimental vine.

After the initial inoculation, vines generally became naturally infected with powdery mildew from conidia that were present in the greenhouse. The vines were arranged into fbur to six blocks based on initiai

Completely Randomized Block Design using Genstat@ (Lawes Agricultural Trust) version 5 release 4.I or version 6.1.0. Eight fully developed leaves, less than 8 weeks old, on each vine were selected, and tagged for later identification. The tags were numbered from l to 8 and placed on randomly selected leaves. The selected leaves were 39 scored for disease prior to treatment, using a 0 to 5 scale based on the percentage of the leaf surface area with visible growth of U. necator, as follows;

0; no obvious disease

1; l- 20% of leaf area covered by sporulating U. necator

2; 2l - 40% of leaf area covered by sporulating U. necator

3; 4l - 60% of leaf area covered by sporulating U. necator

4; 6l - 80% of leaf area covered by sporulating U. necator

5; 81 - 100% of leaf area covered by sporulating U. necator

The test materials were applied to four of the eight leaves on each plant (leaves

1-4), one treatment per plant, each replicated once within each block. The materials were sprayed to run off on the upper and lower surfaces of each leaf individually from a l-L hand-held spray bottle. All test materials were applied as aqueous suspensions, using water from a Milli Q Plus Millipore water filter. No surfactants were added unless otherwise stated. Test materials were applied fortnightly for 6 weeks after the initial disease assessment. All leaves were inoculated with conidia of (J. necator I and 3 weeks after the test materials were first applied if no disease was present on untreated leaves at that time. The vines were separated from each other during treatment to ensure that test materials could not contaminate vines allocated to receive other test materials.

Untreated leaves were shielded using rigid plastic sheets to prevent accidental deposition of sprays, which might affect the severity of powdery mildew and confound the results.

The plants were scored non-destructively prior to the first application of the test materials and then weekly for 5 weeks. Assessment of the leaves during the experiment was made immediately prior to each application of the materials. At the completion of the experiment, the four treated leaves and four untreated leaves were removed from the 40 vines and examined using a binocular dissecting microscope (V/ild Heerbrugg M8) to assess the area of the leaf colonised by (J. necator and the appearance of each colony.

This assessment was made to verify the progressive, non-destructive scores and to count cleistothecia and determine their maturity.

At the completion of each experiment, the vines were pruned to remove any treated leaves and then allowed to grow to the original size. The plants were sprayed with the azadirachiín (I.2%) - sesame oil pesticide, Azarnax@, mixed with Eco-Oil@

(Organic Crop Protectants Pty Ltd) to control outbreaks of phytophagous mites. After application of the miticides, the plants were left untreated for 3 weeks to allow natural infection of (-/. necator to become established. Plants that were unsuitable for

1 , r, l--^ ¿- 1^^1- 1^^.,^^ *^^* .,:^^'.* suDscqugnt gxpgfllllglr[s,- -.-L- uuY tu a- l¿1çlt ur^f ùuùuçPrrure^-,^^^-+:L1^ rr/4vvù, P\rur vré\,ur vr^. PuJorwsr ^L.'.;^.1 damage, were replaced with reserve plants of the same age that had been growing alongside the vines used in the experiments. The reserve vines were watered, fertilized

o+ tha como fimc oc r¡ìnec rrced in fhc cvnerirnenfs tn minimise ânv ulu^-'l --,no¡llJr u¡rvu ú! Lltv differences in age and size of the available leaves.

Data collected from the experiments conducted in 2000 and the first two tn 2001 were analysed with assistance from Michelle Lorimer of BiometricsSA. Analysis of

i L ¡--a at-- f^--^^¿l-^^i^ ¿1-^+ ¿L^-^ L^ .^^ vanance (l{L\uvl{, was uscu tu tçst tIrE Jlylruultrsrs ul4r tilt'rqr -,.^.,11wuutl! utr rru,)iËrtr[v4,,¡^:-:+:^^-+ difference in the mean disease score among test materials. The disease seore for the

differences untreated leaves from treated vines was used as a co-variate to account for ,l in disease severity between plants. Differences in the mean disease scores for untreated

qnd laq.¡ec nn rrnfrcqfcÁ rrinac nn r¡ines receivino___. .--^Þ the tesf materials worlld sllssestaa that data for the latter may have been unreliable or that the particular test material in question may have had systemic disease control properties. That material would, therefore, be tested in later experiments. Many of the treated leaves on vines in 4l experiment 212000 were free of disease so data for that experiment were also analysed using the binary response, infected or not infected (Lorimer 2000 a). The 5% level of significance @:0.05) was used for all experiments. Data from experiment 312001 onwards were analysed in a similar manner, without assistance, using Genstat (Lawes

Agricultural Trust) version 5 release 4.1 or version 6.1.0.

The data from some of the greenhouse experiments were skewed by the large difference in disease severity between the untreated vines and vines that had received the test materials. The large difference in disease score increased the values of the standard error (S.E.) and least significant difference (L.S.D.), which may have masked significant differences in the severity of powdery mildew in response to the various test materials. To allow comparisons amongst test materials, disease severity data for the greenhouse experiments were re-analysed, as before, but without the data for the untreated vines.

Two potential problems \Ã/ere identified with the use of ANOVA for the 'Wines 2002/2003 Temple Bruer vineyard experiment. Inconsistencies in vine vigour occurred across the experimental site and there was a large number of 0 scores, particularly for the sulphur, milk and 45 glL whey treatments. ANOVA is not appropriate when data are not normally distributed and there is a high proportion of 0 scores for some of the treatments (Lim, pers. com. 2003). The data were re-analysed with assistance from Patrick Lim, BiometricsSA, using a linear mixed model to account for variations in the field site and the numerous 0 scores (Lim 2003). The model was fitted with a Spatial Analysis Mixed Model (SAMM, (Butler et al. 2000) with S-Plus

(Insighttul Corp, 2002). 42

2.1.3 Management of pests Chemical treatments such as insecticides were avoided during experiments and for 4 weeks prior to the first application of test materials. Pests were controlled during and between experiments using yellow sticky traps and releases of the parasitoid wasp,

Encarsia formosa (to reduce populations of white fly) and the predatory mites,

Amblyseius victoriensis and Galendromous occidentalis (to reduce phytophagous mites). Regular re-application of the biological control agents was required. When pest mite populations caused noticeable damage, Azamax@ was applied prior to the release

+L^ L^-^{i^:^l *i+^^ - -^*^-/aì *^., L^ â,--;^;,{^l ,{;ã nnf \-rI^f Ltlu uvrlvllvr4l llllLvù, rlù^ ruall4^v^ utqJ uw ¡ u¡érgru4rt v^Pwrr¡r¡wr¡rù^-^-;*--+. uru commence until at least 4 weeks after application of this material.

2.2 Field experiments

2.2.1 FÍeld sites Four sites in South Australia were used for field experiments, namely; vineyards at Temple Bruer Wines Pty Ltd at Langhorne Creek. Glenara Wines Pty Ltd at Upper

Hermitage, Mountadam Vineyard at Eden Valley, and V/arriparinga on the southern

Adeiai

2.2.1.1 Temple Bruer Wines vineyards Temple Bruer Wines provided the largest expeimental site and was used in three consecutive years. V. vinifera cv. Verdelho was used in the 2000/01 and 2002103 seasons (Figure 2.1) and cv. Shiraz was used in the 2001102 season. The vineyard is about 60 km to the south-east of Adelaide, on the Fleurieu Peninsula (Appendix 1, Map

3). Climate data, obtained from the nearest weather station at Strathalbyn, approximately 14 km to the north-west of Temple Bruer Wines vineyards, are presented in Appendix 3. 43

The Verdelho vines, planted in 1996 and the Shiraz, planted in 2000, were drip- irrigated with river and bore water. Lrigation was supplemented in the 200112002 season by floodwaters in September 200L The vine rows were oriented in a north-south direction. The vines were grown on a Smart-Dyson trellis system with the canopy rarely exceeding 30 cm in width. The vines were planted 1.5 m apart in rows 2m apart and grew to a height of approximately 1.8 m. No pruning was required during the growing season. The vineyard has been managed to organic standards since 1991. Sulphur and oil plus bicarbonate mixtures were applied for control of powdery mildew and copper fungicides for downy mildew.

Vines were spur pruned to two shoots in June each year. As many as three flag shoots were observed on individual vines. The Shiraz vines were only 15 months old when included in the field experiment and flag shoots were not evident.

Figure 2.1: Verdelho vines at Temple Bruer Wines, November 2000 44

2.2.1.2 Glenara Wines vineyard The Glenara Wines vineyard is situated approximately 30 km north-east of

Adelaide, at Upper Hermitage in the Adelaide Hills (Appendix 1, Map 1). The long- term climate averages obtained for the Lenswood Research Centre are included in

Appendix 3. Weather data were obtained from Mount Barker, 20 km to the south of

Glenara (Appendix 3). Experiments were established each season using Chardonnay vines on a west-facing slope in deep sandy loam. The Chardonnay vines were planted in

1989 and aligned in an east-west direction. The land has been managed using organic metlrorls since clearins and has l-reen nlanted with vines since 1985. Disease control had been achieved mainly by application of sulphur and mixtures of oil plus bicarbonate.

Vine vigour was high during the experiments and the canopy was pruned to a width of approximately 1 m once or twice during the growing season in an attempt to improve spray penetration and allow easy passage along rows. The vine spacing was approximately 1.5 m and row spacing \¡/as 2 m, with the vines growing to about 1.8 m in height. The vines were pruned to three-four buds per shoot and up to five bunches remained on each vine. Some of these bunches carried cleistothecía of U. necator, and there was approximately one flag shoot per three vines each year.

2.2.1,.-J Mountadam Vineyard Mounta

i' l (Appenalx JJ. I ne unaroorìfiay vlnes were allgneu rlr alr easr-west url'eL:uuil ailu plallrsu on undulating land. They were drip-irrigated using stored rainwater supplemented with bore water. 45

The vines used for the experiments had not previously been managed using

organic protocols and had been treated with DMI fungicides in the previous season. The

vines were planted with a spacing of 1.5 m and ro\MS were 2 m apart, They were pruned

to leave canes bearing up to eight shoots and most vines had bunches remnants, some of

which bore cleistothecia.

2.2.1.4 Warriparinga vineyard Warriparinga is an abandoned vineyard on the southern Adelaide Plains, 12 krrr

south of the central business district of Adelaide, on alluvial sand at least 3 m deep. The

vines, cvs Rondella and Palomino, Ìvere approximately 60 - 70 years old and had not

been tended for at least 10 years. During this period no pesticides had been applied to

the vines, however, some herbicide application and mowing had been conducted to

control weeds and reduce the fire hazard (Figure 2.2). In the 199912000 season

approximately 90o/o of the fruit was severely affected by powdery mildew. The vines

were not irrigated. Long-term climate data for the Waite Campus, approximately 5 km 'Warripannga, to the west of are provided in Appendix 3. Complete weather data are no

longer available at the Waite Campus, so the weather data for 2000-2002 provided in

Appendix 3 are for Kent Town, 12krrr to the north of V/arriparinga.

Vine spacing was 2 m, although some vines had died or collapsed leaving larger

spaces between vines. Rows were set 3 m apart and were aligned in an east-west direction. The trellis system was in a poor state of repair and had collapsed completely in some areas, leaving the vines to grow in an unstructured sprawl. The vines \Mere spur pruned to one or two buds in July of 2000-2002. If left unchecked, the vines would grow across the rows, making access difficult and compromising spray coverage, therefore, the vines were trimmed as required throughout the growing season. 46

Figure 2.2: W aringa field experiment site, September 2000. Under-vine application of herbicide reduced weeds directly under the vines.

2.2.3 Methods for field experiments Methods for application of test materials varied b een site and season in an attempt to optimise coverage while reducing drift of test materials between plots. All

use of one or two buffer rows or by selecting blocks separated from the rest of the vine d by at least 10 m of open space. The only site where buffer zones were not used was W paringa. At this site, spray applications to adjacent rows were directed away from test rows to minimize spray drift and the risk of contamination of experimental vines. Buffer zones were sprayed with Synertrol Horti-Oil@ (2 mllL) plus Ecocarb@ (2 g/L) using either hand-held spray equipment or, where fan-forced equipment was used, nozzles and fans were angled to spray away from treated plots. Surfactants were not added to the test materials unless otherwise stated. 47

Test materials were applied between 10.00 a.m. and 3.00 p.m., in random order

(using Genstat 5 random number generation), so that individual treatments were not applied in the same sequence or at the same time each application. Test materials were applied every 10-14 days, except where it was raining or if rain was expected in the next 24 h, where it was windy and when spray drift onto other plots would have occurred, or if the forecast maximum temperature was above 35oC.

In the 200012001season, test materials were applied using 5 L pressurized hand- held wand sprays, except at Temple Bruer'Wines vineyard where a hydraulic spray cart was used. The spray cart was also used at Temple Bruer Wines for the 2001,12002 season and the first two sprays in200212003, subsequent treatments were applied using a 15 L Solo 475@ backpack with a hand-held wand. In 200112002, treatments at

V/arriparin ga and Glenara vineyards were applied using the Solo 475@ . Spray volumes varied between approximately 300 Llha for the first spray to 800 Llha for the last three sprays at all sites. To prevent any cross-contamination between the test materials, spray equipment was rinsed andnozzles cleaned using rainwater between treatments.

Tests with water-sensitive paper (Novartis Crop Protection AG) were conducted during the first two spray applications in 200212003 to estimate leaf coverage using the hydraulic spray cart. The test papers were folded in half and stapled to 10 randomly selected leaves in each plot so that half the test paper was on the upper surface of the leaf and half on the underside. The spray pattern was assessed at 6 kph and 3 kph and, while coverage of exposed leaf surfaces with the test materials was greater at 3 kph than

6 kph, coverage was no better than 50o/o and, in some instances, leaf surfaces received little or no spray (Figure 2.3). As a result, the Solo 475@ backpack was used for subsequent applications with the aim of improving spray coverage. 48

Figure 2.3. Representative water sensitive papers (Novartis, Switzerland) for A: Milk 'Wines treatment @ 6 kph and B: Whey treatment @ 3 kph at Te le Bruer vineyards for the 200112002 season, using a h aulic spray cart. Papers were folded to record spray patterns for the upper and lower surface of the leaf. Purple colour indicates areas of the paper have been sprayed with test material, yellow areas have not received any spray. All group A received poor coverage, the top left section from B indicates the ideal coverage.

A

r Lower surface

þr

\

Ì':' *:

t

rE 49

The powdery mildew infection on leaves and bunches was assessed visually throughout the three growing seasons, prior to each application of test materials. The number of leaves and bunches assessed per plot varied between sites and seasons, mainly due to the variation in plot size. Leaves and bunches, selected at random, were scored for disease at least 7 days after treatment, using either the 0 to 5 scale as described in section 2.1.2 or a 0 to 10 scale based on the percentage of the leaf surface area with visible growth of U. necator, as follows;

0; no obvious disease found

1; | - ß% of leaf area affected by sporulating U. necator

2; ll - 20% of leaf area affected by sporulating U. necator

3; 2l - 30% of leaf area affected by sporulatíng U. necator

4; 3l - 40% of leaf area affected by sporulating U. necûtor

5; 4I - 50% of leaf area affected by sporulating U. necator

6; 5l - 60% of leaf area affected by sporulating U. necator

7; 6l - 70% of leaf area affected by sporulating U. necator

8; 7l - 80% of leaf area affected by sporulating U. necator

9; 8l - 90% of leaf area affected by sporulaling U. necator

10; 9l - 100% of leaf area affected by sporulating tl necator

Data collected from the experiments were subjected to ANOVA using Genstat

(Lawes Agricultural Trust) version 5 release 4.1 or version 6.1.0. The h1'pothesis tested was that there would be no significant difference in the mean disease score among test materials. The 5o/o level of significance (P:0.05) was used for all experiments. The data from some of the field experiments were skewed by the large difference in disease

severity between the untreated vines and the vines that received the test materials. 50

Therefore the disease severity data were re-analysed, without the data for untreated

vines, as described in section 2.I.2.

As described in section 2.I.2 there were inconsistencies in vine vigour across

the Temple Bruer Wines experimental site in 2002/2003 and numerous 0 scores,

particularly for the sulphur, milk and 45 gL whey treatments were obtained, The data

were re-analysed with assistance from Patrick Lim, BiometricsSA, using a linear mixed

model to account for variations in the field site and large number of 0 scores (Lim

2003). The model rvas fitted with SAMM (Butler et al. 2000) with S-Plus (Insightful

Cotp, 2002), in which the zero scores were addressed by using an average disease score

for all bunches assessed on each vine rather than analysing all bunches as individual

data points.

2.2.4 Management of pests and other diseases No treatments were applied to the vines in the Temple Bruer Wines vineyard to nan+-nl na¡4¡ n+ A^+^^+^.7 l^,,^l +L^+ -,.^..11 L--.^ wv^rr^vr PwrLo v¡ vrlrwl^+L^* urùv4ùuû,'l;-^^ dù lllrrrv w4ù LrvLvtJLte\J ctL^+ 4lt/v91^ tlr¿l vv\Juru rr4vu d'tlrvtrtutl^Cf^^¿^l the results of the experiments. Light brown apple moth (LBAM) (Epiphyas postvittana) was maintained at an average of less than one larva per plot by natural predators and hyperparasites. Blister and rust mites, Colomerus vitis and Caliptremeris vitis respectively, were rare and controiled by naturai predators. There was secondary ant damage to some bunches in untreated plots, however, these bunches were severely affected by powdery mildew and the damage did not affect acceptable yield or disease scores

Other diseases and pests at Warriparinqa were below levels that would havc affected the results and pesticides were not required.

The Mountadam Vineyard experimental site was affected by grapevine moth

(Phalaenoides glycinae), however, the moth did not cause significant defoliation until 51 after data were gathered. Populations (two-three per vine) were consistent across the experimental site and would be unlikely to have affected the results. No other pests or diseases were detected at levels that would cause concern and no treatments were applied other than those included in the experiment.

In the 200112002 season at the Glenara Wines vineyard, six oil spots typical of downy mildew were detected over the experimental site but no action was considered necessary. LBAM was present at low levels in all years, however, damage was minimal due to the action of predators. Rust mite was present on the vines each year but at insignificant levels (average of one per leaf). 52

Chapter 3 - Greenhouse experiments 2000

3.1 Introduction Searches of the literature, Internet sites and discussions with organic growers,

identified a large number of materials with potential as novel and biological agents for

the control of grapevine powdery mildew. These materials included fungi, bacteria, oils,

phosphates and other compounds such as clay, salicylic acid and chitosan. While

numerous potential control measures were discussed in Chapter 1, the efficacy of many

of the cotnpounds and organisms is not proven, particularly in South Australian

conditions.

A series of greenhouse experiments was designed to screen a range of materials

to identifvJ the--- most------r--___'-_"onromisinq ontions-r-'--" fL'r field evaluation. The controlled en.¡irorunent

in the greenhouse provided consistent conditions across experiments all year round. The

greenhouse experiments were also used to provide a preliminary insight into the mode

of actton of some of'the materials that showed potential in thc control of grapcvinc

powdery mildew.

The test materials were selected to provide a guide to a range of groups of

materials. Ecocarb@ (875 glkg potassium bicarbonate), Na2CO3 and K2HPoa were

selected from the inorganic salts discussed in section I.4.5.1. All had previously

provided at least modest control of powdery mildew (Pearson et al. 1990; l{orst et al.

l. 1992; Reuveni et al. 1995; Pasini et aL. 1997; Manandhar et al. 1998), however, growers were concerned that they could cause leaf burn. Aminogro@, a foliar nutrient that had been applied by growers at two of the f,reld experiment sites, eonta-ins ehitosan, a material that has been shown to induce SAR in a range of other plant species (see section 1.4.5.6) (Benhamou ef al.1992). 53

Biological agents or formulations evaluated in the 2001 experiments included

AQ10@ (A. quisqualls, Ecogen Inc., see section 1.4.4.1), B. subtilis (see section 1.4.4.2),

Nutri-life 4l2O@ (Nutri-life Products Pty Ltd, see section 1.4.5.4), Trichoderma virens

DAR 74290 (Etebarian et al. 2000), Pseudomonas syringae and Azotobacter sp.

(Bioplus@, Nutritech Solutions Pty Ltd). Formulations of B. subtilis are registered in some countries for control of powdery mildew in viticulture (see section 7.4.4.2),bat their efficacy in Australia has not been documented. Nutri-life 4120@ was included to represent a compost extract (see section 1.4.5.4). The formulation of A. quisqualis tsed in these experiments was developed to address poor efficacy where humidity is sub- optimal and temperatures exceed 25"C (Philipp et al. 1990). T. virens, and other species of Trichoderma, P. syringae and Azotobacter sp. have been reported to control a number of plant pathogenic fungi (see section 1.4.4) and were considered promising organisms for the control of grapevine powdery mildew. Bioplus@ is a commercial formulation containing Azotobacter sp.

To reduce the risk of negative effects on fermentation associated with the antimicrobial compounds (see section I.4.4.2), the B. subtilis cultures used in these experiments were 48 h old or less. B. subtilis was applied as 1) living cultures grown in either a yeast extract medium or whey, 2) a filtrate of the culture and 3) cultures killed by autoclaving. The yeast extract medium provided a standard medium for B. subtilis.

The whey medium was used to evaluate the potential of whey as a nutrient source for B. subtilis on grapevines in the field. Filter-sterilised and autoclaved B. subtilis cultures were included to evaluate whether the bacteria acted directly on the powdery mildew or if metabolites or cell structures were partially or wholly responsible for the control of powdery mildew reported previously. 54

Synerlrol Horti-Oil@ was included as an example of a vegetable oil-based spray

that is available to organic viticulturists and used to control powdery mildew either

alone or mixed with salts or sulphur (see section 1.5.1).

Milk has been shown to control powdery mildew on other crops in glasshouse

conditions @ettiol 1999) and whey was included, in part to assist with determining

which components of milk may be active against powdery mildew.

Three controls were routinely used: 1) no treatment; 2) reverse osmosis (RO)

watcr only; 3) a standard sulphur treatment. The untreated vines were included to

indicate the severity of the powdery mildew if no action were taken, and the water spray

was included to assess the effect of the spray application on the disease. Sulphur was

inciuded as it is wideiy used in the management of grapevine powdery mildew (see

section 1.4.3.1).

3.2 Múerials and methods

3.2.1 Experiment 112000 Preliminary evaluations were carried out of the efficacy of 12 test materials. All were applied as an aqueous solution. Four blocks were established with each treatment applied to one vine in each block. Viognier vines were used for this experiment.

The following materials were applied;

Manufacturer Rate of product applied/L

1) Untreated

2) Water only RO water

3) Sutphur Garden King, 800 g,&g WP 3 g

4) Aminogro@ Organic Crop Protectants Pty Ltd 2.5 ml

5) Nutri-Life 4120@ Nutri-life Products Pty Ltd 100 ml

6) Synertrol Horti-Oil@ Organic Crop Protectants Pty Ltd 2.5 ml 55

7) Synertrol Horti-Oil@ Organic Crop Protectants Pty Ltd 2.5 mI + Aminogro@ Organic Crop Protectants Pty Ltd 2.5 ml

8) Chitosan Sigma-Aldrich Co. 1g

9) Naz CO¡ Sigma-Aldrich Co. 1g r0) Milk Nestlé, Sunshine Full Cream 15 g powder

11) Whey Bonlac Pty Ltd 15 g powder

12) B. subtilis Live culture in yeast extract medium

13) rçHPO4 Sigma -Aldrich Co. -.")\o ë

14) AQlo@ Ecogen Inc. 75 þe

15) Ecocarb@ Organic Crop Protectants Pty Ltd ?o

Nutri-life 4l2O@ was cultured as per instructions on the label (Appendix 2) and applied as a I : l0 dilution in RO water. The B. subtilis culture used in all experiments was obtained from the Undergraduate Teaching Unit (UTL| of the University of

Adelaide, Waite Campus, and was maintained on nutrient agar (NA) plates at 25oC.

When required as a test material, a single colony was transferred to 100 ml liquid growth medium (Appendix 2), using a sterile loop, and maintained at 20o - 25oC in a

250 ml conical flask on an orbital mixer, 48 h prior to application. ,8. subtilis was quantified and the suspension adjusted to 106- 107 cells per ml immediately prior to application. Materials were applied and disease was assessed as described in section

2.1.2.

3.2.2 Experiment 212000 Experiment212000 was aimed to assess the efficacy of milk and whey at a range of concentrations. Six blocks were established, with each test material applied to one vine in each block. 56

The following materials were applied;

Manufacturer Rate of product applied/L

1) Untreated control

2) Whey Bonlac Pty Ltd 15 g powder

3) Whey Bonlac Pty Ltd 30 g powder

4) Whey Bonlac Pty Ltd 45 g powder

5) Whey + Bonlac Pty Ltd 30 g powder Tween 80@ Sigma-Aldrich Co 1ml

6) Milk Nestle, Sunshine full cream powder 15 g powder

7) Milk Nestle, Sunshine full cream powder 30 g powder

8) Tween 80@ Sigma-Aldrich Co. 1 ml

Tween 80@ þolyoxyethylenesorbitan monooleate) was added to one of the whey treatments to determine if the surfactant affected the efficacy of whey. Tween 80@ alone was also included to assess its potential to reduce the incidence or severity of powdery mildew. Sulphur and water controls were not included in this experiment due to a¡ insr-rfficient number of vines. Materials were applied and disease was assessed as described in section 2.1.2.

3.2.3 Experiment 3 12000 Experiment312000 was designed to evaluate B. subtilis and AQ10@ further and to make preliminary evaluations of other biological agents (T. virens, P. syringae and

Azotobacter sp. (Bioplus@) and an unknown fungus isolated from grapevine leaves at

Warriparinga.

The following materials were applied;

Manufacturer Rate of product applied/L

1) Untreated 57

2) Water Milli Q Plus filter

3) Sulphur Garden King, 800 glkg WP 3 g

4) B. subtilis Live culture in yeast extract medium

5) B. subtilis Autoclaved culture in yeast extract medium

6) B. subtilis Filter sterilised (0.22 ¡m)

7) Yeast extract medium Appendix 2

8) B. subtilis Live culture in whey powder

9) T. harzianum Live culture in yeast extract medium

10) Actizyme@ Southern Cross Laboratories 1 g

11) AQ10@ Ecogen Inc. 0.015 g

12) Bioplus@ + Nutri-tech Solutions Pty Ltd 1 g sucrose Sigma-Aldrich Co. 1 g

13) P. syringae Live culture in yeast extract medium

14) Unknown fungus Live culture in yeast extract medium

I 5) Synertrol Horti-Oil@ Organic Crop Protectants Pty Ltd 2 ml

B. subtilis was applied as a live culture, an autoclaved culture (l2l"C for 20 mins) and a culture passed through a sterilising filter, all grown in the yeast extract medium as used in experiment 1/2000 (Appendix 2). A commercially available strain of

B. subtilis, Actizyme@ (Southem Cross Laboratories), was also included to assess its potential in the control of powdery mildew. This formulation is easily stored, handled and applied, and would be suitable for commercial growers if effective. The yeast extract medium was included to evaluate whether the biological agents or the medium might affect the severity of powdery mildew. 58

B. subtilis, T. harzianum, P. syringae, the unknown fungus and the yeast extract medium were all applied as aqueous suspensions at a dilution of 1:10 in RO water. Z. harzianum DAR 74290 (Etebarian et al. 2000), and P. syringae were cultured for 48 h as for B. subtilis. The inoculated whey medium (100 ml) was incubated for 48 h in a

250 ml conical flask at approximately 25oC on an orbital mixer before use.

The unknown fungus was isolated from the surface of powdery mildew affected berries collected from Warriparinga (see section 2.2.1.4) in February 2000. It was maintained on potato dexhose agar (PDA) plates. and cultured on the yeast extract medium as used for B. subtilis in experiment 1/2000. Sucrose was added to the

Bioplus@ to provide an energy source for the Azotobacter. Materials were applied and disease was asscsscd as described in section 2.1.2.

3.3 Results

3.3.1 Experiment 112000 The mean severiiy oi powciery miitiew on ieaves oi untrealeri vines ¿i tirc completion of the experiment was 4.0, representingS0% of the leaf surface affected by

U. necator (Table 3.1). All test materials applied to the vines in this experiment significantly reduced the severity of powdery mildew on leaves when compared to the untreated controls (p <0.001) (Table 3.1). The analysis of disease scores for the untreated leaves, leaves 5-8 on all vines, showed no significant differences between treatments (P:0.626) and, therefore, the significant differences between treated leaves

(P:0.002) were considered to be due to effects of the treatments (Lorimer 2000 a).

Synertroi i{orti-Oii@ (disease score 0.2), B. subtilis (0.2), suiphur (0.6) and

Synertrol Horti-Oil@ plus Aminogro@ (1.1), were most effective in reducing the severity of powdery mildew in this experiment (Table 3.1). The average disease severity was highest on untreated vines and vines sprayed with Aminogro (disease score 3.6), water 59

(3.5) and NazCO¡ (2.6). Other treatments reduced the average severity of powdery mildew on the treated vine leaves by significant but varying amounts. On vines treated with Synertrol Horti-Oil@, B. subtilis and sulphur, the disease \¡/as limited to curled areas of the leaves that may have been missed during application of the test materials.

When the disease severity data for untreated vines were removed and the remaining data re-analysed, the disease score for vines treated with Synertrol Horti-Oil@ was significantly less than that for the Synertrol Horti-Oil@ plus Aminog¡o@ mixture

(Table 3.1). The other notable change in significant difference is that vines treated with

Ecocarb@ had a significantly lower disease severity than those treated with K2HPO+, the salt that gave the second lowest disease severity score.

The data for vines treated with chitosan were considered unreliable as 75o/o of the leaves that received this treatment showed evidence of phytotoxicity (data not presented). Widespread buming was observed and many leaves fell from the vines. No leaves were lost or severely damaged on vines treated with other test materials in this experiment, however, there was slight scorching of the margins of 25o/o of the leaves sprayed with sulphur. There was sporadic necrosis on other leaves but this had no obvious association with any of the treatments.

Microscopic observations made at the completion of the experiment showed that cleistothecia had developed, at varying densities, on the leaves of vines receiving all test materials except B. subtilis and Synertrol Horti-Oil@. Numbers and density of cleistothecia were closely related to the severity of the disease, with density up to

200lcm2 on untreated leaves. The leaf area infected with (1. necator observed by microscopic evaluation of the treated leaves was equivalent to that recorded by the non- destructive scoring during the experimental period. 60

Table 3.1: Mean disease severity on treated grapevine leaves (cv. Viognier), for greenhouse experiment I/2000, after three fortnightly applications of the test materials" LSD (5%): when data for untreated vines included : 0.982, when data for untreated vines excluded : 0.6784. Score for visual assessment of leaf area with sporulating colonies of powdery mildew; 0 to 1; I - 20yo I to 2;21 - 40yo,2 to 3; 4l - 600/0, 3 to 4; 6I - 8lyq4 to 5; 81 - 100%.o/o of initial infection is the relative severity of powdery mildew on treated leaves at the completion of the experiment in relation to initial severity of infection. Test materials ranked according to mean disease score. Results with the same letter are not significantly different (P <0.05). N/A: not applicable.

Statistical Test Material Mean disease significance when score data for untreated vines excluded Synertrol Horti-Oilc' 0.2 a a

Bacillus subtilis 0.3 a a

Sulphur WP 0.6 ab ab

Synerhol Horti-Oil@ 1.1 abc bc + AminoEro@ Milk 1.4 bcd cd

Ecocarb@ 1.6 bcd cde

Whey 1.7 cde cdef

4/20@ 1.9 cde defg

AQl o@ 2.0 cde efgh

KzHPO+ 2.3 de fsh

NazCO¡ 2.6 ef h

Water 3.5 fs I

Aminogro @ 3.6 fg j

Untreated control 4.0 e N/A

3.3.2 Experiment 2/2000 The mean severity of powdery mildew on leaves of untreated vines at the completion of the experiment was2.4, representing4S% of the leaf surface affected by 6r

U. necator (Table 3.2). Analysis of both disease severity and disease incidence showed

that all test materials reduced powdery mildew on treated leaves significantly (p<0.001)

(Tables 3.2 e,3.3).

Analysis of the disease scores of leaves on treated vines showed no significant

difference between treatments (Lorimer 2000 b) except for Tween 80@, where disease

was significantly more severe than on vines sprayed with milk or whey 45 glL (Table

3.2). While the Tween 80@ plus 30 g/L whey mixture resulted in a lower disease

severity score than either the Tween 80@ or whey powder 30 g/L when applied alone, the difference was not significant. At the completion of the experiment, there was no

disease on leaves treated fortnightly with milk powder at 30 glL or whey powder at 45

g/L. While there was no significant difference in the severity of powdery mildew for vines treated with the different rates of milk or whey, there was a trend for lower disease severity as the concentrations of the test materials increased.

When the data were re-analysed without the disease scores for the untreated vines, the disease severity for vines receiving whey at 45 glL and milk at 30 g/L was

significantly less than for vines treated with 15 glL or 30 glL whey (Table 3.2). Also, the disease severity on leaves treated with whey plus Tween 80@ was siguificantly less than on those treated with Tween 80@ alone.

The incidence of colonies of powdery mildew on the assessed leaves was greatest on the leaves of untreated vines, with all test materials reducing the incidence of powdery mildew on treated leaves (Table 3.3). There was no significant difference between the whey powder at 15 glL and 30 g/L. The incidence of powdery mildew on leaves of vines treated with the Tween@ plus whey 30 glL mixture was significantly lower (P<0.05) than where these materials were applied alone. 62

Table 3.2: Mean disease severity on treated grapevine leaves (cv. Viognier) for greenhouse experiment 212000, after three fortnightly applications of the test materials. LSD (5%): when data for untreated vines included : 0.6726, when data for untreated vines excluded : 0.4366. Score for visual assessment of leaf area affected with sporulating colonies of powdery mildew; 0 to 1; 1 - 20% 1 to 2; 2l - 4lo/o,2 to 3; 4l - 600/0, 3 to 4; 6l - 80yo,4 to 5; 81 - 100%. Test materials ranked according to mean disease score. Results with the same letter are not significantly different (P <0.05). N/A : not applicable.

Statistical Test Material Mean disease Standard error significance when score of means data for untreated vines excluded Milk 30 0a N/A a

Whey 45 0a N/A a

Milk 15 0.1 a 0.1 ab

Whey 30 + Tween 80@ 0.2 ab 0.2 ab

Whey 15 0.5 ab 0.2 bc

Whey 30 0.5 ab 0.2 bc

Tween 80@ 0.9 bc 0.2 c

Untreated 2.4 d 0.3 N/A

There was no evidence of phytotoxicity to the leaves of any of the vines that could be attributed to any of the treatments, except that leaves treated with whey plus

Tween 80@ appeared less healthy (dull and with a papery feel) than those sn other vines. There was a light fihn present on leaves treated with 30 g/L of milk but no obvious damage. Microscopic examination revealed cleistothecia on leaves treated with all test materials except milk at 30 glL and whey at 45 g/L. The powdery mildew was evenly distributed on the vines treated with Tween 80@ and untreated leaves; however, the powdery mildew on leaves treated with the other test materials was limited to folds or sheltered areas. 63

Microscopic observations made at the completion of the experiment showed that the leaf area of treated and untreated leaves infected with U. necator was equivalent to that recorded by the non-destructive scoring during the experimental period.

Table 3.3: Mean disease incidence on treated gtapevine leaves (cv. Viognier), for greenhouse experiment 212000, after three fortnightly applications of the test materials. Test materials ranked according to mean disease score. Results with the same letter are not significantly different (P <0.05).

Test Material Proportion diseased Estimated standard error of means Milk 30 0a 0.0

Whey 45 0a 0.0

Milk 1s 0.1 b 0.1

Whey 30 + Tween 80@ 0.2b 0.1

Whey 15 0.5 c 0.2

Whey 30 0.6 c 0.2

Tween 80@ 0.7 d 0.1

Untreated control 1.0 d 0.0

3.3.3 Experiment 3 12000 The mean severity of powdery mildew on leaves of untreated vines at the completion of the experiment was 2.8, representin g 56% of the leaf surface affected by

U. necator (Table 3.4). The analysis of disease scores for untreated leaves (leaves 5-8 on all vines) showed no significant differences between treatments (P:0.728) and, therefore, any significant differences between treated leaves, 1-4, (P<0.001) were considered to be due to effects of the test materials (Lorimer 2000 b). There were no significant differences in disease severity among vines receiving any of the B. subtilis treatments or the culture medium (Table 3.4). The severity of powdery mildew on vines 64

sprayed \¡iith P. syringae was not significantly different to that on vines sprayed with Z.

Itarzianum, the culfure medium or the B. subtilis treatments except the autoclaved

culture. Powdery mildew on the vines sprayed with T. harzianum was signifrcantly

more severe than on vines sprayed the autoclaved and 48-h B. subtilis cultures but not

significantly different to that on vines sprayed with the filter sterilised.B. subtilis or the

culture medium. Disease severity on vines treated with AQ10@ ldisease score 2.7) and

Bioplus@ (3.1) did not differ significantly from the untreated (2.S) or water-treated (2.4)

control plants.

Analysis of the data following removal of the results for untreated vines showed

a significant difference in powdery mildew severity between the vines treated with ,8. subtilis (autoclaved) and those treated with yeast extract medium or B. subtilis (filter

sterilised) (Table 3.4).

None of the test materials in this experiment caused phytotoxicity. There was a

crystalline residue on some leaves treated with yeast extract medium or with B. subtilis,

except where cultured in whey.

The area infected with tl necator observed during mieroscopie evaluation of the treated leaves after completion of the experiment was equivalent to that recorded by the non-ciestructive scoring during the experimental period.

î 65

Table 3.4: Mean disease severity on treated grapevine leaves (cv. Viognier) for greenhouse experiment 312000, after three fortnightly applications of the test materials. LSD (5%): when data for untreated vines included:0.9725, when data for untreated vines excluded data : 0.6083. Score for visual assessment of leaf area with sporulating colonies of powdery mildew; 0 to 1; | - 20% I to 2;2I - 40%o,2 to 3; 4l - 60yo,3 to 4; 6I - 80yo,4 to 5; 81 - 100%. Test materials ranked according to mean disease score. Results with the same letter are not significantly different (P <0.05). N/A : not applicable.

Statistical significance Test material Mean disease score when data for untreated vines excluded B. subtilis 0.3 a a (autoclaved) Sulphur 0.4 ab ab

B. subtilis 0.5 ab abc (48 hour culture) Unidentified fungus 0.7 abc abcd

Synerhol Horti-Oil@ 0.7 abc abcd

B. subtilis 0.8 abc abcde (whey culture) Culture medium 0.9 abc bcde

B. subtilis 1.1 abc cde (filter sterilised) P. syringae 1.3 bc de

T. harzianum 1.5 c e

Water 2.4 d f

AQlo@ 2.7 d f

Untreated control 2.8 d N/A

(Ð Bioplus 3.1 d f

3.4 Discussion Greenhouse experiments conducted in 2000 showed that several test materials provided excellent control of powdery mildew and that there were significant differences in the efficacy of the test materials. The performance of several of these 66

materials warranted their inclusion in f,reld experiments. These materials, including milk, whey, Synertrol Horti-Oil@, Ecocarb@ and .8. subtilis, controlled powdery mildew to levels not significantly different from those provided by sulphur in at least one

experiment. The second experiment suggested that the efficacy of milk and whey in reducing the severity of powdery mildew was related to the concentration applied. The incidence of powdery mildew was reduced when the surfactant, Tween 80@, was added to whey compared to either treatment applied alone, but disease severity was not rcduced significantly.

Statistical analysis of disease severity on vines revealed differences between some treatments only when the data for the untreated vines were excluded. In particular, this analysis showed that Ecocarb@ was a significantly better treatment than KzHPO¿, and as a result Ecocarb@ was selected for field experiments.

When all data were considered, there was no significant difference in the reduction in severity of powdery mildew on vines sprayed with any of thc B. subtili.s treatments. This suggests that the reduction in disease was not directly related to the living cells of B. subtilis. As the cultures were not old enough to have produced the antimierobial products associated with sporulation, such as iturin A and surfactin, other factors must be active against powdery mildew. The results are similar to those of

Liefert (1995), where irltrates of B. subtil¿s culture were as effective as applications of living cultures for control of powdery mildew. Lytic enzymes produced by B. subtilis, including chitinases and other cell wall degrading enzyrnes, may contribute to its anti- fungal properties (Liefert et al. 1995). Bacillus spp. may also produce elicitors of plant resistance mechanisms. Each active product af B. subtilis appears to have different modes of action and be effective against different fungi (Schreiber et al. 1988). The efficacy of the enzymes woulcl make the preparation, storage and application of B. 67 subtilis sprays simpler than if live cultures were required for powdery mildew control.

However, if the live cultures persist on the surface of vine leaves there is the likelihood that the duration of protection against re-infection by U. necator would be greater than that provided by enzymes alone.

It is likely that the failure of AQ10@, and possibly also of Nutri-Life 4120@, to control powdery mildew effectively was related to the dry conditions in the greenhouse.

A. quisqualis requires an extended period of leaf wetness to become established (see section 1.4.4.I) and it is possible that the performance of Nutri-Life 4120@ may also be enhanced by moist conditions. While it would have been possible to provide conditions more suited to the establishment of these microorganisms by maintaining leaf wetness through the use of misters in the greenhouse, that would not reflect the usual conditions in commercial vineyards in South Australia. Maintaining leaf wetness in the greenhouse could also promote infection by other fungal pathogens, such as B. cinerea. which would have confounded results.

T. harzianum significantly reduced the severity of powdery mildew on treated vines when compared to untreated vines but was less effective than sulphur and some,B. subtilis treatments. Previous research has shown that the production of antibiotic compounds by Z harzianum varies with the age of the culture and appears, like antibiotics produced by B. subtilis, to be related to spore production by Trichoderma spp., and the culture media (Lederer et al. 1992). The efficacy of Trichoderma as a control agent of U. necator vanes between species, with Z. harzianum possibly being the most effective (Lederer et al. 1992). The moderate level of control of powdery mildew achieved in the greenhouse trials, the need for extended culturing periods for production of anti-fungal compounds and variations in efficacy described by Lederer

(1992) indicated that T. harzíanum was not suitable for inclusion in field trials. 68

As expected, all leaves treated with sulphur had detectable residues on the surface at the completion of the experiments. Other test materials that resulted in detectable residues on at least some leaves were milk, whey and B. subtilis (see section

3.4). These residues did not appear to have any phytotoxic effects but may affect photosynthesis by the vine. If there is reduced photosynthesis, there is the potential for reduced yield. The residues of milk and whey may also be a nutrient source for microorganisms that could be antagonistic to (J. necator, reduce berry quality and interfere with the fermentation process.

The application of sulphur or chitosan caused noticeable phytotoxicity to leaves in experiment 1/2000. The sulphur caused a phytotoxic reaction only in the one experiment, and the damage 'was mainly to areas of the leaf where excess liquid accumulated, particularly around the lower edges. Also, the gteenhouse temperature exceeded 25oC on the day of the second application of the treatments in this

q v1\yvÀlllrvrllto-^o-i-o-f Á,¡ausv fn nnr¡¡cr foilrrrc rnrl this mav hawe contrihrterf to the-- nhvtotoxicitv-I J - J

In later experiments, care was taken to avoid over-spraying sulphur and any excess liquid was shaken from the leaves.

Chitosan dissolves only in acid and the solution had to be maintained at pH 5.5 or less to prevent precipitation. Treating leaves with the acidic spray caused scorching of most of the leaves and 50o/o of them dropped within 2 weeks of the first application.

As a result, chitosan, in this form was not considered for field experiments. Other forms of chitosan, as in some foliar nutrients, e.g. Aminogro@, were evaluated in later experiments.

There was no significant difference in the mean powdery mildew severity between untreated leaves of vines treated with the test materials and untreated control vines for any of the greenhouse experiments conducted in 2000. This suggests that none 69 of the test materials induced systemic resistance to powdery mildew in the test vines, or that levels of resistance induced were not sufficient to provide effective protection from

U. necator. The presence of powdery mildew colonies on protected areas of treated leaves supported this hypothesis.

Cleistothecia formed on all leaves where substantial colonies of powdery mildew had developed. The cleistothecia appeared normal and none of the test materials appeared to have an impact on their production, however, the viability of cleistothecia on treated leaves was not assessed. In situations where cleistothecia were not present, e.g., leaves treated with B. subtilis and Synertrol Horti-Oil@ in experiment 112000, powdery mildew colonies were small, isolated and could have developed from single conidia, which would preclude the formation of cleistothecia.

In the case of the some of the most effective test materials, namely milk, whey,

B. subtilis and Synertrol Horti-Oil@, the powdery mildew infection at the completion of the experiments was less severe than prior to the initial application. The reduction in severity over time indicated that these test materials might have curative properties.

Further greenhouse and laboratory experiments, using leaves inoculated after application of the test materials, should be conducted to evaluate the period of protection from new infection of powdery mildew, if any, provided by the test materials.

A fourth greenhouse experiment was established in 2000 to compare the effect of lactose, the major component of whey, at 15 g/L and 1 1 .25 ElL, with whey but was abandoned after the vines suffered severe damage from two spotted mite, Tetranychus urticae group, and broad mite, Polyphagotarsonemus latus. The vines were discarded and replaced for the experiments conducted in 2001. 70

Chapter 4 - Field experiments 2000/2001

4.1 Introduction Test materials that reduced the incidence or severity of powdery mildew in the

greenhouse experiments were evaluated in the vineyard to assess their efficacy in a

commercial environment. Variation in relative humidity, temperature, lfV light and

other environmental factors may have significant impacts on the performance of the materials. Other issues that may arise in field conditions include phytotoxicity at high temperatures and in slow dryrng conditions, uncvcn spray coverage, and thE establishment of other fungi on nutrient-rich materials such as milk or whey.

Bicarbonates have been reported to cause damage if temperatures exceed 30oC in association with hot, dry wincls (see section 1,4.5.1), con

AQ10@ and Nutri-Life 4120@, which reduced the severity of powdery mildew by approximately 50o/o in the greenhouse (see section 3.3.1), were included in the field experiments, as there was the possibility that moisture fiom dew and rainfall could improve their efficacy in the vineyard. In addition two materials that had not been tested in the greenhouse, wheast (a whey/yeast extract mix, Appendix 2), that is used to

aLLraaL antl sustain beneficial artluopocls, and Neemtech@ (Moeco Pty Ltd Australia), a potassium insecticidal soaB used to reduce pest arthropods. Information on the effects of these materials on powdery mildew could assist with development of progtams to control powdery mildew and insect pests. 7l

The aims of these experiments were to provide data on the efficacy in the field

of the most promising treatments from greenhouse experiments, so that one or two

could be selected to investigate the mode of action and to develop protocols for use in

commercial vineyards. Also, the experiments were designed to determine if any

products were phytotoxic and if the level of spray coverage limited efficacy.

4.2 Methods and materials Test materials selected for the 200012001 field experiments were 1) Synertrol

Horti-Oil@ (2 ml/L),2) Ecocarbt (¡ g/t), 3) milk (t:10 dilurion), 4) whey (15 g/L), 5)

B. subtilis (1:2 dilution), 6) yeast extract medium (1:2 dilution),7) AQ10@ Q.075 glL),

8) Nutri-Life 4120@,9) wheast and 10) Neemtech@ (2 mllL). Rates of application of the

test materials were based on label rates and rates that had reduced the severity of

powdery mildew in the earlier greenhouse trials. The B. subtilis l:2 dilution was based

on the greenhouse trials where it had been the most effective test material in reducing

the severity of powdery mildew. The yeast extract medium was applied at the same

dilution as the B. subtilis so that a reliable comparison of the impact of the cultures and medium on the severity of powdery mildew could be made. The Nutri-Life 4/20@ was prepared and diluted applied as per the manufacturer's instructions and wheast (15 glL of whey and 15 glL yeast extract) was mixed and applied at rates commonly used in organic viticulture.

Test materials 1 - 8 had significantly reduced the severity of powdery mildew in greenhouse experiments conducted in 2000 (see sections 3.3.1, 3.3.2 and 3.3.3).

Materials 9 and 10 were untested at that stage. While it would have been ideal to test all treatments at each site, the number of vines available at each site dictated the number of test materials evaluated. In addition, the number of vines and time involved in applying 72

materials and assessing vines made this impractical. For this reason sulphur was not

included in 200012001 field experiments.

4.2.1Temple Bruer Wines vineyard The experiment at Temple Bruer Wines vineyard comprised four blocks of five

plots with four vines of cv. Verdelho vines in each plot. Plots were separated by four

untreated vines to minimise spray drift from neighbouring plots. Test materials were

applied on seven occasions (Table 4.1) at approximately 10 - 14 day intervals,

depending on weather. Rates ancl spray volumes applied at various grorvth stages are

shown in Table 4.1. Disease severity was evaluated monthly, with the first assessment

immediately prior to the first application of test materials. Fifteen leaves and bunches

per vine were assesseci (when availabie) using tÌre i-iû scaie giveir in section 2.¿.:.

Table 4.1: Application dates and rates for test materials applied to grapevines, cv. Verdelho, at the Temple Bruer Wines field experiment site in the 200012001 vintage. All treatments applied using a 5 L pressurized spray pack and hand-held wand. Application rate is the water equivalent rate. Dilution : dilution in RO water. Results ¿L^ 1^++^- ..^a ¡rD.-fì fì{\ --.:¿LWltll lffu ùallllu^^*^ llvLLtrl Clltv^*^ ll\JL ùlÈílllllw4rrlrJ^;-;{:^^-+1.' 'liffo¡onfurrrwrw¡r! \r -v'vJ r'

Application Date 3lr0l 1,8lt0l 301101 TtrU 24llll 6lr2l 29n21 Treatment 2000 2000 2000 2UOU ¿OUU 200û 2ûû0 Ijntreated

Milk (Dilution) 1 :10 1:10 I 1 0 l:10 l:10 l:10 1:10

Whey (eil) l5 15 15 l5 l5 15 15

B. subtilis I:2 l:2 l:2 l:2 1:2 l:2 l:2 (Dilution) Yeast extract I:2 I:2 l:2 7:2 I:2 1:2 l:2 medium (Dilution) Application rate 300 300 400 400 600 600 600 (Llha\

4.2.2 Glenara Wines vineyard The test materials applied to grapevines of cv. Chardonnay at the Glenara Wines

vineyard were identical to those applied at Temple Bruer Wines. Four blocks of five IJ

plots each containing two vines were established with a two-vine buffer to minimise

cross-contamination between materials. Test materials were applied on seven occasions

(Table 4.2) at approximately 10 - 14 day intervals, depending on weather. Rates and

spray volumes applied at various growth stages are shown in Table 4.2. Higher water

equivalent rates were required in the Glenara Wines vineyard than at Temple Bruer

Wines due to the size and density of the vine canopy. Disease severity was evaluated

monthly, with the first assessment immediately prior to the first application. Ten leaves

and bunches per vine (when available) were assessed using the 1-10 scale given in

section 2.2.3. The untreated control vines rtrere sprayed with 4 glL sulphur (Garden

King wettable powder) for the last two applications as powdery mildew on the

untreated vines had become severe.

Table 4.2: Application dates and rates for test materials applied to grapevines, cv. Chardonnay, at the Glenara Wines field experiment site in the 20001200I vintage. All treatments applied using a 5 L pressuized spray pack and hand-held wand. Application rate is the water equivalent rate. Dilution: dilution in RO water. Results with the same letter are not significantly different (P <0.05).

Application date Treatment 13/91 27tgt tllt0l 26110t t0llU tTltU 30lru 2000 2000 2000 2000 2000 2000 2000 Water Sulphur Sulphur

Milk (Dilution) 1:10 1: l0 1:10 1:10 1:10 1:10 1:10

Whey (g/L) l5 15 15 15 15 15 15

B. subtilis 7:2 l:2 l:2 l:2 l:2 l:2 l:2 (Dilution) Yeast extact l:2 7:2 I:2 1.:2 7:2 I:2 7:2 medium (Dilution) Application 400 400 500 500 600 800 800 rate (L/ha)

4.2.3 Mountadam Vineyard The test materials at the Mountadam Vineyard site were applied to cv.

Chardonnay vines. Four blocks of eight plots were established, with five vines in each 74 plot, with a two-vine buffer separating plots to minimise spray drift from neighbouring plots. Test materials were applied on seven occasions (Table 4.3), as at the other experimental sites. At this site, two controls were included; an untreated plot and a water-only control. The water control was included to assess if a water spray affected the severity of powdery mildew on grapevines in the field.

Disease severity was evaluated monthly, with the first assessment immediately prior to the hrst application of test materials. Twenty five leaves and bunches (when available) were randomly selected from each vine, five from each vine in the plot ancl assessed, from each plot using the 1-10 scale given in section 2.2.3. Five bunches were harvested from each vine, on 27 }rlarch200I, and weighed to assess any differenees in f t ' t ¿ | l--.:11- lifj-^-^--¿¿^^¿.--^+^.^i-l^ DUnCn SIZC OetWCçIl VlIlçS tt9At9U WITII UllltrItrllt ttrSt lllaltçlralù.

Table 4.3: Application dates and rates for test materials applied to grapevines, cv. Chardonnay, at the Mountadam Vineyards field experiment site in the 200A12001 vintage. All treatments applied using a 5 L pressurized spray pack and hand-held wanel. Application rate is the water equivalent rate. Dilution : dilution in RO water. Results +1"^ qra ¡rD "'i+Lvv lLtr Llrv úolrtv.oma lvlLvrlalfar @lw nnftlvc urb^¡rrtv4ulrcimifinonfl.¡ rlìffaranf

Application date 22191 6lr0l 23ltjt r4ltr/ 30/rU r0ltzl 2Ur2l Treatment 2000 2000 2000 2000 2000 2000 2000 Untreated control

RO water

a Synertroi Ì10nI- z z ¿ z ¿ L L oil@ (ml/L) , (R-) , a .| .' 1 Þcocarb - lgtL) J J J J

B. subtilis l:2 l:2l:2 l:2 l:2 l:2 (Dilution) Neemtech@ (ml/L) 2 2 2 2 2 2 2

Yeast extract l:'¿ i:2 i:2 r'.¿ i:2 t:¿ medium (Dilution) Whey powder 15 i5 15 15 15 15 15 G./L) Application tate 300 300 400 500 600 600 600 (Llha) 75

4.2.4Warriparinga At the Warriparinga vineyard the test materials were applied to cv. Rondella and

cv. Palomino vines. Four blocks of 10 plots each containing four vines were

established. A four-vine buffer was established between each plot to limit spray drift from neighbouring plots. Test materials were applied on seven occasions (Table 4.4), as for the other experimental sites. Disease severity was evaluated monthly, by assessing

20 leaves and bunches (when available) using the 1-10 scale given in section 2.2.3.

At V/arriparinga vineyard in 200012001 no powdery mildew colonies were found during the regular assessments so 20 leaves and one bunch were taken at random from each vine in the untreated control plots in November and December 2000. The leaves and bunches were incubated in the laboratory in plastic bags at between 20 and,

25o C for 24 h to promote sporulation of (J. necator, and then examined under a dissecting microscope (Wild Heerbrugg M8) to check for the presence of powdery mildew. The leaves were also examined for mycophagous arthropods such as tydeid mites that may have been responsible for the absence of powdery mildew on leaves at

Warriparinga. In November 2000 and January 2001, samples were taken from all plots at the site, then incubated and examined in a similar manner.

4.3 Results

4.3.1 Temple Bruer'Wines vineyard The grapes at this site were mechanically harvested before final assessments could be made; therefore, data obtained on 19 January 2001 were used in the analysis.

The earlier than expected harvest was required as an extended period of temperatures over 35oC resulted in a rapid increase in Baumé. 76

Table 4.4: Application dates and rates for test materials applied to grapevines, cv. -Warriparinga Rondelia anri cv. Paiomino, at the freld experimeui site irr the 2000/2001 vintage. All treatments applied using a 5 L pressurized spray pack and hand-held wand' Application rate is the water equivalent rate. Dilution : dilution in RO water. Results with the same letter are not significantly different (P <0.05). App 2: Appendix 2.

Application date 28t91 r2lr0l 25ltÙl 811.y 22111.1 7n2l t9lr2l Treatment 2000 2000 2000 2000 2000 2000 2000 Untreated control B. subtilis l:2 l:2 1:2 1:2 l:2 7:2 I:2 (Dilution) Wheast Ãpp 2 App 2 App 2 App 2 App2 App 2 App2

a Syncrhol Horti- J J J J 3 3 nil@ r-llTvL"' v \t Ecocarb@ (g/L) J J 3 J -) J J

AQ10@ (s/L) 0.07s 0.075 0.075 0.075 0.075 0.075 0.075

1-1^ 1.1^ 1.1rì 't .1^ 1.1rì Viiik (Diiution) I:IU l: lu l;lv l.lv I.I\J l.lv whey (g/L) 15 15 l5 l5 15 l5 15

Neemtech@ 2 2 2 2 2 ), 2 (m1/L) ¡ ¡¡n@ rn:l,,+j 1.4 1.) 1.:2 1) 1:2 I:2 +/ LV \L/rrUrrurr/^-\

Application rate 300 400 500 500 600 800 900 (Llha)

The mean severity of powdery miidew on'ounches on untteated vines at Temple

Rrrrcru¡ uvl lrvineer r r¡rvu r¡ine.-¡erdI ¡rrvJ sr st nn vr¡ lQ lanllarv 200!- was 4-2- reoresenting- 42o of the berry surface affected by U.necator. All materials applied at Temple Bruer Wines vineyard significantly reduced the severity of powdery mildew on bunches when compared to the untreated controls (p <0.001). There was no significant difference in the severity of powdery mildew on vines treated with milk (1.5) and whey (1.8). These materials were the most effective in reducing the severity of powdery mildew in this experiment (Table

4.5). The average disease severity on vines sprayed with the yeast extract medium and

B. subtilis was 3.1 and 2.5 respectively. Re-analysis of the

The powdery mildew on bunches \¡/as most severe on the side of the bunch closest to the centre of the vine and on berries that were sheltered by leaves or other bunches and which did not receive adequate spray. However, there were some inconsistencies in the disease development on vines treated with B. subtilis and the yeast extract medium, in that some of the colonies developed in areas of the bunches that would have been sprayed with the test materials (Figure 4.1).

Exposed bunches treated with milk or whey were generally either free of powdery mildew or had scarring where colonies of powdery mildew had developed and subsequently been killed by the test material (Figure 4.1). Similarly, exposed untreated bunches had severe powdery mildew infection on all surfaces of the berries and by mid-

January 2001 berries were split, discoloured and showed uneven maturity (Figure 4.1).

Table 4.5: Mean disease severity on bunches on treated grapevines (19 January 200I), cv. Verdelho for the Temple Bruer Wines field experiment site 20001200I, after seven applications of the test materials. LSD (5%): when data for untreated vines included : 0.4384; when data for untreated vines excluded : 0. Score for visual assessment of bunch area affected with sporulating colonies of powdery mildew; 0 to 1; I - l\Yo I to 2; Il - 20yo,2 Io 3;21 - 30o/o, 3 to 4; 3l - 40yo, etc. N/A : not applicable. Results with the same letter are not significantly different (P <0.05).

Statistical sÍgnificance Treatment Mean disease when data for severity untreated vines excluded Whey 1.5 a a

Milk 1.5 a a

B. subtilis 2.sb b

Yeast medium 3.1 c c

Untreated control 4.2 d N/A 78

Figure 4.1: Development of powdery mildew on bunches of cv. Verdelho grapes at the

(B) eated, (C) milk and (D) B. subtilis.

4.3.2 Glenara Wines vineyard Powdery mildew was first detected prior to treatment on 10 October 2000, on the untreated vines, and was present in all plots during inspection of the vines prior to application of test materials on26 October 2000. The disease developed most rapidly in the centre of the canopy where spray coverage was inadequate and conditions were more favourable for powdery mildew. The experiment at Glenara vineyard was abandoned after application 5 on 30 November 2000, as powdery mildew had developed extensively in the experimental area and was providing inoculum for the rest 79

of the vineyard. All vines were sprayed with sulphur at arate of 4ky'haon 4 December

2000.

4.3.3 Mountadam Vineyard The mean severity of powdery mildew on bunches on untreated vines at

Mountadam Vineyard was 1.1 on the 13 December 2000, representing 11% of the

berries, rising to 3.9 or 39o/o at the completion of the experiment on 27 March 2001.

The severity of powdery mildew on vines treated with the test materials was

significantly less than that on the untreated vines (p <0.001), except for vines treated

with B. subtilis, which had disease scores on 27 March 2001 of 4.3, repres enting 43%o

of berries affected (Table 4.6).

On 13 December 2000, there was no significant difference in the severity of

powdery mildew on bunches sprayed with the test materials other than B. subtilis. By

27 March 2001, the severity of powdery mildew on the bunches of vines treated with

Synertrol Horti-Oil@ was 1.8, representing 18% of beries, significantly less than on bunches on vines receiving all other test materials except Neemtech@ (22% of berries).

The powdery mildew severity on vines sprayed with water alone was

significantly lower than on untreated vines for both assessments (9% and 2lYo of berries), and was similar to that of vines treated with Synertrol Horti-Oil@ or

Neemtech@ at the completion of the experiment. However, the disease severity on the vines receiving water only and on untreated vines was artificially low as the most severely affected leaves had dropped off, leaving only a few leaves that could be assessed. This effect can be seen in Figure 4.2 where an untreated vine (D) appears defoliated compared to vines that recieved Ecocarb@ and B. subtilis (A and B respectively). Water sensitive papers indicated that areas closest to the centre of the canopy received irregular coverage, varying between 80% to none of the leaf surface. 80

The average bunch weight varied significantly between treatments, with the

heaviest developing on vines treated with Ecocarb@ (77 g), water only (74.3 g),

Neemtech@ (64.7 g), the yeast extract medium 6a.a Ð and B. subtilis (67.1 g). The

average bunch size did not reflect total yield, as the number of infected bunches varied

between treatments and most bunches on the untreated vines were severely infected and

unsuitable for harvest (Figure 4.2). The removal of data for untreated vines from the

analysis, for the 27 l|darch 200I, had no impact on the significance of differences between test matedals.

Table 4.6: Mean disease severity on bunches on treated grapevines, cv. Chardonnay for the Mountadam field experiment site 20001200I, after seven applications of the test materials. LSD (5%): 13 Dec (when data for untreated vines included) : 0.183, 27 March (when data for untreated vines included) : 0.4662, 27 March (when data for untreate

Statistical Average õ-,- -1_- -l. r €attrtcllt i3 Dec 20ûíi 27 ivlar 2it0i significance bunch Mean disease Mean disease when data for weight severity severity untreated vines (grams) excluded Synertrol 4.6 a 1.8 a a 59.6 bcd Horti-Oil@ Water 0.9 b 2.1 ab ab 74.3 ab

Neemtech@ 0.5 a 2.2 ab ab 64.7 abc

Ecocarb@ 0.5 a 2.3b b 77.0 a

\ühey 0.6 a 2.3b b 52.lcd

Yeast extract 0.5 a 2.4b b 64.4 abc medium ÀE E I Untreated 1't c -?.9 c

B. subtìlís 0.8 b 4.3 d c 67.1 abc 81

Figure 4.2: Representative images of vines at the Mountadam Vineyard experimental site on 27 March2O0l, after seven applications of test materials. Treatments clockwise from top left: (A) Ecocarb@, (B) B. subtilis, (C) water and (D) untreated.

4.3.4 Warriparinga Powdery mildew was not detected orr leaves or bunches at V/arripannga

vineyard except for one bunch, which was obviously infected (sampled on 22

November 2000). Microscopic examination of leaves sampled from untreated vines on

22 November 2000 showed large populations, over 100 mites per leaf of an

unidentified species of Orthotydeid mite. Further examination of leaves sampled from

all plots on24 November 2000 confirmed the presence of the mite at similar densities

in all plots. Populations varied from no mites to over 100 per leaf: the populations

consisted of at least 50% juveniles; unhatched eggs were present on > 50olo of leaves. 82

4.4 Discussion The coverage of vine leaf area achieved with the spraypacks and spray cart used

was assessed using water sensitive papers. The coverage achieved by the spray pack was inconsistent and varied from excess spray on some leaves to none on others. This was due to difficulties associated with maintaining pressure as spraying progressed and poor spray pattern. The coverage achieved by the spray cart was ideal on exposed

leaves, at speeds of 3 kph or lower, but the spray did not penetrate into the canopy or on

to the back of leaves (see section 2.2.3),leaving much of the leaf surface untreated.

the At Temple Br";er Wines vine'yard, milk aüd whey were most effective in reducing the severity of powdery mildew (see section 4.3.r). powdery mildew developed only in areas where the spray coverage of leaves and bunches was

compromised, such as on the sheltered side of bunches or where bunches were covererl

by leaves. The test materials appeared to kill existing colonies of the pathogen as

effectively as they had in the greenhouse. The success of these materials at this site and in the greenhouse warrants their inclusion in further experiments on the control of powdery mildew.

B. subtilis reduced the severity of po',vdery mildew at the Temple Bmer Wines

vineyard, but was less successful than the milk and whey treatments and did not reduce

tire severity of pow

areas, povødery milde-w developed on leaves and bunches that had been exposed to the

spray and would have reoeived adequate coverage. There was no significant difference between the B. subtilis treatments and the yeast extract medium in which rhe B. subtilis rrau'^^ã L^^-urverr ^.,1+..-^l uurtursu' rfle lnconslstency suggests tactors other than Spray coverage may affect the efficacy of B. subtilis as a control of powdery mildew in the field experiments. The reduced efficacy of B. subtil¿s in the field experiments, compared to that the greenhouse in experiments, could have been due to environmental conditions, 83 such high temperatures and lower humidity in the field, incompatibility with the spray equipment or the inability of B. subtilis to compete with other phylloplane microorganisms.

The survival of B. subtilis on grapevine leaves in the field needs to be investigated as it may explain the difference in efficacy in powdery mildew control under field and greenhouse conditions. If the difference in environmental conditions between the greenhouse and field was responsible for reduced efficacy, selecting different strains of the bacterium or altering the culture medium may improve efficacy.

Strains of B. subtil¿s isolated from vines may be better suited to the environmental conditions prevailing in the field and provide better control of powdery mildew than the laboratory strain.

The failure of the test materials to control powdery mildew at the Glenara Wines vineyard was partly due to poor spray coverage on vine leaves and bunches. Vy'ater sensitive papers placed in the centre of the canopy indicated that no better than llYo coverage had been achieved. The canopy structure and density of shoot growth resulted in less than 50% coverage of leaves and bunches, particularly in the interior of the vines. The poor disease control due to inadequate coverage was further compounded by the vigour of the vines, which resulted in large areas of new, highly susceptible growth developing between spray applications. However, development of powdery mildew on leaves that received adequate coverage appeared to be lowest on vines treated with milk and whey. The experiment was abandoned, as large amounts of inoculum produced in the trial areas could have spread to other areas of the vineyard.

Results from the Mountadam Vineyard were confounded by two unforeseen factors, a) loss of leaves on the most severely affected plots and b) an area of the experiment site with soil that remained wet for most of the growing season. Moisture 84

from the soil in this area resulted in increased canopy vigour and humidity, creating

conditions suited to the development of powdery mildew. These two factors need to be

considered when interpreting the results from this experiment. At this site spray

coverage of the leaves and bunches was also inadequate, and powdery mildew was

more severe in sheltered areas of the canopy than on leaves and bunches that were

exposed to spray of the test materials.

The infestation of powdery mildew on the untreated and water-treated vines at

Mountadam Vineyarrl was severe ancl leaf clrop of the most severely infected leaves

occurred prior to the final assessment. As a result, only less severely infected leaves

remained for assessment, giving a low disease severity score and increasing the

êvñ^cìrrê l-o-.ioo +n +Lo c"n 'f'L;. L^",^ l,:11^l rL^ *^,.,1^-,, vr^f uvrrrvo rv rr¡w ùu¡r. r rrrJ w^yvùulv^--^- rlral rrútvv Àlllvtl. ù\Jltlu^^*^ uI^f Llru P\.rw\rçt J

mildew on bunches, affecting disease assessments, supported by scarring on many of the berries where powdery mildew had established but later died.

The wet area wâs not noticeahle prior to establishment of the sites a.nd the first application of test materials and was, therefore, not factored into the design of the experiment. Despite the ranciom ailocatlon of'the test materials to the experiment plots, three of the fbur B. subtilis-treated plots were in the wet area, whereas no other test mateial was applied to more than one plot in the wet area. The significantly greater disease severity on vines treated with B. subtilis compared to vines treated with the yeast extract medium was not seen in the greenhouse or at other field sites, suggesting that the extra soil moisture around the vines treated with B. subtilis may have led to the increase in both disease severity and bunch size.

Despite the difficulties experienced at the Mountadam Vineyard most treatments reduced the severity of powdery mildew. Neemtech@ significantly reduced the severity of the powdery mildew on the vines, which would make it unsuitable for use as an 85 insect control if required in later experiments, however, it has application in vineyards where there both insect and disease levels need to be addressed, potentially reducing the number of materials to be applied.

Powdery mildew severity on vines sprayed with whey, Ecocarb@ and yeast extract medium was not significantly different to that on the vines treated with

Neemtech@. The reduction in the severity of powdery mildew on vines sprayed with whey supported the results from Temple Bruer Wines. At both Mountadam Vineyard and Temple Bruer Wines the powdery mildew on vines treated with whey was more severe (23% and 15% respectively) than considered acceptable for commercial harvest

(5%) and higher concentrations of whey were assessed in later trials.

The most unexpected result came from the Warriparinga site where, despite severe infection the previous season, powdery mildew did not develop even on untreated control vines. There are a number of possible explanations for this, including unfavourable environmental conditions for development of powdery mildew, high populations of tydeid mites (see section 1.4.4.6), and coating of leaves and berries with dust from nearby construction work.

While the weather conditions at.Warriparinga were possibly less favourable in the 200012001 season than in the previous season, powdery mildew did develop on some vines of an unknown cultivar 10 m from the experiment site. Powdery mildew developed on untreated vines at all other experimental sites in 200012001 and while there are climatic differences between experiment sites, it is unlikely that the difference would be large enough to have had such a great influence on disease levels.

Leaves examined late in January 2000 had mixed populations of phytophagous, predatory (Phytoseiidae, Typhlodromus doreenae) and tydeid mites. Examination of the leaves in November 2000 revealed a different acarine community structure, with 86

significant populations only of phytophagous mites and tydeid mites. The tydeid mites

were a species of Orthotydeus, but not O. lambii, which reduces the severity of

powdery mildew in vines (see section I.4.4.6) (Krantz 1978; Agrawal 1997; English-

Loeb et al. 1998; Jones 2000; Norton et al. 2000). It is possible that populations of

Orthotydeus sp. could have contributed to the low levels of powdery mildew in the

vines but, unless the population was unusually high in the 200012001 season, the

inability of the mites to control the powdery mildew in the previous season casts doubt

on this explanation.

One method of controlling powdery mildew mentioned in chapter I (section

1.5.5) is application of whitewash or fine clay to the leaves and bunches (Marco et al.

I ôfì/'l\ n-,-:.^- +L^ 1^rìrì/a^^l *-^--.:-- l---L C.-^,-- --^^--1^-- L7>+). L)utttté Ltrç LvvvtLvul ËruwrrrË ¡itr¿lsurr, uust rluln a^ rrg¡lruy uuuuuuL:uuil^¿j^,^ sil,E ^jt^

coated most of the leaves and bunches, and could have been responsible for the

reduction in powdery mildew on the vines. The adjacent vines that developed powdery

tnilrlp'r¡¡ in fhc )nñ /)(\^1 cêâc^ñ r¡¡crc alcn of'fa¡tprl lrr¡ fha rlrrcf lrrrf 4yHv4rvuannaorp¡l f^ ha lacc

heavily coated,

The test materials most successful in reducing the severity of powdery mildew were milk, whey, Synertrol Horti-Oil@ and Neemtech@. The latter was included only to test whether its use as an insecticide wouid affect the severity of powciery miidew on vines. However, one of the aims of the project was to develop powdery mildew control protocols that have minimal impact on other organisms in the vine ecosystem, particularly beneficial arthropods. The use of insecticidal soap does not fit this criterion;

+L^-^f^-^ ¿Li^ ---^^ --^L L^-L^) E-11-^-^ À till- ---1^------I o---- -,),^-1 rr- ,r: n:r(E rrrçlEltJln, ulrù lilill,çtl¡41-^^¿^-i^l wab llut ttrstgu lululçl . lvlilK, wltvy ¿lilu ù-ylrgrtIUr ¡1ulu-\Jll , however, were deemed worthy of study in subsequent experiments. 87

Chapter 5 - Mode of action of selected materials

5.L Introduction While the mode of action of bicarbonate sprays is well known (Homma et a1.,

1981), the mode of action of milk, whey and the canola oil-based sprays is not well understood (see sections 1.4.5.3 and 1.5.1). There have been a number of explanations for the action of milk, including the anti-fungal action of the fatty acids in milk, or the creation of osmotic imbalance due to salts and other components of milk. The induction of systemic resistance was suggested by Bettiol (1999) as a possible explanation for the reduction in powdery mildew on zucchini plants sprayed with various concentrations of milk. There is also evidence that exposure of milk to ultra-violet radiation, in sunlight, resulted in the production of oxygen radicals that interfered with the cell membranes of

P. infestans (Jordan et al. 1992). Materials such as methionine and riboflavin have been shown to control powdery mildew through the production of free radicals (Tzeng et al.

19S9) (see section 1.4.5.7). Free radical production can be measured using electron spin resonance spectrometry (ESR) (Tzenget al. 1984; Tzenget al. 1989).

Another possibility is that milk may act as a source of nutrients for non- pathogenic microorganisms, which may then compete with U. necator (M. Ryder, pers. com., 2000). If milk or whey can assist non-pathogenic microorganisms to establish and spread on leaves, conidia of U. necator may not be able to establish as readily. The nutrients may also assist antagonists of U. necator to persist on the leaf surface when normally they would not be able to survive. The presence of such antagonists prior to establishment of powdery mildew may inhibit germination of conidia through sequestration of micronutrients, such as iron, or antagonists may attack vegetative structures of (J. necator and thus limit the size and rate of spread of colonies. 88

Components of milk and whey, particularly lactoferrin and lactoperoxidase,

have been extensively researched as antimicrobial and antiviral agents for use in human

medicine and for the reduction of food spoilage (Batish et al. 1988; Modler et al.;

Kuipers et al.1999; Sallmann et al. 1999; Kanyshkova et al. 2000; Samaranayake et al.

2001). Lactoferrin, an 80 kd iron-binding glycoprotein, was originally thought to inhibit

microbial development by scavenging any free iron molecules required for germination

of spores. However, subsequent research showed that lactoferrin binds to porins of E.

coli, resulting in chatrges tt¡ thc stability aurd permeability of cell membranes (Sallmann

et al. 1999). Lactoferrin binds to the membranes of a number of other bacteria and

fungi, causing damage to membranes and loss of cytoplasmic fluids (Batish et al. 1988;

Kuipers et ai. 1999; Saiimann ef al. 1999; Samaranayake et al. 2001).

Research into lactoferrin's antibacterial properties has concentrated on Gram-

negative bacteria and other species prirnarily involved in food spoilage, whereas much

oi tiie researuh iliu iis anii-iurrgai ¡rru¡reriies iras been reiaiecì to human heaith, with

Candida spp. of particular interest. In addition to direct anti-fungal effects on Candidq

spp., lactofen-in also appears to boost the hruman immune system making it ideal for

patients compromised immune systems such as those receiving chemotherapy or "l'ith suffering HIV (Kuipers et al. 7999; Kanyshkova et ai. 200A; Samaranayake et al. 200i).

The concentration of lactofèrrin ranges from 20 to 200 pdml in bovine milk and

56 to 164 pdml in whey (Riechel et al. 1998). The optimal concentration of lactoferrin

for control of E. coli and Scilmonella typhi is 0.2 mglml (Batish et al. 1988). In field and

^-.-^-2-^^,-r- ,--:11- ---^- ^,-,-l:-l L a . I l^ i'1 ,' I I Ërççruruuùç-^^-L^--^^ rrÀIjçlurtuu[s, rrllü( was apprrcu at a t:J or t:lu ollullon ano wney at between 1:3 and 1:6 dilutions. A concentration o120 ¡t"g/ml of lactoferrin was selected for use in the scanning electron microscopy (SEM) experiments, as being representative 89 of the concentration in milk and whey used previously (see sections 3.3.1, 3.3.2, 4.3.I and 4.3.3).

Lactoperoxidase is a known antimicrobial protein that is present in milk at approximately 30 mg/L (Modler et al.). The ability of lactoperoxidase to control E. coli,

Lactococcus lactis and some other bacteria varied with the concentration of hydrogen peroxide and thiocyanate, with low or excessive levels of hydrogen peroxide inhibiting the antibacterial effects of lactoperoxidase (Modler et al.). However, research into the ability of lactoperoxidase to control fungi appears limited. The application rate selected for lactoperoxidase (10 ¡rg/ml) was based on the concentration of lactoperoxidase in bovine milk, and adjusted for the dilution rate of milk used in the field.

SEM can provide detailed images of the surface of fungal hyphae and conidia.

(J. necator lives mainly superficially (see section 1.3.2), ensuring exposure to the test materials and making SEM a suitable method of examining physical changes caused by the materials. The cryogenic technique, involving minimal specimen preparation, was selected to reduce the likelihood of damage to the hyphae and conidia during preparation.

5.2 Materials and methods

5.2.1 Detection of free radical activity Several potential novel control agents were assessed for the presence and production of active oxygen radicals using ESR. A Varian E-12 EPR spectrometer, operating at X-band (-9.1 GHz) at the Department of Physics at Monash University,

Melbourne, Victoria, was used. All measurements were performed at room temperature

(approx. 22' C). The specimens were placed in standard, special quartz EPR tubes, internal diameter approx. 2 mm. The sensitivity of the apparatus was tested using the standard "weak pitch" sample supplied by Varian. 90

All dry samples, whether "pure" (e.9. milk powder) or "mixtures" (e.g. MR

formulations) were tested, undiluted, for free radical signals. The apparatus was not

sufficiently sensitive to observe free radical signals at the dilutions used in the field.

The materials assessed in this manner were: full cream milk powder; whey powder;

whey protein; three MR formulations based on Tzeng and DeVay (1989); and lactose.

The three MR formulations ,ü/ere: 1) the formulation listed in appendix 2, section 2.3;2)

the MR without laurel sulphate; 3) the MR with copper sulphate replaced by ferrous

sulphate.

5.2.2 Scanning electron microscopy Infected leaf tissue treated with various test materials was examined in a Philips

XL30 ficld cmission SEM at Adelaide Microscopy. The microscope was equipped with

an Oxford Instruments CT1500 HF Cryo.

The treatments examined in this series of experiments were: milk (l:5 and 1:10

dilutions of fi-rll cream milk); .¡,he), (30 gl¡ .vhe), porvder); .,vhey protein (30 glL);

lactoferrin (from bovine colostrum, 20 pglml, Sigma Chemicals); lactoperoxidase (from

bovine colostrum 10 mglml glL, Sigma Chemicals); hydrogen peroxide (0.3%

solution); Synertrol Horti-Oil@ (2 mUL); and Synertrol Horti-Oil@ (2 ml/L) mixed with potassium bicarbonate (a lL Ecocarb@, Organic Crop Protectants Pty Ltd). Lactoferrin and hydrogen peroxide were included to determine if they might be active components of milk and whey.

The application rates of the test materials were selected to emulate rates that were used in the greenhouse or field experiments, aithough in some later experiments field rates were adjusted to improve efficacy or reduce costs. The eoncentration of hydrogen peroxide used for these experiments was based on measurements by Modler 9T et al. (1996), who found hydrogen peroxide concentrations of 5.6 - 28.6 pglml in samples of whey.

Young, near-fuIly expanded leaves with no noticeable powdery mildew were excised from vines of V. vinifera cv. Viognier or cv. Cabernet Sauvignon that had been grown in the greenhouse. The leaves were surface sterilized by soaking in a solution of

White Kittgt domestic bleach (0.5 glL sodium hypochlorite) and Tween 80@ for 3 min.

After surface sterilization the leaves were rinsed in sterile distilled water three times and allowed to dry on sterile paper towels before being placed in Falcon tissue culture dishes, 100 ûtm diameter, 20 Inm deep containing 1.5% agú (Bitek, Difco

Laboratories, Michigan, USA). Leaves were placed adaxial side uppermost on four sterile tootþicks with the petiole embedded in the agar (Evans et al. 1996). Spores of

(1. necator, collected from vines maintained in tissue culture (see section 2.1.2) using a modified cyclone separator, were brushed onto the leaves using a sterile artist's brush

(Evans et al. 1996). The leaves were then incubated in a growth cabinet for 2 weeks at

25oC with a l2-hour light/dark rotation to allow the fungus to grow on the leaf surface and form conidia. Leaves on which no powdery mildew developed were discarded.

Leaves treated with milk 30 min, 6, 12,18 and 48 h prior to examination were also observed to assess response over time. Additional leaves treated with whey, whey protein and lactoferrin were incubated in natural light for 48 h prior to examination.

Infected leaves, untreated or sprayed with water 24hpnor to examination, were used as controls.

Test materials were applied using an Atomizer reagent sprayer (Alltech

Associates Inc., Belgium), to achieve as close as possible to I00o/o coverage while applying a dose of the test material similar to that applied in greenhouse and field experiments. As in the greenhouse and field experiments, no surfactants were used. The 92

Falcon dishes were left open in a laminar flow cabinet to allow the leaves to dry, the

lids were replaced and the dishes were stored at room temperature, approximately 15 /

25"C night lday, in natural light. Plates to be incubated in darkness were wrapped in foil

and incubated alongside leaves exposed to light. Leaves were examined 24 h after treatment, unless otherwise stated. Leaf segments, approximately 10 x 5mm, were cut from the treated leaves and frozen in nitrogen slush, then transferred under vacuum to the preparation chamber. The sample was coated with platinum, then placed on the microscopc stagc (hcld at < -150oC) and examined.

Observations of the hyphae and conidia of U. necator on untreated leaves and on leaves sprayed with sterile RO water only were used as a reference when observing fungai structures on ieaves spraye

l^--^-^ L^ .1-- Í, , 1 'T'L^rrr.v þsLrrrrcrtuù^^+:-^+^^ ur^f Lr4rrr4Bs ru urs L,urllul¿l--,-:t:- tJL-c u. necul.ur oL lgavcs sljÌaycu wluì ulg tcst materials were based on counts of conidia that appeared damaged and those that appear similar to those on untreated and water-treated leaves.

A second series of experiments was conducted using leaves with older colonies ai U. necalor and cleistothecia, which were coliected tiom ihe greenhouse, piaced directly into Falcon dishes, as above, and treated 24 h pnor to examination. Untreated leaves were used as a reference for the appearance of natural deterioration of hyphae and conidia, to allow comparison with those stnlctures following exposlrre to test

*^t-;-l- 'I-h^-^ .,^^l r^ --.L^JL Lt- ¡rr4rwrr4rù. lllvùv v^purrrrrçtrLù^--^..j*^-r wçlç 4rùL,^1^^ ubçLl tu alùsgss^^^^-^ wtlçtllgl^- ulE;^ LE;st Iflalçftals^,i^1- uausgu ^^--^^l any visible damage to cleistothecia of (J. necator. 93

5.3 Results

5.3.1 Electron spin spectrometry EPR spectrometer signals and ouþuts are qualitative rather than quantitative and, while showing the presence or absence of free radical species, do not provide information on their relative abundance. All samples tested in the EPR spectrometer showed a free radical signal with no structure. The three MR samples, containing Cu** or Fe**, also showed other EPR signals, including a signal tlpical of Cu** or Fe***, respectively.

5.3.2 Scanning electron microscopy Using SEM, approx. 90Yo of (J. necator hl,phae from 2-week-old colonies on untreated and water-treated leaf segments appeared turgid and undamaged, with the remaining 10% either collapsed or otherwise damaged (Figures 5.1, 5.2 A). Similarly, more than 90o/o of conidia appeared undamaged after spraying with water. There \ryas no

obvious difference in the appearance of conidia and hlphae on samples collected from

control leaves incubated in natural light or where light was excluded. Conidia produced

germ tubes at one end and close to the leaf surface (Figure 5.2 B). Colonies that were

more than 6 weeks old displayed large areas of collapsed h¡phae, and conidia were

often collapsed and cracked (Figure 5.3). Cleistothecia on untreated leaves were

roughly spherical in shape and appeared turgid (Figure 5.a). 94

Figure 5.1: SEM image U. necator of on the upper surface of a dctached leaf of V. vinifera cv. Viognier 15 days after inoculation (untreated control). A) hyphae and appressoria ( ws) B) hyphae and conidium (arrow)

I, I .5 lB I ¡l \ I \ \ \ r\r

('on itl i iu r r

--" = \ \v \-- 1 \ Â<:r: V Íjpol Maqrr Dcl WL) txp 20 pni 2 00 kV 1Ì 0 3200x llF ItA 9s

Figure 5.2: SEM image of U. necator onthe upper surface of a detached 1r-af of V. vinifera cv. Viognier 15 days after inoculation, sprayed with sterile distilled water 24h prior to examination and incubated in natural light at 20 - 25o C. A) hyphae and appressoria (arrows) B) germinating conidium

-\.28 ¿.#.-

(ìcnr tLrbc I L ¿

*) ¿

Acc V Det WD l00kvsE 15 5 96

Figure 5.3: SEM image (J. of untreated hyphae of necator on the upper surface of a detached leaf of V. vinifera cv. Viognier 42 days after inoculation (untreated control).

-) \ lf / ¡ f -r {

't 4,.- I v. ')

I)ctcnrlrrrf

Ç¡ t ,/: )P. lc elt I J çonrtlr¿t ,l -{ I

A<;c -Dct fff ri0 \ .';l l0 0, firilieateil

Figure 5.4: Cleistothecium, appressorium and hyphae (arrows) of (J. necator on the upper sur e of a detached leaf of V. vinifera cv. Cabemet Sauvignon from the greenholtse (natttql irrnnrr \¡¡-i!!r^tr^ lqfin- ¡, rrnfraoralu¡rlrv4lwu ^^-+*^t\wu¡rll\rlr.

k 97

Milk (1:10 dilution) or whey, applied to leaves incubated in natural light 24 h prior to examination, resulted in extensive damage to both hlphae and conidia but not to cleistothecia. Most (80-90%) of the hlphae were collapsed and, in some cases, appeared to have ruptured (Figure 5.54). A similar proportion of conidia were collapsed, cracked or ruptured with "hernia-like" protrusions of cell contents along the cracks (Figure 5.5B). However, undamaged conidia were able to germinate. No

symptoms were noted on fungal structures on untreated areas of the same leaves. For leaves treated with milk and kept in the dark for lhe 24 h prior to examination, the

appeffance of the conidia was similar to that on leaf segments exposed to light, but only

20To of the hlphae were damaged (Figure 5.6). Cleistothecia appeared undamaged and

similar in appearance to those on untreated leaves.

The extent of damage to fungal structures on leaf samples observed 30 min after

spraying with milk was difficult to assess accurately as the samples were not

completely dry and milk droplets obscured much of the leaf surface. The "hernia-1ike"

features were present on approximately 20%o of conidia on leaf samples sprayed with

milk 6 h prior to assessment. Up to 80% of the hyphae were damaged in observations

made 6 h after treatment, however, many of the hyphae were only partially rather than

completely collapsed as was the case with samples treated 12,24 and 48 h previously.

There were no obvious differences in the extent of damage to conidia and hyphae of U.

necator on samples from leaves sprayed with milk and observed either 12,24 or 48 h

after treatment. 98

(J. FÍgure 5.5: SEM of necator onthe er surface of detached leaf on V. vinifera cv. Viognier i5 days after inoculation, sprayed with a 1:10 dilution of milk 24hpnor to

Collapsed hlphae and conidia and ruptured conidia are indicated by arrows. B) ruptured conidium. Note crack continuing away from rupture and appearance of cell material within the cell.

+

I Acc V l)ol 5 500kvst ¡rrrr 99

Figure 5.6: SEM of hlphae and conidia of (J. necator on the upper surface of detached leaf of on V. vinifera cv. Viognier 15 days after inoculation, sprayed with a 1:10 dilution of milk 24hpriror to examination. The dish containing the leaf was wrapped in foil to exclude light after milk was applied and maintained at 20 - 25" C.

Application of whey protein to the leaves, exposed to natural light and

maintained at 20 - 25oC for 24 h pnor to examination, resulted in damage to fungal

structures similar to that observed on leaves that had been sprayed with milk and

incubated in darkness. The majority of h¡phae (>S0%) were turgid and similar in

appearance to those on untreated leaves. Conidia displayed cracking and rupturing

(Figure 5.7). Cleistothecia appeared slightly collapsed and dehydrated (Figure 5.8).

Examination of samples 48 h after application of whey protein showed complete

collapse of conidia and collapse of up to 50o/o of the hlphae. 100

FÍgure 5'7: SEM of (J. necator conidia ancl hyphae on the upper surfacc of detached

20 - 25'C.

F'ipure 5-ß:-'-' SFM nf nlpicr^nl"^^:,.*v'vrurvur¡ww¡urtr ^c , t ---^.., ,1 - ð--' ur .J. nccutul'on Uìe upper surlace of detached leaf or v. vinifera cv. viognier collected from the greenhouse, sprayed with L whey protein 24hpnor to examination. After treatmeãt, leaf was incuúated in light at 20 - 25'C. n

-5.8

ntacl u¡l¡lcrrtlagcs

/* --* ^1 ¡1 ''*{ -¿,'i /

M, rqn '. J bo ¡un ll05x Wheiy Prulr¡rrr 4 .luly"20U) 101

Examination of leaves sprayed with lactoferrin 24 h prior to assessment and incubated in natural light at 20 - 25oC, showed that approximately 70%o of the hlphae of

(J. necator appeared undamaged. The remaining 30%o had lost turgidity and, in some places, appeared to have ruptured (Figure 5.94). As was the case for U. necator on

approximately 90% of the conidia were cracked and ruptured, with leakage of cell contents (Figures 5.9B and 5.9C). Examination of leaves sprayed with lactoferrin 48 h prior to assessment and incubated in natural light at 20 - 25oC, showed that the collapse of hyphae had increased to approximately 50o/o and conidia rwere completely collapsed

(Figure 5.10).

Figure 5.9: SEM of (J. necator on the upper surface of detached leaf of V. vinifera cv. Viognier 15 days after inoculation, sprayed with 20 Vglml aqueous lactofernn 24 h prior to examination. After treatment, leaf was incubated in natural light at 20 - 25o C. A) ruptured hyphae B) conidia and hlphae C) ruptured conidium showing crack in cell wall and "hernia-1ike" structure.

5.eA Danragctl yphac

t

\ / Ap¡rrcssor-ilt

-t I

Acc V Maqn Det WD 10 ¡-rnr l00kV50B8x SE 1/b 2 a¡O2 z0r 103

Figure 5.10: SEM of conidia of (J. necator on the upper surface of detached leaf of V. vinifera cv. Viognier 15 days after inoculation, sprayed with 20 pglml lactoferrin 48 h prior to examination. After treatment, leaf was incubated in natural light at 20 - 25o C.

Examination of leaves 24 h after treatment with lactoperoxidase and incubated

in natural light at 20 - 25oC, revealed that up to 20 Yo of conidia had split open, but the

remaining 80o/o appeared undamaged and, in some cases, appeared to have germinated

normally (Figure 5.114). Approximately 70% of hl,phae appeared turgid and

undamaged, the remaining3}o/o had lost turgidity and collapsed (Figure 5.118).

The application of mixtures of Synertrol Horti-Oil@ and Ecocarb@ to infected leaves

resulted in the collapse of hlphae and approximately 50% of conidia appeared

disfigured (Figures 5.12A). The integrity of hyphae ìwas maintained only in areas that

were not treated. The conidia did not show "hernia-1ike" features. Germ tubes had

collapsed (Figure 5.128). Residues were more noticeable on leaves sprayed with

Synertrol Horti-Oil@ plus Ecocarb@ than on leaves sprayed with other materials in the

experiments. 104

Figure 5.11: (J. S of necator on the upper surface of detached leaves of I/. vinifera cv' Viognier 15 days after inoculation, sprayed with 10 pdml aqueous lactoperoxidase

C. A) conidia and hyphae B) conidia and hlphae s llA

.F -à'.-

J ltrlit t nrqr:rl con jrìi ¿r

f S¡rlìt t'olrirlia

L

4:

Maqrr l00gm udan raqctl hy¡rlrrc 531x l. acto roxldase l2 Junrr 2ÒO? 105

Figure 5.12: SEM of U. necator on the upper surface of detached leaves of V. vinifera cv. Viognier 15 days after inoculation, sprayed with 2 ml/L Synertrol Horti-Oil@ plus 2 glLBcocarbt 24 hours prior to examination. After treatment, the leaf was incubated in natural light at 20 - 25o C. A) hyphae and conidia B) hy,phae and conidia at higher magnification.

-J** 5.1 ì

! Ð i ('o I lapsccl ? hyphac ancl ¿l p[)fessofl Ll Gcnrinatir,* couicliunr -

ll a- ('oll tl gcrnr conidium tttbe

Acc V Det 20 pm lOOKVSE 106

but the h1'phae had collapsed and ruptured (Figure 5.134). The remaining conidia had

co letely collapsed. Over 50% of the intact conidia on the hydrogen peroxide-treated leaf segments appeared to have ge inated (Figure 5.13A), compared to approx. l0% of conidia on untreated leaf segments. "Hernia-1ike" structures were visible on some pafüally collapsed hyphae on leaf segments treated with hydrogen peroxide.

appeared to have collapsed when examined 48 h after the application of the hydrogen

Figure 5.13: SEM of hyphae and conidi ge nating conidia. After treatment, leaf was incubated in natural light at 20 - 25. C. A) 24 h after inoculation B) 48 h after inoculation

\ llA +---J'ollapscd rel'lìtlU lrcs itlÌlfcssoflUnl

('ollir¡-rs<'rl conirliurr nnrl ( 'ollapsctl gr:t'rlr 1rlhc

hyplrac I v

( ollapsc(l uonrii¡nln

M;rQrr t-- J ¡i¡,!..-... I t1¡\' lllll b60x lly

5.4 Discussion A number of the materials tested caused collapse of conidia and or hyphae of U. necator after incubation for 24hin natural light. Milk, whey and whey protein affected both hlphae and conidia of the frrngus, while damage 24h after exposure to lactoferrin appeared to be restricted to conidia and to h¡1phae in the case of hydrogen peroxide.

Both hyphae and conidia were affected 48 h after exposure to lactoferrin or hydrogen peroxide. When powdery mildew-affected leaves were incubated in the dark after exposuro to a 1:10 dilution of milk, the damage to conidia was unchanged but the damage to hyphae was greatly reduced compared to those incubated in natural light.

Free radicals were produced by all samples when exposed to natural light. Free radicals have been shown to reduce the severity of powdery mildew (see section

I.4.5.7) and may have contributed to the reduction observed in greenhouse and field experiments (see sections 3.3.1, 3.3.2,3.3.3, 4.3.1. and 4.3.3). Tzeng and DeVay (1989) found that exposure of methionine, riboflavin and sulphur-rich amino acids to light 108

resulted in the production of free radicals, which was associated with redu-oed severity

of powdery mildew on grapevines.

The level of production of free radicals from the test materials is dependent on

light and temperature in the leaf canopy. In the field, solar frV radiation would increase

the production of free radicals from the original solids (G. Troup, pers com. 2002). The influence of light intensity and temperature on free radical production has implications for the timing of spray applications in the field. Spraying on bright, warïn days could result in greater free radical production thali applioation on cooler, cloudy clays. This may also have implications for the efficacy of the test materials in cool climates where the intensity of natural light is lower, leading to a need to increase concentrations of

*^+^-:^l- -- 1. rrraLçrrill¡i ur^ tuuuge-- - sptay lnlervals.

Canopy structure and alignment can have a major impact on the temperature and light intensity within the canopy and would influence the production of free radicals

frnrn fccf \IfL.;l^ r mafar-iol.¡¡rervtrorú' vY ^^-+ ^----^:- ^- r rrtrv lIUùt urË4lrrtrus llav9 tlleçnanlsms ïOf Copmg Wtth lOw levels of free radicals, high levels can result in damage to cells. Similarly, large, dense vine canopies not only reduce spray coverage and provide conditions conducive to powdery mildew (see section I.4.1,1) but also provide shade withiir the canopy, which would reduce fr'ee radical production and hence the eflcacy of the treatments. East-west

alignment of vine rows may fuither reduce the exposure to direct sunlight of leaves that

are in the middle of, or low in, the canopy.

Exposure of powdery mildew-affectecl leaves to hydrogen peroxidc (0.I% sollltion) rnrl incnholi^^ f^- 1A l;^ *^+,,-^r t!^1^¿,-- - tt r . rrvrt ¡vr 'a tL ttt ttø'Lutiet lrËlrt I'9SU|[e(l ln OetgflOfatlOn Ol thg hyphae but there appeared to be minimal damage to conidia. In some cases the conidia of y necator appeared to have germinated. However, examination of powdery mildew affected leaf-segments 48 h after application of 0.1%o hydrogen peroxide revealed that 109 approximately 50o/o of the germ tubes emerging from the germinating conidia had collapsed. Whether this was a direct effect of the hydrogen peroxide, failure of the germ tubes to form penetration pegs or appressoria, the induction of a plant defense response or some other effect is unknown.

SEM observations of powdery mildew on leaves exposed to a 1:5 or 1:10 dilution of milk and incubated in natural light for 6, 12,24 and 48 h revealed damage to both hyphae and conidia, that was not evident on leaves sprayed with water or left untreated. The damage to hlphae of (1. necqtor on leaf segments from leaves sprayed with a 1:10 dilution of milk or Io/o hydrogen peroxide 24 or 48 h prior to examination was similar. However, the conidia on leaf segments exposed to hydrogen peroxide appeared undamaged and those on leaf segments exposed to a l:10 dilution of milk appeared damaged, i.e., the milk had fungicidal properties. This was supported by the results in greenhouse trials, in which not only was the severity of the disease on leaves sprayed with milk and whey less than on untreated leaves, but the severity of powdery mildew on leaves at the completion of the experiments was less than that prior to the first application of test materials.

When light was excluded after exposing powdery mildew-affected leaves to a

1:10 dilution of milk, the damage to hyphae was considerably less than when leaves were incubated in natural light. The reduced damage to hyphae in the absence of light suggested that the fractions of milk active against hyphae and conidia in the 24 h after application are different or that they act in a different manner. If the damage to hyphae was caused by induction of resistance in the plant due to the application of milk and whey, exclusion of light should not reduce the efficacy of the treatments. The apparent decreased efficacy of milk in darkness has implications for the timing of sprays in vineyards and canopy management as discussed in relation to free radical production. 110

There are a numher of possible soruces of free radicals in milk and whey; sulphur-rich

amino acids (Tzeng et al. 1989), bacteria and enzyrne activity. Application of milk or

milk components such as whey to vines early in the day would allow maximum

exposure of the materials to sunlight, potentially producing more free radicals within

the canopy.

The "hernia-like" rupturing of conidia of (J. necator was seen on leaves sprayed

with milk, whether incubated in natural light or in darkness, and on leaves exposed to

whey, ',¡¿hey proteiu and lactoferrin. trl lastolerrin bounrl to membranes of {/. necator jn

these experiments and altered the permeability of the conidia, possibly disrupting the

osmotic balance of the cells (O. Schmidt, pers. coÍxn. 2003), increased intemal pressure couici ieaci to ciamage of the walls of' the conidia and the "hernia-like" structures observed by SEM. Further research is required to establish if lactoferrin binds to the cell membranes of U. necato¡" and is responsible lor the damage. The visually il^--¿i^^l ,c Ll I r ruErlrrual rr¡1rurÉ ol llle ualuagtr uauseu [() conl(tta oy lactolemn, mllK and wney suggested that lactoferrin is an active component of milk and whey. While lactoferrin caused more collapse of U. necator hyphae ai 48 h than at 24 h after exposure, the majority of the damage caused to hyphae after exposure to milk, in light or dark, whey or hydrogen peroxide was within 24 h of application of the matenals. Possible explanations for this are that components of milk other than lactoferrin also cause damage to the hyphae, or that interaction with other components of milk increased the activity of lactoferrin against hyphae.

Â..*li^^¡l^- 1^^+^-.^..^--:l^-^ ta: ' t r-ì'PPrru4trurr ur^f r¿lutuPçlur(Iuastr, ---1-i1-wllllç I-€SUltlng-- ln Oamage IO appfOx. lU"/o OIIflC conidia and 30%o of the hyphae of (J. necator on sprayed leaf segments, caused less damage than did milk or whey. However, the ability of lactoperoxidase to control .8. coli, L. lactis and other bacteria varied with the concentration of hydrogen peroxide and 111 thiocyanate added to treatments (Modler et a1.). Damage may increase if optimal amounts of thiocyanate or hydrogen peroxide were included with lactoperoxidase sprays. However, excessive levels of hydrogen peroxide inhibit the antibacterial effects of lactoperoxidase (Modler et al.). The extent of damage to U. necalor observed in the experiments reported here suggest that lactoperoxidase is an active component of milk and whey in the control of powdery mildew and, as thiocyanate and hydrogen peroxide are present in milk and whey, the effects on powdery mildew may be greater than when lactoperoxidase is applied alone. Further in vitro and greenhouse experiments are needed to identify optimal levels of the additives if lactoperoxidase is to be used in the control of powdery mildew.

The observations of U. necator on leaves exposed to mixtures of Synertrol

Horti-Oil@ and Ecocarb@ revealed that the hyphae, conidia and germ tubes were collapsed. There also appeared to be a residual film of oil coating the hyphae. "Hernia- like" structures were not visible. Time restrictions prevented examination of leaves

sprayed with Synertrol Horti-Oil@ or Ecocarb@ alone and further experiments to

evaluate the individual components need to be carried out.

These experiments showed that milk, some components of milk and oil plus bicarbonate mixtures have fungicidal effects on U. necator. The reduction in the

severity of powdery mildew on vines sprayed with these test materials in greenhouse

and field experiments may be attributed directly to the application of these materials.

While most treatments resulted in some damage to either or both the hyphae and

conidia, none appeared to damage the cleistothecia, except, perhaps, the whey protein,

which was associated with some loss of turgidity.

Milk, whey, lactoferrin, and Synertrol-Horti-Oi1@ plus Ecocarb@ mixtures have

potential as altematives to sulphur and synthetic fungicides for the control of powdery t12 mildew in grapevines. The experiments also show that light influences the efficacy of milk and, therefore, spraying on clear, bright days may improve the efficacy of the treatments. This should be considered when developing spray programs. The cost of lactoferrin makes it uneconomical for use as a spray on its own, but blends of lactoferrin and oil may be an effective and economical option. The most desirable option for spray programs for field situations will be programs using milk or whey and oil plus bicarbonate mixtures. These would reduce concerns about the perceived negative impacts of relying on applications of either oil or bicarbonate all season and reduce the risk of U. necalor developing resistance or tolerance to one or other of the materials. 113

Chapter 6 - Greenhouse experiments 2001 and 2002

6.1 Introduction The greenhouse experiments conducted in 2001 were designed to evaluate fi.uther the most promising treatments for the control of powdery mildew from the field experiments in 2000/2OOl, and to evaluate some new materials. Due to the need to replace the vines at the end of 2000 (see section 3.4) and the lack of suitable rooted cuttings of cv. Viognier, cv. Cabernet Sauvignon vines were used for the greenhouse experiments in 2001. This allowed evaluation of effects of test materials on the severity of powdery mildew on a second grapevine cultivar. Experiments were also designed to determine which components of milk may have contributed to the control of powdery mildew observed in previous experiments.

Only one greenhouse experiment was conducted in 2002, which was designed to assess whey further as a "soft fungicide" for the control of grapevine powdery mildew and to assess a number of compounds, mainly "plant strengtheners", that were as yet untested.

6.2 Methods and materials In 2001, cv. Cabemet Sauvignon vines obtained from Temple Bruer Wines vineyard nursery were established in the greenhouse as described in section 2.1.1.

Control treatment vines were sprayed with water and sulphur was applied as the standard treatment. Not all experiments conducted in 2001 and2002 included untreated controls, as there was no significant difference in powdery mildew severity between vines treated with water and untreated vines in previous greenhouse experiments, and where the number of suitable vines was limited it was deemed more useful to omit the untreated control and include an additional test material. The concentration of wettable sulphur was changed from 3 glL to 2 glL to reduce the risk of phytotoxicity, It4

experienced in experiment 1/2000. In 2001 and2002, vines were not inoculated with U.

necator, as natural infection in the greenhouse was comprehensive.

The vines used in the 2001 greenhouse experiments were replaced, prior to the

conunencement of the 2002 experiment, with V. vinifera cv. Viognier from Temple

Bruer Wines vineyard mrsery. These were established in the greenhouse as described

in section 2.1.1. Two "plant strengtheners" that contain chitosan, and are claimed to

reduce plant disease by enhancing plant defence systems, Vitec Kelp@ and Vitec

Combo@ (Vitec Pty ttd) were included in thc 2002 cxpcriments. The Vitec@ products ,

which are fish and kelp extracts, were tested to provide an alternative to the original

phytotoxic formulation of chitosan (see section 3.3.1). If either of the Vitec@ products

reduceci the seventy or incidence of oowdery mildew, further testing could be

conducted to isolate the active components and determine the role of chitosan in

controlling powdery mildew. Two mixtures, Nu-Film I7@, a surfactant, plus whey

powder and Synertrol Horti-Oil@ plus whcy protein were also tested. The two nixtures

were included to assess tne potential of surfactants and oils to improve the efficacy of

either whey or whey protein in the control of powdery mildew.

Treatments were applied at recommended rates or, in the case of whey, a rate

established in earlier experiments and for Coaton ILP@ an estimated rate was used.

Coaton ILP@ is sold as an attractant for lepidopteran pests such as Helicoverpa zea and

H. armigera (Murray et al. 2000) to improve the efficacy of Bacillus thuringiensis and other biopesticide treatments and the recoÍrmended rate of 2 mllL was considered too low for control of powdery mildew.

6.2.1 Experiment l/2001 The first greenhouse experiment in 2001 was designed to test the efficacy of a mixture consisting mainly of methionine and riboflavin (MR formulation) (Tzeng et al. 115

1989) (see section 1.4.5.7), and to compare two canola oil-based products, Synertrol

Horti-Oil@ (see sections 3.2.1 and 3.2.3) and Biotrol@. Treatments were applied to single vines, with seven replicates for each product per treatment, and ananged in a completely randomised block design.

The following materials were applied;

Manufacturer Rate of product applied/L

1) Water

2) MR formulation see Appendix 2 section 42.3

3) Milk Nestlé, Sunshine full cream powder 15g

4) Synertrol Horti-Oil@ Organic Crop Protectants Pty Ltd 2.5 ml

5) Biotrol@ Gullf Ag Pty Ltd 2.0 ml

6) Sulphur Garden King wettable powder 2.0 ml

Materials were applied and disease was assessed as described in section 2.1.2

6.2.2 Experiment 2 I 2001 MR formulation, whey and Biotrol@ were tested in various combinations (see below). The surfactant Penatrat 11020 g/L polyether modified polysiloxane, Gullf Ag

Pty Ltd), which is registered for use in organic viticulture in Australia, was included in this experiment to evaluate the effect of a surfactant on oils applied for the control of powdery mildew. Lime sulphur was used in this experiment in place of wettable sulphur, as some organic and biodynamic viticulturists use it to control powdery mildew, particularly in cooler climates. Biotrol@ was added to whey and to MR formulation to assess the effect of these mixtures on powdery mildew and to assess their compatibility if they were to be used in tank mixes in field situations. There were six replicate vines per treatment, arranged in a completely randomised block design. tt6

The following materials were applied;

Manufacturer Rate of product applied/L

1) Water

2)Lime sulphur Arthur Yates and Co. Ltd 50 rnl

3) Biotrol@ Gullf Ag Pty Ltd 2.0 ml

@ 4) Biotrol Gullf Ag Pty Ltd 4.0 ml

5) Biotrol@ plus Gullf Ag Pty Ltd 2.0 rnl whey Bonlac Pty Ltd 3og

6\'W-[rer¡ Bonlac Pty Ltd 3og

7) Biotrol@ plus Gullf Ag Pty Ltd 2.0 ml Penatra@ Gullf Ag Pty Ltd 0.4 Íìl

8) Fenatra@ Gullf Ag Pty Ltd ^A^1W.T IIII

9) Biotrol@ plus Gullf Ag Pty Ltd 2.0 ml MR formulation see Appendix 2 section A2.3

10) MR formulation see Appendix 2 section A23

Materials were applied and disease was assessed as described in section 2.1.2

6.2.3 Experiment 3 l200l This experiment was designed to investigate the mode of action of milk and to

elucidate which components contribute to the suppression of powdery mildew. Full cream, skim milk and whey were included to evaluate if fats and fatty acids in milk control the disease. The two main components of whey (lactose, which constitutes approximately 75%o of whey powder, and whey protein) were included. V/ater was applied to vines as a control and a wettable powder formulation of sulphur applied as a standard. All milk-based treatments were applied at the same concentration, 15 g/L, rather than at relative rates. tt7

An additional assessment of disease was conducted24 h after application of the first treatment to assess the reduction in disease compared to the pre-treatment level.

The following materials were applied;

Manufacturer Rate of product applied/L

1) Water

2) Sulphur Garden King Wettable Powder )o

3) Milk 4o/o fat Nestlé Sunshine full cream milk powder 15g

4) Milk lo/o fat Dairy Vale "Skimmer" 15g

5) Whey powder Warmambool Cheese and Butter Factory Co. 15g

6) Whey protein Warmambool Cheese and Butter Factory Co. 15g

7) Lactose Sigma-Aldrich Co. 15 g

Materials were applied and disease was assessed as described in section 2.1.2.

To assess the relative effect of each component of milk on powdery mildew the reduction in disease score in relation to untreated vines was converted to a percentage, and then adjusted so that high fat milk was given a score of 100. The adjusted score was then divided by the relative concentration of the material in whole milk, such that high fat milk: l, low fat milk 1.02, whey : l.66,lactose :2.22 and whey protein: 6.66 (S.

Billington, Warrnambool Cheese and Butter Factory Co. Ltd, pers. com. 2001).

6.2.4 Experiment 112002 The treatments selected for the greenhouse experiment conducted in 2002 included Coaton ILP@ (Biostarch Australia Pty Ltd), Nu-Film 17@ (CWC Chemical

In"), a surfactant approved for organic agriculture in Australia and overseas, a wheyA,lu-Film 17@ mixture and several "plant strengtheners". Sulphur was omitted from this experiment and replaced with the synthetic fungicide Amistar@ (Syngenta

Corp., Switzerland), a strobilurin, so that a comparison between whey and a 118

commercially available synthetic fungicide could be made. Five blocks were

established for this experiment.

The following materials were applied;

Manufacturer Rate of product applied/L;

1) Untreated

2) Amistar@ Syngenta

3) Whey Dairy Gold Pty Ltd 15 g

4) Nu-Film@ CV/C Chemical lnc. 2ml

5) Whey Dairy Gold Pty Ltd 15g Nu-Film@ CWC Chemical Inc. 2ml

6) Coaton ILP@ Biostarch Australia Pty Ltd 5ml

7) Aminofit flower@ Industrial Products Marketing 5 ml

8) Aminofit finish@ lndustrial Products Marketing 5 ml

9) Vitec Kelp@ Vitec Pty Ltd 5ml

10) Vitec Combo@ Vitec Pty Ltd 5ml

6.3 Results

6.3.1 Experiment ll200l The analysis of disease scores for untreated leaves showed no significant differences between treatments (P:0.308) ffid, therefore, significant differences between treated leaves (P>0.001) were considered to be due to effects of the test materials (Lorimer 2000 a). At the completion of the experiment the rnean severity score of powdery mildew on leaves of vines sprayed with water was 1.4, representing

28o/o of the leaf surface affected by U. necator. All vines treated with the test materials had signifrcantly less powdery mildew on the treated leaves than the water control 119

(Table 6.1). The lowest level of powdery mildew was found in the sulphur treatment but 'When this was not significantly different from the other products (Lorimer 2000 b). the results for the vines treated with water-only were excluded from the analysis, the

disease severity score for vines treated with either Biotrol@ or the MR formulation was

significantly gteater than that for vines treated with sulphur (Table 6.1).

The treated leaves on the vines treated with sulphur suffered scorching on up to

80% of the leaf area. The sulphur and MR formulation both left visible residues on the

leaf surface; the former, off-white patches and the latter, small shiny spots over the

entire surface of the leaf. Deposits of milk and oil residues were not detectable. The

area of leaves infected with U. necator observed during microscopic evaluation of the

treated leaves after completion of the experiment was equivalent to that recorded by the

non-destructive scoring during the experimental period. Cleistothecia were present on at

least two of the treated leaves for all materials. However, in 2001 there were fewer

cleistothecia present than in experiments conducted in 2000.

6.3.2 Experiment 212001 The analysis for disease scores of untreated leaves showed no significant

differences between treatments (P:0.692), and therefore the significant differences

between treated leaves (P:0.003) were considered to be due to effects of the test

materials (Lorimer 2000 a). At the completion of the experiment the mean severity of

powdery mildew on leaves of vines sprayed with water was 1.3, representing26%o of

the leaf surface affected by U. necator. Leaves on vines treated with the test materials

all had significantly less disease than the water controls (Table 6.2).

The MR formulation plus Biotrol@ 2.0 mVL resulted in less disease than the MR

formulation alone. However, when the data for the water control were included in the

analysis, the mixture did not give significantly better control than Biotrot@ 1Z.O mVL) 120

alone. The addition of Biotrol@ to whey powdery or Penatra@ also resulted in slightly

lower, but not significantly, mean disease scores than the components alone. When the

data for the water treatment were excluded from the analysis, the vines treated with

Biotrol@ plus MR formulation or Biotrol@ plus whey had significantly less disease than

those treated with Biotrol@ 2 ml/L alone. The addition of Penatra@ to Biotr ol@ 2 mW

did not alter the effect of Biotrol@. The area of leaves infected with U. necator observed

during microscopic evaluation of the treated leaves after completion of the experiment

was equivalent to that recorded by the non-destructive scoring during the experimental

period.

Table 6.1 Mean severity of powdery mildew on treated grapevine leaves (cv. Viognier) after three fortnightly applications of test materials for greenhouse experiment 212002. LSD (5%); when data for untreated vines are included: 0.385, when data for untreated vines are excluded : 0.4206. Score for visual assessment 0 - 5 based on the area affected; 0 to 1; I - 20% of leaf area, I to 2; 2r - 40o/o, 2 to 3; 4r - 600/0, 3 to 4; 6r - 80yq 4 to 5; 81 - I00%. Test materials ranked according to mean disease score. Treatments with the same letter are not significantly different (P<0.05). N/A : not applicable.

Statistical 'fr-^^¿*.^-¿r r ç¿lttttrllt" rvtçau[f--- urüçaùc l:-^^-- st:ufË-----_ significance when data for untreated vines excluded Sulphur 0.1 a a

Milk 0.2 a ab

Synertrol Horti -Oil@ 0.3 a ab

Biotrol@ 0.4 a b

MR formulation 0.4 a b

Water t.4b N/A l2l

Table 6.2: Mean severity of powdery mildew on treated grapevine leaves, cv. Cabernet Sauvignon, after three fortnightly applications of test materials in greenhouse experiment 212001. LSD (5%); when data for water treated vines a¡e included:0.446; when data for water treated vines are excluded: 0.378. Score for visual assessment 0 - 5 based on the area affected; 0 to 1; 1 - 20Yo of leafarea, I to 2;21 - 40yo,2 to 3; 4l - 60yo,3 to 4;61 - 80yo,4 to 5; 8l - 100%. Test materials ranked according to mean disease score. Treatments with the same letter are not signifrcantly different (p<0.05). N/A: not applicable.

Statistical significance Treatment Mean disease score when data for water treated vines excluded Biotrol@ (2 mVL) plus 0.3 a a MR formulation Biotrol@ (2 m/L) plus 0.3 a a whey Sulphur 0.4 a a

Biotrol@ (2 mVL) plus 0.5 ab ab Penatra@ Whey 0.5 ab ab

Biotrol@ @ mUL) 0.6 ab ab

Penatra@ 0.6 ab ab

Biotrol@ (2mUL) 0.8 ab b

MR formulation 0.9 b b

Water 1.3 c N/A

6.3.3 Experiment 3 12001 Analysis of disease scores for untreated leaves showed no significant differences between treatments (P:0.110), and therefore the significant differences between treated leaves (P:0.001) at the completion of the experiment were considered to be due to effects of the test materials. The difference in mean disease scores for all leaves prior to treatment was not significant (P:0.376).

The mean disease scores 24 h after treatment were not significantly different

(P:0.057) (Table 6.3). The mean disease severity score for all test materials was lower then prior to application of the materials, but disease severity increased slightly on the 122

controls sprayed with water or not treated. Residues of lactose, sulphur and whey

protein on leaves made the assessment at 24 h difficult. While the full fat and skim milk

left a light residue, this did not affect disease assessment.

Leaves on treated vines all had significantly less disease 35 days after the first treatment than the leaves sprayed with water only or left untreated (Table 6.3). The full cream milk reduced the severity of powdery mildew compared to that following the lactose or whey treatments (p<0.05), and the disease severity on leaves sprayed with whey protein was less than that of the whey treatment. The powrlery mildew severity on vines treated with whey protein or sulphur treatments was significantly less than that on vines treated with lactose or whey when the data for the untreated control were

çÃryruunL¡^-,^l-.1^J c.-^-^iluill +1-^Liltr ¿1ltiltysrs.---^1---:-

At completion of the experiment, residue on the leaves treated with sulphur, whey and lactose was obvious and there was a light f,rlm on the leaves treated with rnill¿ f-hlnrnfin lcci^no rlcr¡plnnarl nn fhp loa.roo t¡aola¡7 vrr Lrrv rv4 v vu ùr v4Lvu "';+LvY I tlt '.,^f--vv 4!wr 4tlu^ñ,{ vll^n "-+¡an+az{ ulLt w4twu leaves where the powdery mildew infection was most severe. Up to 50% of the leaf area on vines treated with sulphur was necrotic.

The area of leaves infected with L/. necator observed during microscopic evaluatioü after completion of the experiment was sirnilar to that recorded by the non- destructive scoring during the experimental period, except for the leaves treated with lactose. Colonies of mainly non-sporulating powdery mildew hyphae, covering 10-50% of the leaf surface, developed on leaves sprayed with lactose. Where conidia had been nrorllrcerl nn lear¡ec Jhql lr.q¡l hcen cnrq.rcrl rrrifh lqnfnce fho., rrro.o looo flran nn rr v¡v rvuù ^^-,-nnvutrurrvrt Lrrúrr vrr untreated leaves and leaves receiving other materials. r23

Table 6.3: Mean severity of powdery mildew on treated grapevine leaves, cv. Cabernet Sauvignon, pre-treatment, 24 h after the first treatment and after three forlnightly applications of test materials (35 days after the first treatment) for greenhouse experiment 312001. LSD (5%) 24 h, when data for untreated vines included :0.446;35 days after first treatment when data for untreated vines included :0.402) 35 days after first treatment where data for untreated vines excluded : 0.349. Score for visual assessment 0 - 5 based on the area affected; 0 to l; 1 - 20Yo of leaf area, I to 2; 2I - 40yo,2 to 3; 4l - 60yo, 3 to 4; 6l - 80o/o, 4 to 5;81 - 100%. Test materials ranked according to mean disease score. Treatments with the same letter are not significantly different (P<0.05).

Statistical Mean disease Mean disease Mean disease significance when Treatment score score score data for untreated Pre-treatment 24 h after first 35 days after vines excludedr 35 treatment first treatment days after first treatment Milk - High Fat 0.9 a 0.5 a 0.1 a a

Whey protein 0.6 a 0.2 a 0.2 ab a

Sulphur 0.7 a 0.6 a 0.2 ab a

Milk - Skim 0.8 a 0.5 a 0.2 abc ab

Lactose 0.6 a 0.4 a 0.5 bc bc

Whey 0.7 a 0.3 a 0.6 c c

Water 0.6 a 0.6 a 1.s d N/A

Untreated 0.7 a 0.7 a 2.0 e N/A

When the data were converted to reflect the reduction in severity of powdery

mildew based on the composition of milk, high fat milk was the most effective,

followed by low fat milk, whey lactose and whey protein (Figure 6.1). t24

components of milk in experiment3l200I. Equal concentration; all materials sprayed at

actual concentration of the test materials in milk. The reduction in the severity of powdery mildew from pre-treatment to completion of the experiment was calculated, and then adjusted so that high fat milk was given a score of 100. The adjusted score was then divided by the relative concentration of the material, high-fat milk : 1, low-fat milk: 1.02, whey: 1.66, lactose :2.22 and wheyprotein :6.66.

100 90

oËzo 860 3uo È40 õ30o É, 20 10 0 Milk (high fat) Milk (low fat) Whey Lactose Whey protein Treatment

¡| Equal concentration E Concentration adjusted

6.3.4 Experiment ll2Û02 The analysis of disease scores for leaves prior to the initial treatment was not significant

were considered to be due to effects of the treatments. The mean severity of powdery mildew on leaves of untreated control vines, at the completion of the experiment, was

1.6, representing32Yo of the leaf surface affected by (J. necator. Alltreatments in experiment 212002 had significantly less powdery mildew on treated leaves than the water control (Table 6.4). Vines treated with Amistar@ and wheyÆ.{u-fikn 17@ had the lowest disease score at the completion of the experiment but these were not significantly different from those treated with whey alone, the Vitec Combo@ or Nu- film 17@ only. r25

Table 6.4: Mean severity of powdery mildew on treated grapevine leaves (cv. Viognier) after three fortnightly applications of test materials for greenhouse experiment ll200l. LSD: (5%) when data for untreated vines included : 0.385, when data for untreated vines excluded : 0.4206. Score for visual assessment 0 - 5 based on theareaaffected;0to 1; l-20%of leaf area,lto2;21 -40o/o,2to3;41 -60yo,3to4; 6l - 80yo,4 to 5; 8l - 100%. Test materials ranked according to mean disease score. Treatments with the same letter are not significantly different (P<0.05).

Treatment Mean disease Excluding data score for Untreated Vines Amistar@ 0.1 a a

Whey/Ì.{u-film 17@ 0.1 a a

Whey 0.3 ab ab

Vitec Combo@ 0.4 abc abc

Nu-film 17@ 0.4 abc abc

Vitec Kelp@ 0.6 bcd bcd

Coaton ILP@ 0.6 bcd bcd

Amino flower@ 0.8 cd cd

Amino finish@ r.0 d d

Control 1.6 e N/A

6.4 I)iscussion

6.4.1 Discussion 2001 The disease on leaves treated with milk and whey was consistently significantly less severe than that for untreated and water treatments. The 1:10 dilution of milk was the lowest rate used by et al. (1999) in greenhouse experiments to investigate the potential of milk as a means of control of powdery mildew on zucchini squash. Bettiol

(1999) found that dilutions of milk between 1:5 and l:10 provided control of powdery t26

mildew similar to that provided by the fungicides, fenarimol and benomyl. The

reduction in powdery mildew severity on vines assessed afr.er 24 h in experiment

312001suggests that milk and whey acted within24h of application.

All the fractions of milk evaluated in the greenhouse reduced the severity of

powdery mildew when compared to untreated or water-treated control vines. While

there is some research into uses of whey or whey fractions for the treatment of human

fungal pathogens (Batish et al. 1988; De Lucca et al. 1999; Kuipers et al. 1999), no

reports of the use of whey for the control of pathogens in agricultural systems could be

found.

Experiment 312001was designed to provide information on which components

nf rnill¿ mqr¡ hqr¡o ltoo- ooo^^iaforl rrrifh a -o'1,,^+ì^- i- +L- .^',^;+., *:1J^,,, suuvvrervu vr rL¡l s ¡vvuvurv¡r rrr L¡19 ÐwYwrILJ \rr^f PUwLrur ^^,,,J^*., j ttut\!vw

on grape leaves. This was intended to assist with determination of the mode of action of

milk and whey. Some of the differences in powdery mildew severity for the different

comoonents of milk were not signifrcant, however, a,s selectcql components of núlk were removed, the level of disease control decreased if the disease score was adjusted to represent the reiative proportion of that component rn whole milk (Figure 6.1). Milk was applied at the equivalent of a l:10 dilution, whey at approximately 1:6, lactose

l:4.5, and whey protein at 1:1.5 (Billington, S., Warrnambool Cheese and Butter

Factory Co Ltd, pers. com. , 2001). The 46%o reduction in severity of powdery mildew by whey was similar to the reduction observed following the application of its two components, lactose and whey protein (34.14 Yo and 14.28% of milk), combined. This suggests that each of the major groups of compounds in milk, namely fafs, proteins and lactose, contribute to the control of powdery mildew. Similarly, a possible link between milk and whey concentration and reduction in the severity of powdery mildew was observed in the results for greenhouse experiment212000 (see section 3.3.2). r27

The severity of powdery mildew on vines treated with the oil products,

Biotrol@ and Synertrol Horti-Oil@, was significantly reduced compared to that on untreated vines and vines sprayed with water alone. The control of powdery mildew by oils, both vegetable and mineral, first reported by Barker and Lees in 1914 has since been reported by others (Rovesti et al. 1992; Ziv et al. 1993; Wicks et al. 1998). While reports on the level of control have varied widely, particularly in held experiments (see section 1.5), results achieved in these experiments and in previous research indicate that the inclusion of oils in field experiments is warranted. The phytotoxic effects of oil reported by Calpouzos (1996) were not evident in the greenhouse experiments in either

2000 or 2001. The rate at which Synertrol Horti-Oil@ and Biotrol@ was applied to vines in these experiments was 0.2yo, or ll5 the rate used by Calpouzos (1996) where phytotoxicity was recorded.

The MR formulation used in the first and second experiments reduced the severity of powdery mildew on treated leaves and, when mixed with Biotrol@, the mean disease severity was reduced significantly compared to MR formulation alone.

The efficacy of MR formulation in the greenhouse experiments, combined with the results of the experiments conducted by Tzeng and DeVay in 1989 (see section 1.4.5.7) was considered sufficient to warrant inclusion of this material in field experiments.

The addition of the canola oil-based Biotrol@ to whey powder or the surfactant

Penatra@ also resulted in improved control compared to that achieved with either of the components used alone, although the reduction was not statistically significant in the

greenhouse experiments. The consistent pattern suggested that the use of mixtures and

surfactants might be more effective in reducing disease severity than of the materials

alone. This is supported by previous research that has shown that mixing oils with t28

surfactants (Schneider et al. 1990) or bicarbonates (Ziv et al. 1993) significantly

reduced the severity of powdery mildew compared with the materials alone.

The improvement in the control of powdery mildew resulting from addition of

Biotrol@ to whey is most likely the result of improved coverage of the leaves by whey

although a synergistic association between the two materials is possible. There were no

obvious phytotoxic effects caused by mixing Biotrol@ with either whey or the MR

formulation.

The assessment cf lea'¿es during Lhc experimental period was based on area of

the leaf with sporulating colonies of (J. necator.The reduction in disease severity for

lactose-treated vines was the only case where an obvious discrepancy between visual

anci microscopic evaiuation was apparent. Two factors may have contributed to the

discrepancy; first the lactose residue may have reduced the accuracy of the visual

assessments due to deposits on the leaf lanúna, or second, the visual assessments, based

^- --1 : I I urr sPurul¿ltlrrg-,^^---1-Li,-- uururllçs, wolllu ilol rËveal powoery mlldew colonles On laotose-treateCl leaves that were not sporulating. In the first case, conidia would have been observed during the microscopic evaluation at the completion of the experiment; however, few of the powdery mildew colonies on the treated leaves produced conidia.

This lack of, coiticiial prociuction may have been due to the immaturity of the colonies and./or inhibition of conidial development. Conidia are produced on mature hyphae of U. necator (see section I.3.2) and it is possible that many of the powdery mildew colonies on the lactose-treated leaves were newly established and not

."f{ì^i^-+I., .-^+,,-^ {^ s: Tl : r r ùurrrvrçrrLry lrr4Lultr ru --^J--^^pruuuus L;ullula.^^,^: - rrtrs goulo occur rI tng lactosg treatmeni was killing the hyphae of U. necator, but not affecting the viability of existing eonidia, which could then germinate and establish new colonies between applications of lactose.

The ESR experiments showed that lactose produces oxygen radicals (see section 5.3.1), 129 which have been shown to reduce the severity of powdery mildews (Tzeng et al. 1989).

The SEM images showed that free radicals killed hyphae but did not appear to damage conidia, which were able to germinate (see section 5.3.2).

The death of older colonies and subsequent germination of conidia and development of new colonies explains the reduced conidia on leaves from vines sprayed with lactose. The microscopic examination of leaves was carried out 7 days after the final treatment. Given the environmental conditions in the greenhouse, while extensive hlphal growth had time to develop, there was insufficient time for large numbers of conidia to be produced. However, with l4-day intervals between spray applications in the greenhouse sufficient conidia would have developed to allow persistence of powdery mildew on the treated vines. .

The results of the greenhouse experiments conducted in 2001 supported those of greenhouse experiments in 2000 and the field experiments in 200012001, showing that milk, whey and the vegetable oil-based materials, Biotrol@ or Synertrol Horti-Oil@, were suitable options for controlling powdery mildew. The suppression of powdery mildew by milk and whey, which contain several different components, suggests that

the control is achieved by multiple modes of action. All the above materials reduced the

severity of powdery mildew on treated leaves over the 7-week experimental period to

below pre-treatment levels. The degree of control of powdery mildew on vines treated

with these materials warranted further testing of them in field trials.

6.4.2 Discussion 2002 While the disease severity on vines sprayed with Nu-film 17@, whey and whey

plus Nu-film 17@ treatments was not significantly different, the mixtwe appeared more

effective and provided the same amount of disease control as the fi.urgicide Amistar@.

The result indicates that adding a wetter/extender such as Nu-Film 17@ to milk and 130

whey may improve their efficacy as a control of powdery mildew under field conditions

and this was evaluated in field experiments conducted in200212003.

While reducing the severity of powdery mildew, the Amino@ products did not

provide commercially acceptable control of powdery mildew. However, their use as

foliar nutrients could help in reducing powdery mildew infection. In this experiment,

Vitec Combo@ and Vitec Kelp@ treatments reduced the level of powdery mildew to

levels not significantly different from those on vines sprayed with whey. However, this result was not confinned in the 200112002 field experirnc¡rts where powtlery mildcw

severity on vines sprayed with Vitec Combo@ was higher than on untreated vines.

The application rate for Coaton ILP@ was an estimate as the recoÍtmended rate applies to its role as an insect attractant. Higher application rates could provide of disease control, therefore, Coaton ILP@ was included in field experiments in2002/2003. 131

Chapter 7 - F'ield experiments 2001/2002

7.1 Introduction The second series of field experiments was conducted at three of the sites used 'Wines in 200012001, namely Temple Bruer Wines vineyard, Glenara vineyard and

Warriparinga. These experiments were designed to evaluate materials such as milk and whey in a second season, Synerhol Horti-Oil@ in combination with Ecocarb@, and

Hayrite@, a coÍtmercial preparation of Bacillus spp. (Bio-care Technology Pty Ltd,

Australia). Additionally, some test materials that showed promise in experiments conducted in the greenhouse after the commencement of the 200012001 field experiments were subjected to evaluation in the field. The experiments at Temple Bruer

Wines vineyard were conducted on l2-month-old vines of cv. Shiraz that were sufficiently isolated from the rest of the vineyard to allow inclusion of the DMI fungicide Topas@ for comparison without compromising the organic status of the vineyard.

At Warriparinga, the layout of the field experiments was adapted to allow

University of Adelaide Honows student Robert Marlow to assess the impact of the test materials on arthropods within the vine canopy. His arthropod survey, while on a limited scale, provided information that contributed to the evaluation of test materials for suitability for use in organic agriculture. Test materials with a negative impact on the ecosystem within the vine canopy would be incompatible with the aims of the project.

Mixed programs of whey or milk altemating with Synertrol Horti-Oil@ plus

Ecocarb@ were not used in the 200112002 experiments, so that the efficacy of the test materials and mixtures as controls of powdery mildew could be evaluated on a second 132

cultivar, Shiraz, in the Temple Bruer Wines vineyard. IJsing individual test rnaterials or

mixtures rather than mixed programs allowed evaluation as controls of powdery mildew

of the materials in a second season with a different degree of disease pressure due to

variations in weather patterns.

7.2 Múerials and methods Materials included in the field experiments conducted in200112002 were milk,

whey, canola oil-based sprays, MR formulation. B. subtilis, Ecocarb@ an<1 Hayrite@

(Bacillus spp.), two foliar nutrients, SM6ae, a concentrated seaweed product, and Vitec

Combo@, and some mixtures of the oils and either Ecocarb@ or whey protein. Hayrite@,

a blend of two Bacillus spp. marketed to reduce fungal spoilage of hay, was included to

assess whether this prociuct was also an effective control agent of grapevine powdery

mildew. As Hayrite@ has been developed for agricultural use under Australian

conditions it rnay perform better under field conditions than the strain of B. subtil¿s used

in the 200012001 field experiments (see sections 4.2.1 and 4.2.3). The two foliar

nutrients were included to assess the impact of these types of materials on the severitv

of powdery mildew in the vineyard.

MR formulation was included for lwo reasons, to assess its efhcacy as a novel nnrrfrnl *:11^.-. ^.^l +^ ^--:-L ---:Lt- r r l r vvrrLrvr vr^f yvvvuurJ ^^".1^*.' r[lr\rçw allu LU asslst wltft eslaDllsnment Of tne mo(le OI aotlon Ot other test materials. Ecocarb@ was applied as a mixture witli Syrer1rri Horii-Oil@ in these experiments, as recoÍtmended by the manufacturer.

The use of sulphur and Topas@ as standard treatments in two of the 2001/2002

^--- ^--,-^ ^,-t- -ii expeÍli-nenis aiioweci a comparison of ihe relative efÏcacy of the test materials with tr¡¡o chemicals widely used in Australian viticulture for control of powdery mildew. 133

7.2.1 Temple Bruer Wines The 2001/2002 experiments at Temple Bruer Wines were conducted on Z vinifera cv. Shiraz. Three blocks of seven plots with eight vines in each plot were established, v/'ith plots separated by four untreated vines to minimise cross- contamination. Test materials were applied on seven occasions (Table 7.1), at 10 - 14 day intervals, depending on weather conditions and time constraints. Disease severity was evaluated by assessing 10 fully expanded leaves selected randomly from each of the six vines in the centre of each plot, using the 1-10 scale given in section 2.2.3.

Table 7.1: Dates and rates at which test materials were applied to V. vinifera cv. Shiraz at the Temple Bruer Wines vineyard ín 200t12002. All treatments applied using a hydraulic spray cart. Application rate is the water equivalent rate. Dil'n: dilution in RO water, App 2 refers to Appendix 2. Milk was pasteurized (fat content 3.25yo), sulphw was applied as a wettable powder (Garden King 800 dkg), whey as a spray-dried powder (Dairy Gold, Australia).

Date 3n0l t8lt0l 30ltDl 7nt/ 24ltU 6/t2l 29/12/ 2001 2001 2001 2001 2001 2001 Treatment 2001 Untreated

Sulphur (g/L) J J J J J J

Milk (Dil'n) 1:10 l:10 1:10 l:10 1:10 1:10 1:10

whey (g/L) 15 l5 l5 l5 l5 l5 l5

Hayrite@ (g/L) 1 I I I 1 I 1

MR App2 App2 App 2 App 2 App2 App2 App 2 formulation Topas@ (mVL) 0.2 0.2 0.2 0.2 0.2 0.2 0.2 EC IOO Application 300 300 400 400 600 600 600 rate (L/tra)

T.2.2Warriparinga

7.2.2.1 Warriparinga experiment I The first of two experiments at Warriparinga 1n 200112002 was conducted in

association with the evaluation of the impact of test materials on arthropod populations 734

within the vine canopy and included; milk, B. suhtilis, sulphur, Topas@ and tluee

combinations of Synefrol Horti-Oil@ plus Ecocarb@. The latter were used to ascertain

the impact of increasing the rate of Ecocarb@ on the severity of powdery mildew on

treated vines and to assess the risk of phytotoxicity of higher rates. The test materials

were appliedto V. vinifera cv Rondella using a Solo 475@ backpack sprayer with a hand

held wand' The experimental design comprised four blocks of eight rectangular plots

each containing 15 vines. Test materials were appliecl on seven occasions (Table7.2),

intencied to be at lO - 14 day intervals, however, this varied as before. Disease severity

was evaluated by assessing 20 leaves and bunches from each of the three centre vines in

each plot using the 1-10 scale given in section 2.2.3.

,l. 'f-oÌ.!^ 7 f\^+^^ ^- J ^L ---1- | 1 , i.¡uiu ¡.4, i¿;cLçs arrû -^¿^-i-ales aï w-iilcn i.esi matenais appiteci to /. viniJera cv. Rondeila at the Warriparinga vineyard in 200112002. All treatments applied using a Solo 475@ pressurized spray-pack and hand : wand. Application rate is-the water equivalent rate. Dil'n dilution in RO water. Milk was pasteurized (fat eontent 3.25%). Sulphur was applied as a wettable powder (Garden King 800 CkS). B. subtilis was cultured as described in appendix 2.

Date 28/9/ t2l10t 25/10/ 8/tU 22/tt/ 7lt2l t9/12/ Treatment 2001 2001 2001 2001 2001 2001 2001 iintreateci

Sulphur (g/L) -3 3 3 3 3 3 J

Topas EC I (mYL) 0.2 0.2 0.2 0.2 0.2 0.2 0.2

Swerfrnl ¡/ñ'l /I \ ) t I ô L ¿ ¿ 2 ¿ Ecocarb@ 2 2 2 2 2 2 2 Synerlrol (mYL) 2 2 2 2 2 2 2 l,cocarb" 3 3 3 3 J J J @ Synertrol Horti-Oi I (mlil-) 2 2 2 2 2 2 2 plus Ecocarbt (øf) 4 4 4 4 4 4 4 Milk(Dil'n) 1: l0 1:10 1:10 l: l0 1: l0 l: l0 1: l0

R ttth¡ili" ¡/l-ìil'n\ 1,4 1 .a t.L t.L L:¿ I:¿ li¿ l:¿ Application rcfe (Llha) 300 400 500 500 600 800 900 135

7.2.2.2 Warriparinga experiment 2 The second experiment at Warriparinga was aimed at evaluating a range of test materials, including Biotrol@ and Synertrol Horti-Oil@, whey, whey protein, alone and mixed with Biotrol@, Azamax@ and SM6@ and Vitec Combo@. The treatments were applied to vines divided into four blocks of ten plots with eight vines per plot. Test materials were applied on seven occasions (Table 7.3) intended to be at l0 - 14 day intervals, however, this varied as before. Disease severity was evaluated by assessing

10 leaves from each of the six vines at the centre of each plot, using the 1-10 scale given in section 2.2.3.

Table 7.3: Dates and rates at which test materials applied to V. vinifera cv. Rondella at the Warriparinga field experiment site in 200112002. All treatments applied using a Solo 475@ pressurized spray pack and hand held wand. Application rate is the water equivalent rate. 'Whey was spray-dried powder (Dairy Gold, Australia).

Date 28/91 t2/l0l 2sltÙl Sltt/ 22/tt/ 7/12/ 19lt2l 2001 2001 2001 2001 2001 2001 Treatment 2001 Untreated

Whey (g/L) l5 15 15 l5 15 15 l5

Biotrol@ (mVL) 4 4 4 4 4 4 4

Biotrol@ (mVL) 2 2 2 2 2 2 2

Biotrol@ (mVL) plus 2 2 2 2 2 2 2 whey protein (e/L) l5 15 15 l5 l5 t5 15 Whey protein l5 l5 15 15 l5 15 l5

Synerûol Horti-Oil@ 2 2 2 2 2 2 2 (mVL) SM6 (mVL) l5 l5 l5 l5 l5 15 l5

Azamax'(mVL) 2 2 2 2 2 2 2

Vltec Uombo"^.tR) l:2 7:2 l:2 l:2 l:2 1:2 l:2

Application rate 300 400 500 500 600 800 900 lLlha) 136

7.2.3 Glenara Wines The experiment at Glenara in200I12002 aimed to evaluate the mixture of whey

plus Synerhol Horti-Oil@ to control powdery mildew and to compare the control with

the vineyard owner's program, which comprised mixtures of Synertrol Horti-Oil@ and

sulphur. A Hardy Maxi@ tractor-drawn air-blast sprayer was used to apply materials to

the vines. Test materials were to be applied on seven or eight occasions, intended to be

at 10 - 14 day intervals. However, after the first four applications, powdery mildew was

observed in the central areas of the canopy in vines treated with whey plus S¡mertrol

Horti-Oil@. As a result the experiment was abandoned and all vines were treated with

wettable sulphur at 4 g/L.

7.3 Results

7.3.1 Temple Bruer Wines

The mean severity of powdery mildew on the leaves of untreated vines at

'T.^*-1^i emptc õruer D-,^- \rr.i-^-iv ines. -r rr, r ,. at ilie compieiion o f ihe experiment , was 0.'17 , representin g 7 .7%

of the leaf surface affected by U.necator. All test materials applied to the vines in this

experiment significantly reduced the severity of powdery mildew on leaves when

compared to the untreated controls þ <0.001). There was no significant difference in the severity of powdery mildew on vines treated with Topast, milk or whey (Table 7.4).

There was also no significant difference in powdery mildew on the vines that were treated with milk, whey or sulphur, but there was significantly less disease on the vines treated with Topas@ than on those treated with sulphur (Table 7.4). Re-analysis of the data excluding results from the untreated control vines did not change the outcome. The vines used in this experiment were only 12 months old at the commencement of the experiment and any bunches that developed were removed during the season to encourage vegetative growth. As a result no data were available for the fruit. 137

Table 7.4: Mean disease severity on leaves on treated grapevines (30 January 2002), cv. Shiraz for the Temple Bruer Wines field trial site 2001/2002, after seven applications of the test materials. LSD (5%); when data for untreated vines included: 0.0982; when data for untreated vines excluded : 0.0949. Score for visual assessment of leaf area affected with sporulating colonies of powdery mildew; 0 to l; I - 10% I to 2; Il - 20yo,2 to 3;2I - 30yo,3 to 4;31 - 40yo, etc. N/A : not applicable. Treatments with the same letter are not significantly different (P<0.05).

Treatment Mean disease Statistical severity on leaves significance when data for untreated vines excluded Topas@ 0.2 a a

Mitk 0.3 ab ab

\ilhey 0.3 ab ab

Sulphur 0.4 bc bc

MR formulation 0.4 cd cd

Hayrite@ 0.5 d d

Untreated 0.8 e N/A

7 .3.2.1 Warriparinga experiment I The mean severity of powdery mildew on the leaves of untreated vines at

'Warriparinga was 1.8 and 1.3 for bunches at the completion of experiment l, representin g 18% of the leaf and I 3 % of bunch surfaces affected by U. necator. All test materials significantly reduced the severity of powdery mildew on leaves bunches when compared to the untreated controls (p <0.001).

On leaves, the disease severity was signihcantly less on vines treated with

Topas@ than for any other treatment other than sulphur (Table 7.5). Powdery mildew on vines treated with Synertrol Horti-Oil@ plus Ecocarb@ at 4 glL was significantly less than on vines receiving the mixture with the two lower rates of Ecocarb@. In this 138

experiment, the leaves of vines sprayed with milk had significantly more disease than

vines treated with sulphur or Topas@. The re-analysis of leaf data with the results for

untreated control vines excluded did not change the outcome.

However, there was no significant difference in the severity of powdery mildew

on bunches sprayed with Topas@, sulphur, milk or Synerhol Horti-Oil@ plus Ecocarb@ 4

g/L (Table 7.5)' As was the case for the severity of disease on leaves there was no

significant difference between the Synerhol Horti-Oil@ plus Ecocarb@ mixtures

containing Ecocarb@ at 2 and,3 g/L. As with leaves there was a significant difference in

disease severity on vines treated with the 4 dL and 3 g/L Ecocarb@ plus Synertrol

Horti-Oil@ mixtures. The re-analysis of bunch data with the results for untreated control

vines exciudeci did not change the outcome.

The severity of powdery mildew on leaves and bunches on vines treated with B.

subtilis was significantly greater than on vines sprayed with the other test materials

used in this experiment.

7.3.2.2'Warriparinga experimenr 2 The mean severity of powdery mildew on the leaves of untreated vines at

Warriparinga at the completion of experiment 2 was 2.3, representing 23%o of the leaf

qrrrfnnc qffpoÍcrl lr.tt f I øô^nr^v /'T^1^1^'7 AY¡L^ :1 1 l uJ v' ,.çwutvt \raurv t'u), Lilç ùEvçrrry^^,.^..:*-. ur^r--^---r-,---,- Puwugry llillucw on leaves of vines sprayed with whey, Biotrol@ 4 mllL and the whey protein plus Biotrol@ 2 mllL in experiment 2 at V/arriparinga in 200I/2002 was significantly less than on untreated controls (p <0.001). Severity on vines treated with Vitec Combo@ was significantly greater than on unireateci vines. The Biotrol@ iZ m7f) plus whey protein mixture significantly reduced powdery mildew on treated vines compared to vines treated with

Biotrol@ (2 mUL) alone. The re-analysis of leaf data with the results for untreated control vines excluded did not change the outcomes. t39

Table 7.5: Mean disease severity on leaves and bunches on treated grapevines, cv. Rondella, for experiment 1 at the Warriparinga field site 2001/2002, after seven applications of the test materials (20 January 2002). LSD (5%); when data for ru"rtreated vine leaves included :0.2716; when data for untreated vine leaves excluded :0.2473; where data for untreated grape bunches included : 0.2578; where data for untreated gape bunches excluded :0.2484. S + E is Synertrol Horti-Oil@ 1Z mVI-¡ plus Ecocarb@ at rate in brackets. Score for visual assessment of bunch area affected with sporulating colonies of powdery mildew; 0 to I ; I - l0% I to 2; lI - 20yo, 2 to 3; 2I - 30yo, 3 to 4; 3l - 40yq4 to 5; 4l - 50% etc. N/A: not applicable. Treatments with the same letter are not significantly different (P<0.05).

Treatment Mean Statistical Mean Statistical disease significance disease significance severity on when data for severity on when data for leaves untreated bunches untreated vines excluded vines excluded Topas @ 0.2 a a 0.4 a a

Sulphur 0.3 ab ab 0.5 a a

S+E(gtL) 0.5 bc bc 0.6 ab ab

Mitk 0.7 cd cd 0.5 a a

S+E(3gll,) 0.9 de de 0.9 cd cd

s+E (2etL) 0.9 de de 0.8 bc bc

B. subtílís 1.1 e e 1.1 d d

Untreated 1.8 f NiA 1.3 e N/A

7.4 Discussion The most effective test materials at the Temple Bruer Wines vineyard were milk

and whey. Milk also proved effective at reducing the severity of powdery mildew on

vines, particularly on bturches, in experiment 1 at V/arriparinga. The Synertrol Horti-

Oil@ plus Ecocarb@ mixtures signifìcantly reduced the severity of powdery mildew, and

disease levels following application of the mixture with the highest concentration of

Ecocarb@ were not signifrcantly different from those after sulphur. 140

Table 7.6: Mean clisease sev evlnes' cv. Warriparinga field site Rondclla, for the 2û0i s of the (5%); wheñ ¿utu fo. unrreate test materials. LSD en data for excluded : 0.3506. Score fo untreated vines coronies orpowdeiv 0 ro r; t - t0% t to 3,1 - 40yo,4 _rr9.*; 2; tt z',iit"iï"åïl;t:iår";,Tîliî to S; 4l 50%eíc. N/AInot applicable. Treatments with the same letter ur" not.igoih.*tifaifrerent (p<0.05).

Statistical significance Treatment Mean dÍsease when data for severity untreated vines Whey excluded 1.7 a a Biotrol @ mVL) 1.7 a a

Q ml/L) plus 1.8 ab whey protein ab SM 2.0 abc abc Aza 2.0 abc abc wh ey protein 2.1bc bc Synertrol 2.1bc bc BÍotrol (2 mUL) 2.2 c c Untreated 2.3 .NT/ c at/^ A VÍtec Combo 2.s d d

'l'L^rne ^--.1: - apphcaiion of Fiayrite@ and B. subtilis, while reducing the severity of powdery mildew when compared to untreated vines, did not control the powdery mildew infection to levels considered acceptable for harvest. The failure to control powdery mildew sufficiently in these experiments and those conducted in 2000120a1 (see sections 4'3'1 and 4'3'3) meant that Bacillusproducts were not included in further field experiments conducted in this project. There was a reduction in the efficacy of ^8. subtilis in the control of powdery mildew in field experiments compared to greenhouse expenments. t4l

The MR formulation reduced the severity of powdery mildew in field

experiments conducted at the Temple Bruer Wines vineyard when compared to untreated controls. The reduction in severity of powdery mildew on vines sprayed with

MR formulation suggested that free radicals and active oxygen species can reduce the

severity of powdery mildew in the field.

The results for experiment 1 at Warriparinga suggest that milk and the Ecocarb@

4 glL plus Synertrol Horti-Oil@ mixture controlled powdery mildew as effectively as

Topas@ and sulphur on bunches but they were less successful at reducing the level of

powdery mildew on leaves. Powdery mildew was less severe on bunches than on leaves

on milk-treated vines and more severe on bunches than leaves on Topas@- and sulphur-

treated vines. These differences may be related to coverage, shading or interaction of

the materials with the swface of berries. The smooth waxy surface of the berries may

lead to better coverage of berries than of leaves where hairs may reduce coverage. Also

the bunches at both sites were relatively well exposed compared to leaves near the

centre of the canopy and, as leaf severity is scored using leaves through the entire

canopy greater disease severity on inner leaves resulted in higher average disease scores

for leaves treated with milk. As spray coverage is less critical for Topas@, due to

translaminar movement the disease severity on leaves within the canopy may be lower

than on leaves in the centre of the canopy of vines sprayed with milk. The additional

shading on leaves near the centre of the canopy may also lead to reduced efficacy of

milk on those leaves and an increased average disease severity for leaves treated with

milk.

The severity of powdery mildew at the completion of experiment 2 at

Warriparinga was greater than in experiment 1, however, there were still significant

differences between some of the treatments. The vines treated with the whey protein t42

plus Biotrot@ mvt¡ 1z mixture had significantly less powdery milde,w than vi'es treateri with Biotrol@ 2 muL alone' There was no significant difference in severity of powdery mildew on vines treated with either Biotrol@ or synertrol Horti-oil@ when both applied at 2 mI/L, confirming the results achieved in greenhouse experim ent r/200r. The efficacy of the test materials varied fiom matching the efficacy of sulphur in controlling powdery mildew at the Temple Bruer wines vineyard and warriparinga to lack of control at Glenara wines vineyard. while a number of factors may rrave contributcd to ihe differences in the level of control of powdery mildew. such as microclimate' shading, cultivar and coverage, the most critical appeared to be coverage. This was most obvious at warriparinga where the two experiments were divided by a Íbnse: on one sicie' (experiment 1,), the canopi/',¡v'as opcn and sparse while on the other (experimenl' 2)' the canopy was much denser and attaining the same leaf and bunch eoverage was not possible with the available equipment. 'I-L^r'u u'relcrlcoli¡¡ ln canopy structure and density at warriparinga appeared to be related to the different weed management strategies. weeds around the vines in experiment were l ailowed to grow armost unchecked and were only mown oncc through the experimental periocl. The resulting competition for water and nuhients appeared to reciuce ihe vigour of the vines. In comparison, the vines in expenmeniai area 2' where the weeds were sprayed at the beginning of the season and a 1_5 cm layer of tree mulch applied to the area produced dense canopies and vigorous growth. The increased density of the canopy not only provides a more favourable envirn--oh+v¡"¡lv¡u¡¡wrrL i('r f^- ursease ri- ceveropment but also reduces the le.,,er of, spra), co\,,crage achieved' Light intensity also influences the efficacy of milk (see sectio n 5.3.2) and, whey and the increased shading in the centre the of canopy may have reduced the control of powdery mildew. canopy design, density and alignment and the eapacity of 143 spray equipment available need to be considered when selecting spray volumes. At

Glenara Wines vineyard, to improve the efficacy of this spray program and other programs using contact fungicides, action needs to be taken to reduce the density of the canopy and reduce vine vigour.

The canopy of the l2-month-old Shiraz used in experiments in the Temple

Bruer Wines vineyards was small and open, exposing all leaves to light and improving spray coverage. The combination of less favourable conditions for powdery mildew, improved spray coverage and the increased resistance to powdery mildew compared to the Chardonnay vines used in experiments at Glenara Wines vineyards, would have all contributed to the difference in the control of powdery mildew seen between the two sites. These differences highlight the need for a fully integrated approach to powdery mildew in vineyards that includes canopy management, spray application and vigour optimisation. Further commercial scale testing is required on vines with large dense canopies to assess the suitability of these materials for large-scale use under conditions that may not favour their application.

The results of the field experiments conducted, so far, suggested that a program consisting of applications of Synerhol Horti-Oil@ plus Ecocarb@ in rotation with milk or whey could provide control of powdery mildew in commercial vineyards provided a coverage rate of over 90o/o can be achieved. There is reluctance among many growers to use an oil plus bicarbonate mix all season, due to reports of phytotoxicity, reduced yield and reduced fruit quality (see section 1.5), and whey or milk provide effective altemative treatment. 144

Chapter 8 - Field experiments 20022003

8.1 Introduction The field experiments in 200212003 were restricted to two sites, Temple Bruer

V/ines vineyard and Glenara Wines vineyard, after the vines at V/arriparinga were

removed. The experiments were established to evaluate the most successful individual

treatments and the potential of programs developed over the past two seasons for the

control of powdery mildew. The programs were desig¡red for use in organic or

chemically assisted vineyards.

In addition, the effects of the materials on yield and juice quality were

investigated. Variations in the acidity ofjuice and wine, measured as pH and titratable

acidity (TA), affect the fermentation process and can affect the colour, flavour and oBrix, stability of the wine (Iland 1993; Iland et al. 2000). or Baumé, a measure of the

total soluble solids, is a common guide to ripeness of the grapes and an indication of the

final alcohol content of the wine (Ilancl et al. 2000). There is some evidence that oBrix application of oil to vines can delay ripening and affect the of juice (Finger et al.

2002) (see section 1.5.1).

8.2 Methods and materials

8.2.1 Temple Bruer'Wines vineyard Two experiments \ryere conducted at Temple Bruer Wines vineyard in

200212003 on V. vinifera. cv. Verdelho vines. Making wine from the grapes harvested from vines sprayed with the selected test materials, while the best means for quality assessment, was not practical due to time and budget constraints. Therefore, pH, TA oBrix and of the juices were assessed as a guide to the impact of the test materials and programs on fruit quality. t4s

8.2.1.1 Experiment I

The first and largest experiment was organized into seven blocks of nine plots, each consisting of two panels (a panel is the space between two trellis support posts).

Most plots consisted of eight vines, four per panel; however, some plots contained only seven vines. In these cases, a neighbouring vine had been trained across the gap to minimise the impact of the missing vine on canopy size and yield. The disease severity on ten randomly selected fully expanded leaves and bunches on the centre six vines in each plot, five from each side (east and west), was assessed using the 1-10 scale given in section 2.2.3. The final assessment of leaves was on 8 January 2003, after which time loss of the most severely affected leaves and the development of age-related resistance to infection would have confounded of the results.

The test materials applied in this experiment were: mixtures of Synertrol Horti-

Oil@ plus Ecocarb@ in rotation with whey; or sulphur; milk; whey; Ecocarb@; and

Synertrol Horti-Oil@ (Table S.1). A mixture of whey plus Synertrol Horti-Oil@ was also evaluated to assess the effrcacy of mixtures compared to oil alone (see section 6.3.2).

Whey alone was applied af 45 lL rather than 15 glL to examine the potential for phytotoxicity, residue or secondary microbial infection that may result from the higher application rate. The final whey-only spray was applied at 30 glL as it was considered that disease pressure was low and a lower rate could be used atthat time of year without compromising control.

Test materials and standard treatments were applied using a hydraulic spray cart for the first two sprays, but coverage was no better than 50o/o of the leaf surface (see section 2.2.3), and subsequent applications were made with a Solo 475@ backpack spray. An assessment of the coverage achieved with the Solo 475@ backpack spray was conducted using water sensitive papers using the same methods as for the spray cart t46

(see section 2-2.3). Coverage varied from flrll coverage of at least one surface and >

50%o of the >80% other surface of of leaves to approxim ately 50%io of both surfaces;

there were no surfaces on the assessed leaves that did not receive at least 30%o coverage. Approximately 20%o of leaves, usually on the outer margins on the canopy, received

spray levels that would be considered excessive. Spray volumes varied from 300 Llha at the beginning of the season to 900 L/hafor the final five applications. While use of the

Solo 475@ improved coverage on exposed surfaces, coverage in protected areas was still

less '*vith than l00yu, soure leaves and bunches receivirrg more spray than others. Yield data were obtained for bunches harvested from all vines in each plot. All

bunches were removed from the vines and sorted into "acceptable,,, i.e. < 5% of the bunch surfàce affected by oowdery nuldew, and unacceptable i.e. > 5% powderir

mildew based on the assessment key developed by R. Emmett and T. V/icks (pers. eom. 2000). The total weight of acceptable and unacceptable bunches for each plot was

rrtcasureri using fieici scaies. Two bunches, approximat ely 250 g in total, wcre selected at random from the acceptable fmit harvested from each plot for assessment of quality parameters. In the ease of one untreateci control plot where the total weight of acceptable bunches was only 60 g, benies affected by powdery mildew were excised frorrr unacceptable bunches to bring the weight of acceptable berries to approximately

250 g.

Flagshoots are a source of primary inoculum of LJ. necator at the beginning of each growing season (see section I.3.2). To assess the impact of the test materials apptied^,-,-1: - -1 ) to vines in the 2002,2003 season on the density of flagshoots in the following season' the vines in the experimental area \¡/ere examined for flagshoots in spring 2003

(October 1, 8 and 13). The numbers of flagshoots were recorded for each treatment as applied in the 200212003 experiment. t47

Table 8.1: Dates and rates at which test materials were applied to grapevines, cv. Verdelho, at the Temple Bruer Wines field trial site in 200212003. SH - Synertrol Horti- Oil@ appliedat2nll.lL, E - Ecocarb@ applied at3 mlll-, Su - sulphur, W - whey at 15 glL. Tlne milk used was pasteurized (îat content of 3.25%). The whey was spray-dried powder and the sulphur rvas a wettable powder (Garden King 800 dtg). First two applications \ilere applied using a hydraulic spray cart, subsequent applications were with a Solo 475@ backpack.

Date 3019t t0/tol 24lr0l 6ltll 20/tU 5/12 t7lt2l 3t/tzl Treaünent 2002 2002 2002 2002 2002 2002 2002 2002 Program I SIVE SHÆ w SHÆ w SIVE w w

Program 2 STI/E SHÆ SIVE w SH/E Su Su V/

Milk l: l0 l:10 l:10 l: l0 l:5 l:10 l:10 l:10 (Dil'n)

Whey 45 45 45 45 45 45 45 30 GIL) Whey + SH t5l2 r5/2 1sl2 t5/2 t5l2 r512 ts12 t5l2 (slL / nntL)

Su (g/L) J 3 .J J 4 3 J 3

Unheated

8.2.1.2 Yield and quality assessment oBrix Bunches collected for assessment of pH, TA and were placed in paper bags in cooled foam boxes for transport and stored at approximately 4o C for up to 24h prior to assessment. The bunches from each plot were placed into separate plastic bags and squeezed by hand. The resulting juice was then poured into 50 ml plastic centrifuge oBrix tubes and centrifuged at 3500 rpm at 20o C for 5 min. TA, pH and of the supernatant were measured as described by Iland et al. (2000). Brix ,was measured using a Atago hand-held refractometer, with readings adjusted for temperature. pH was determined using a Metrohm 691 pH meter, calibrated at pH 4 and 7 pnor to use.

Assessments were made on the supernatant for each individual plot, and data were then subject to ANOVA using Genstat 5. 148

8.2.1.3 Experiment 2 The second experiment at the Temple Bruer Wines vineyard was also conducted

on cv' Verdelho vines, in this case ¿uïanged in three blocks of six single-panel plots,

and all treatments were applied using the Solo 475@ backpack. Spray volumes varied

from 300 Llhta at the beginning of the season to 900 Lftta for the final five applications.

In experiment, this the disease severity on 10 leaves and bunches on three vines in each plot was assessed using the 1-10 scale given in section 2.2.3. Yield and quality were not

assessed.

This experiment was established to evaluate four nrixtures, including whey or

whey protein, and a commercial whey product, Coaton ILP@ (Tabl e B.Z), for the control

of powdery mildew on grapevines. The mixtures were applied to allow comparison of

Nu-film 17@ with Synertrol Horti-Oil@ as adjuvants to improve the efficacy of whey.

The efficacy of whey and whey plus Synertrol Horti-Oil@ was compared in the larger

experiment (see section 8.2.1.1). Whey. at 15 glL and,30 glL,was mired with Synenrol

Horti-Oil@ to evaluate the effect of concentration on the severity of powdery mildew in the fieid anci aiso to evaiuate phytotoxicity ot'the mixtures at the higher concentration of whey. The fourth mixture included in the experiment was whey protein plus

Slmertrol Horti-Oil@.

8.2.2 Glenara Wines vineyard The experiment at the Glenara Wines vineyard tn 200212003 was conducted on

V. vinifera cv. Chardonnay vines, and \Mas designed to evaluate the program 1 used at the Temple Bruer Wines vineyard (program l, see section 5.2.I). The main purpose of the experiment was to determine if the program could overcome the difficulties in controlling powdery mildew, such as inadequate coverage and resultant poor control of powdery mildew, encountered in previous experiments at the Glenara Wines site. The r49 test materials were applied using a Hardy Maxi@ fan-assisted hydraulic spray cart. Six blocks were established, each divided into two plots of six vines. The severity of powdery mildew on 20 bunches and leaves for each of the fow centre vines in each plot was assessed using the 1-10 scale given in section 2.2.3.It was intended to apply test materials on seven or eight occasions at 10 - 14 day intervals. However, after the first three applications, powdery mildew was observed in the central areas of the canopy on vines treated with the test materials and the experiment was again abandoned. All vines were treated with sulphur at 4 glLmixed with Synertrol Horti-Oil@.

Table 8.2: Dates and rates at which test materials were applied to grapevines cv. Verdelho, at the Temple Bruer'Wines vineyard in200212003. SH - Synerlrol Horti-Oil@ applied at 2 mllL; W - whey at 15 or 30 dL. Test materials were applied using a Solo 475@ backpack. Whey was spray-dried powder (Dairy Gold, Australia); whey protein was supplied by Warrnambool Cheese and Butter Factory, Victoria.

Date 30lt2l t0ltol 24/tO/ 6/tt/ 20/tI 5/t2l l7 /t2l 3|12/ 2002 Treatrnent 2002 2002 2002 2002 2002 2002 2002 Whey (g/L) plus 15 l5 15 l5 15 l5 15 l5 Nu-film 17@ (mYL) 2 2 2 2 2 2 2 2

Coaton ILP*' (mVL) STVE SHÆ SHÆ w STVE Su Su w

whey (g/L) 30 30 30 30 30 30 30 30 plus SH (mVL) 2 2 2 2 2 2 2 2 whey (g/L) l5 l5 l5 l5 l5 15 l5 15 plus SH (mVL) 2 2 2 2 2 2 2 2

Whey protein (g/L) 15 l5 15 l5 t5 l5 15 15 plus SH (mVL) 2 2 2 2 2 2 2 2 Untreated

8.3 Results

8.3.1 Temple Bruer Wines vineyard experiment 1 The mean severity of powdery mildew on the leaves of untreated vines at the

Temple Bruer Wines vineyard on 8 January 2003 was 2.05, indicating that 20.5% of the leaf strrface was affected by U. necator (Table 8.3). All test materials applied to the vines in this experiment significantly reduced the severity of powdery mildew on leaves when compared to the untreated controls (p <0.001) (Table 8.3). There was no 150

significant difference in the severity of powdery mildew on leaves sprayed with whey

45 glL, milk, sulphur, program 1 or progran 2. Disease on vines sprayed with the

Synertrol Horti-Oil@ plus whey 15 {L mixture was significantly less than on vines

treated with Synertrol Horti-Oil@ alone. Disease severity on leaves sprayed with either

Ecocarb@ or Synertrol Horti-Oil@ alone was significantly greater than on those

receiving the mixed programs, milk, whey 45 glL or sulphur.

In October 2003 the total number of flagshoots removed from vines in all plots

sprayed with the test materials in 200312003 varied from three to seven (approximately

0'5 and 1 per block) compared to a total of 22 (three per block) for untreated vines. 'When the data for untreated vines were excluded from the analysis of results, a

significant clifference in fhe severifrr nf nn*¡dp"-r nn.,i-oo t*^^+^Ã,-,.:iL ,,,L^,, /< -il'{o.',vv vrr v ¡ltvù tr w4!vu w tltl wll9y .+J

glL and program 2became apparent, with the former resulting in less disease. Also, the

severity of powdery mildew on vines treated with whey 15 EIL plus Synertrol Horti-

Oil@ rvas significantly morc than on the vines treaterl with whey 45 glL,milk, sulphur orprogram 1.

The mean severity of powdery mildew on bunches of untreated vines at the

Tempie Bruer Wines vineyard on 19 February 2003 was 4.6, indicating that 46%o of rhe bunch area was affected by U.necator (Table 8.4 ancl Figure g.1). All test materials applied to the vines in this experiment signihcantly reduced the severity of powdery mildew on bunches when comparecl to the tmtreated controls (p <0.001) (Table 8.4).

There was no signifìcant difference in the severity of powdery mildew on bunches on

'¿ines sprayed with whey 45 glL, milk, sulphur. program 1 or program 2. The scventy of powdery mildew on bunches sprayed with Ecocarb@ or Synertrol Horti-Oil@ alone was significantly greater than on bunches sprayed with the other materials and programs. As was the case with leaves, the severity of powdery mildew on bunches 151 sprayed with whey 15 glLplus Synertrol Horti-Oil@ was less than on bunches sprayed with Synertrol Horti-Oil@ alone. Disease on berries and bunches of untreated vines and typical disease-free bunches from vines treated with milk, whey and program I are shown in Figure 8.1. Secondary damage was evident on some of the bunches on untreated vines where berries that had split as a result of powdery mildew were further damaged by ants (Figure 8.1).

Table 8.3: Mean disease severity (8 January 2003) on leaves on treated grapevines, cv. Verdelho, for the Temple Bruer Wines field trial site in 200212003, after eight applications of the test materials. LSD (5%): when data for untreated vines included : 0.18; when data for untreated vines excluded:0.11. Score for visual assessment of bunch area with sporulating powdery mildew; 0 to 1.; | - l0% I to 2; lI - 20yo,2 to 3; 2l - 30yo,3 to 4; 3l - 40yo, etc. N/A: not applicable. Flagshoots were collected on 1, 8 and 13 October 2003 and data pooled. Treatments with the same letter are not significantly different (P<0. 05 ).

Statistical Treatment Mean disease significance Total severity when data for flagshoots October on leaves untreated vines 2003 excluded Whey 0.1 a a 7

Sulphur 0.2 ab ab 5

Program 1 0.2 ab ab 7

Milk 0.2 ab ab 4

Program 2 0.3 ab bc J

Wheyplus 0.3 bc cd 5 Synertrol Horti-Oil@ a Ecocarb@ 0.5 c d J

Synertrol Horti-Oil@ 0.7 d e 5

Untreated 2.0 e N/A 22 r52

When the data for trntreated vines were excluded from the analysis, the disease

severity on bunches on vines treated with program I was significantly greater than on

vines sprayed with either milk or sulphur. Otherwise, the results were similar to those

for the complete data set.

The average severity of powdery mildew on bunches on 19 February 2003 was

slightly less when the linear model was used to analyse data than when ANOVA was

used. The relative severity of powdery mildew for the various test materials was the

same (Table 8.4) regardless of which analysis was used. Analysis of the data using the

linear model showed no plot effect and a small block effect of 0.00048 (Lim 2003).

At harvest, there were visible residues on vines that received the whey and

sulphur sprays. The residuo rvas most obvious on the vi¡res sprayed with whey aL 45 gL

(Figure 8.1), and was present on most leaves and bunches. This residue was similar in

appearance to residues on vines treated with whey or lactose in previous greenhouse

trials (see section 3 3 3) Isolated patches of the residue .ù/ere secn on leavcs spraycd

'Where with whey plus Synertrol Horti-Oil@, and programs I or 2. the residue was most pronounced, the leaves felt dry and papery, but there 'were no obvious signs of phytotoxicity. Leaves on vines sprayed with milk had no obvious residue at harvest, and the leaves felt softer than those on untreated vines or vines sprayed with other materials or programs. 153

Table 8.4: Mean disease severity on bunches (19 February 2003) on treated grapevines, cv. Verdelho, for the Temple Bruer Wines vineyard 200212003, after eight applications of the test materials. LSD (5%) for ANOVA; when data for untreated vines included : 0.2274; when data for untreated vines excluded : 0.1748. Score for visual assessment of bunch area with sporulating colonies of powdery mildew; 0 to L; I - l0%l to 2; 17 - 20yo,2 to 3;21 - 30yo,3 to 4;3I - 40o/o, 4 to 5; 4I - 50% etc. N/A : not applicable. Treatments with the same letter are not significantly different (P<0.05). Linear mixed model was fitted with SAMM (Butler et al. 2000) with S-Plus (hsightful Corp,2002) (see section 2.2.3).

Treatment Mean disease Statistical Mean disease severity significance severity on bunches when data for on bunches (ANOVA) untreated vines (Linear mixed excluded model) (ANOVA) Milk 0.3 a a 0.1

Sulphur 0.3 a a 0.2

Whey 0.3 a ab 0.2

Program2 0.4 a abc 0.3

Program 1 0.5 a c 0.3

'Wheyplus S/H 0.7 b d 0.5

. rRl -Bcocarb" 1.0 c e 0.7

S¡mertrol Horti- 1.6 d f r.2 oil@ Untreated 4.6 e N/A 4.0 t54

Figure 8.1: Representative images of bunches of cv. Verdelho grapes af the Te le Bruer Wines experiment site on 16 February 2003, after seven applications of test

Secondary damage by ants on untreated vines (E). Residue on leaves and bunches following treatment with whey 45 g/L (F). 155

8.3.2 Yield and quality assessment The average total yield from the untreated vines at the completion of this experiment at Temple Bruer Wines vineyard was 9.3 kg per plot; the average yield from all plots receiving the test materials was significantly greater than the untreated controls

O:0.015). The average yield from plots of vines sprayed with the sulphur (15.2 kg) treatment was significantly greater than for any of the test materials (Table 8.5). There was no significant difference in total average yield between the whey plus Synertrol

Horti-Oil@, whey 45 glL, Synertrol Horti-Oil@, milk or program 1. However, the average total yield harvested from vines sprayed with Ecocarb@ and program 2 was significantly less than that harvested from the vines treated with whey plus Synertrol

Horti-Oil@ or whey 45 glL.

The average acceptable yield from untreated vines at the completion of this experiment was 4.0 kg per plot, and the average acceptable yield from all plots receiving the test materials was significantly higher than the untreated controls (p

<0.001) (Table 8.5). There was no significant difference in the average acceptable yield of grapes harvested from vines sprayed with sulphur and whey 45 gL. There was also no significant difference in the acceptable yield harvested from vines treated with whey

45 glL,whey plus Synertrol Horti-Oil@, milk, proglam 1, Ecocarb@, or program 2. The average acceptable yield harvested from vines sprayed with Synertrol Horti-Oil@ alone was significantly less than from vines sprayed with sulphur, milk or whey 45 glL-

The proportion of bunches harvested from untreated vines that was considered acceptable for winemaking (< 5% powdery mildew infection) was 39.7%. The proportion of bunches harvested from all plots receiving the test materials, except

Synertrol Horti-Oil@, was significantly greater than the untreated controls þ <0.001)

(Table 8.5). There was no significant difference in the usable proportion of yield that 156

v/as considered acceptable for winemaking from vines treatecl with sulphur, whey, milk, program I orprogram2.

Table 8.5: Mean total yield, acceptable yield (< 5% powdery mildew) and ro acceptable yield for bunches (3 March 2003) on treated gràpevines, cv. Veráelho, for the Temple Bruer Wines vineyard in 2002/2003, after àight applications of the test materials. LSD (5%): yield : 1.43; acceptable yreld : i.lt y"- acceptable : 19.23. Treatments with the same letter are not significantþ different (p<0.05).

Treatmenf Total yield Acceptable 7o acceptable (ke) yÍetd (ks) Sulphur 15.2 a 1,3.2 a 86.9 a

Whey plus SÆI 13.7 b 8.9 bc 66.1 bc

Whey 13.5 b 10.3 ab 75.7 ab

Synertrol Horti-Oi 12.7 hc 6) ¡- 48.5 cd

Milk 12.5 bcd 9.8 b 78.8 ab

Program 1 l2.4bcd 9,1 bc 71.6 ab

o¡1 ..EL^ Ecocarh@ 11 6 7

Program2 tt.2 d 8.5 bc 75.5 ab

Untreated 9.3 e 4.0 d 39.7 d

There lvas no significant difference in the pH of the juices extracted from bunches harvested from any of the vines sprayed with the test materials and untreated vines (p : 0-532) (Table 8.6). Also, there was no significant difference in the TA (p : 0'3300) oBrix : or (p 0.307) of the juices extracted from grapes harvested from thc vines used in this experiment. ts7

oBrix Table 8.6: Mean pH, TA and for juice extracted from bunches on treated grapevines (3 March 2003), cv. Verdelho, for the Temple Bruer Wines field trial site 20021200} after eight applications of the test materials. All bunches had < 5%o of oBrix berries affected by powdery mildew. LSD (5%) pH : 0.0608, TA : 0.4508, : 0.7725.

oBrix Treatment pH Titratable aciditv Milk 3.03 7.0 23.9

Sulphur 2.96 7.r 24.2

Whey 3.01 7.4 24.1

Prograrn Z 3.00 7.3 24.1

Program 1 3.02 7.0 23.9

Whey plus SÆI 2.99 7.2 23.5

r (R) l1cocaro - 3.02 7.3 24.5

Synertrol Horti-Oil@ 3.02 7.3 24.2

Untreated 3.00 7.4 24.3

8.3.3 Temple Bruer Wines vineyard experiment 2 The mean severity of powdery mildew on the leaves of untreated vines at

Temple Bruer Wines vineyard on 19 February 2003 was 2.5, indicating that 25 Yo of the leaf surface was affected by U. necator. All test materials applied to the vines in this experiment significantly reduced the severity of powdery mildew on leaves when compared to the untreated controls (p <0.001) (Table 8.7). There were no significant differences in the disease severity on vines sprayed with the various test materials.

However, when the data for untreated vines were excluded from the results, the disease severity on vines sprayed with whey protein plus Synertrol Horti-Oil@ *as significantly less than on vines receiving whey 15 glLplus Nu-film 17@. 1s8

There was no evidence of phytotoxicity on any of the vines used in experiment 2. There was a white crystalline residue on leaves and bunches of vines sprayed with the mixtures that included whey. The residues were not observed on vines sprayed with

Coaton LP@, whey protein plus Synerhol Horti-Oil@ or untreated control vines.

Table 8.7: Mean disease severity on leaves (19 February 2003) on treated grapevines, cv. Verdelho for the Temple Bruer Wines vineyarás in 200212003, ãftêr eight applications of the test materials. LSD for ANOV A (5%) when data for untreated vines included : 0.3191; when data for untreated vines excludéd : 0.II77. Score for leaf area affected with sporulating colonies of powdery mildew; 0 to l; r- rc% I b 2; lr- 20o/o, 2 to 3; 2r- 30u./0, 3 to 4; 3l - 40yo,4 to 5; 4r - 50% etc. N/A : not applicable. Trcatments with the sanlc lerter are not significantly differeni (p<û.05).

Statistical Treatment Mean disease significance when severity data for on leaves untreated vines evnf rr ¡l o¡l Whey protein plus 0.1 a a Synertrol Horti-Oil@

Coaton ILPG) 0.1 a ab

Whey 30 e/L plus 0.1 a ab Slatertrol Horti-Oil@ Whey 15 g/L plus 0.1 a ab Synertrol Hoti-Oil@ Whey 15 g/L plus 0.2 a b Nu-film 17@ {lntreated 2.5 b

The mean severity of powdery mildew in bunches on untreated vines at Temple

Bruer Wines vineyard on 19 February 2003 was 1.8, indicating that lB %o ofthe bunch surface was affected by U. necator. All test materials applied to the vines in this experirneni significantiy reciuceci the seventy of powdery mildew on bunches when compared to the untreated controls (p <0.001) (Table 8.8). There was no significant difference in the disease severity in bunches on vines sprayed with the various test materials' However, when the data for untreated vines were excluded from the analysis, 159 the disease severity on vines sprayed with whey 15 glL plus Nu-film 17@ was significantly greater than that on vines sprayed with the other mixtures.

Figure 8.8: Mean disease severity on bunches (19 February 2003) on treated grãpevines, cv. Verdelho, for the Temple Bruer Wines vineyard in 200212003, after ðight apptications of the test materials. LSD for ANOV A (5%) when data for untreated vines i*l.td.¿ :0.2746; when data for untreated vines excluded:0.1369. Score for visual assessment of bunch area affected with sporulating colonies of powdery mildew; 0 to 1; I - l0% I to 2; ll - 20o/o,2 to 3; 2I - 30Vq3 to 4; 3l - 40o/o,4 to 5; 4L - 50% etc. N/A: not applicable. Treatments with the same letter are not significantly different (P<0.0s).

Statistical Treatment Mean disease significance when severity data for on bunches untreated vines excluded Whey 30 g/L plus 0.03 a a Synertrol Horti-Oil@ Whey 15 g/L plus 0.1 a a Synertrol Horti-Oil@ Whey protein plus 0.1 a a Synertrol Hofi-Oil@ Coaton ILP@ 0.1 a a

Whey 15 g/L plus 0.3 a b Nu-film 17@ Untreated 1.8 b N/A

8.4 Discussion All test materials and spray programs applied to vines at the Temple Bruer

Wines vineyard in the 200212003 season significantly reduced the severity of powdery mildew on both leaves and bunches. There was no noticeable impact of the test oBrix, materials on pH and TA nor was there any visible sign of phytotoxicity on any of the treated vines.

In the 200212003 season there was a noticeable variation in the size and health of the vine canopy from the northern end of the experimental site to the southern end, and that was unrelated to the materials applied to the vines. The vines at the northem 160

end were noticeably less vigorous than those at the southern enel of the experimental

plot and the leaves on vines to the north appeared yellow when compared to those further along the rows. Such variation was not evident in previous seasons, when

rainfall had been higher, and was possibly due to a difference in soil depth and available

water that had a noticeable impact on the vines in this drought year. The change was

progressive over the length of the experiment. However, while there was a small block

effect (see section 8.3.1), it did not significantly affect results. Disease scores and yields

on the northemmost vincs were less thatt Llrt¡se to thc south, leading to large variation in results within treatments, and the relatively high LSD values may have hidden some

differences between test materials.

ivfany of the bunches rejecteci as having 5% powdery mildew infection or more,

from vines treated with sulphur, whey 45 g/L, milk, program 1 and program 2, would normally have been included in the halest, as the iarge proportion of disease-free

hrtn^hoo .r¡^,,1.1vYvuru L^,,^rrdvu -^-t--^^l .t, ruuLlL;çu tlle ovel'all (llSeASe levgl to below 5o/o" The losS of production caused powdery by mildew thus would have been minimal for these treatments. The highest total yield of glapes was harvested from vines sprayed with sulphur, and resulted from consistently high yields across nrost of the blocks (i3.3 -

17 '4 kg per block) rather than from orre or two very high yielding plots. possible explanations for the sulphur treated vines returning the highest yield include a) the inconsistencies across the experimental site affecting the average yields, b) sulphur sprays encouraging higher yield or, c) the other test materials inhibiting fruit set. While

Ê'llil qet qnd fhe nrnnn-ri^- (Jr L^,--:- Havt,vrrrurr ^t'^L^¿^-:4uurttiLl usl.I'lgs were not assgssed, thgre was no obvious difference between vines treated with the different materials. Canola oils have been associated with reduced yields (Schneider et al. 1990; pasini et al. 1997) (see section l'5'1), However, in the experiments conducted at Temple Bruer Wines vineyard the oil 161 was applied at volumes and concentrations lower than those shown to affect yield. The yields from vines treated with the test materials and programs were lower than those harvested from vines treated with sulphur, but large variations (up to 100%) between

the highest and lowest yield harvested from individual plots lowered the averages.

The smallest total yield and acceptable yield for all blocks was ha¡vested from

the untreated vines (total yield: 5.6 - 13.3 kglplot; acceptable yield 0.06 - 8.6 kg/plot),

a result which supported previous reports on the impact of powdery mildew on yields

and profitability of vineyards (Chellemi et a1., 1992, Reuveni et al., 1995, Wicks et a1.,

lggT). All test materials and programs lead to an increased yield (total yield :7.8 - 20.6

kg/plot; acceptable yield L2 - 18.4 kg/plot). The largest yield obtained from a single

plot was from vines sprayed with whey 45 glL (total yreld : 9.7 - 20.6 kg/plot;

acceptable yield:6.6 - 18.4 kgiplot). While the reduced yield of untreated vines was

expected, further evaluation is needed to clarify the effects of the test materials on yield

of Verdelho and other cultivars.

ln these experiments, the application rate of canola oil products did not exceed

0.2%o at 900 L/ha or 1.8 L of oil per hectare. Canola oil has been reported to cause leaf

burn if applied in full sun when the temperature exceeds 30oC (Martin et al. 1931;

Schneider et al. 1990; Pasini et al. 1997). In other experiments, phytotoxicity was

recorded only when the oil was applied at rates of 8 Llha, four times the highest tate

used in these experiments, and only in areas where the oil pooled (Finger et aI.2002).

In these experiments, all spray applications were made in late morning to early

afternoon and, on occasions, the temperature did exceed 30oC during spraying. There

was no evidence of phytotoxicity attributable to either Synertrol Horti-Oil@ or Biotrol@,

which supports the findings of Finger et al. (2002) that the rates and concentration of

canola oil required to cause phytotoxicity are much higher than rates normally applied r62 in commercial vineyards. Pasini et al. (1997) found that applications of Synertrol Horti-

Oil@ at 2%o restlted in phytotoxic damage to rose leaves in greenhouse experiments.

This rate is 10 times that used in the field and greenhouse experiments in this study, and may account for the observed differences in phytotoxicity.

Other potential impacts of the application of horticultural oils to grapevines are reduced photosynthesis, delayed maturity and lower yields (Finger et al. 2002)' These effects were related to the volume of oil applied. Differences in photosynthesis and oBrix maturity would lre expected to result in lower measurerrlcllts, not obser-¿ed in this experiment. Finger et al. (2002) applied up to 6,200 Llha of l.5o/o vlv oil treatments or g.3 L of oil per hectale and, at rates equivalent to those used in the experiments at

photosyrthesis Temnle - vinevards. observed no significant ditl-'erences in ^-^_^r-- -Rmer - Wines or maturity between grapes from sprayed and untreated vines.

Early formulations of eanola oil-based sprays dirJ not control U. necator at rates below 2% (y1afün et al. 1931). 'l'he 0.'2Yo Synertroi Florti-Oii@ üeaimenl appiicti io vines at Temple Bruer Wines vineyard did not give adequate control of powdery mildew for a commercial vineyard. However, when used at 0.2Yo mixed with 2 glL

Ecocarb@ and alternated with whey, control of powdery miidew was not significantly different to that provided by application of sulphur af 3 glL. Mixing oil and bicarbonate has previously been shown to improve control of powdery mildew (Ziv et al' 1993).

The failure of Synertrol Horti-Oil@, applied alone at 0.zyo, was probably due, at least in part, to the failure to achieve 100'/0 leaf coverage, a necessity for oils to be effective in the control of foliar diseases (lvfartin et al. 1931).

Whey, applied at 45 glL,left a noticeable residue on leaves and bunches similar

in appearance to residues on vines sprayed with lactose in the greenhouse. While this

residue did not cause phyotoxicity, the leaves had a dry, papery feel when compared to t63 leaves sprayed with other test materials. A low level of a similar residue was noticeable on vines sprayed with lower rates of whey or where whey was part of a mixed program. oBrix, The residue persisted to harvest in 2003 but did not appear to have an impact on pH or TA. Chardonnay berries, sprayed with whey powder three times in the 4 weeks prior to harvest, did have a detectable off flavour attributed to whey residues but this did not persist into the juice (M. Arney, pers. com., 2002).

No residue was obvious on leaves or bunches sprayed with milk, and leaves appeared softer and smoother than those sprayed with the other test materials. Whether this was a physiological effect or was due to an accumulation of milk fats and residues was not established. In some wineries whey and milk powder are used in the late stages of wine production as a fining agent. However, research on the possible effects of the test materials on fermentation and wine quality is required. Research is also needed to assess the impacts of the test materials on the microbiota of the canopy, in particular, wine spoilage microorganisms and those that can influence the fermentation process, particularly if the winery relies on naturally occurring or wild yeasts rather than inoculation.

In experiment2 at Temple Bruer Wines vineyards, all of the test materials and mixtures reduced the severity of powdery mildew on leaves of treated vines when compared to the untreated vines. As was the case in experiment 1, a white residue was observed on vines sprayed with whey, but the residue was not observed on vines sprayed with the whey protein plus Synertrol Horti-Oil@ mixture. The major component removed in the reduction of whey to whey protein is lactose, so that the absence of residue on vines sprayed with whey protein suggested that lactose may be the major component of the residue. lnterestingly, residue on vines sprayed with whey was similar to that on vines sprayed with lactose in greenhouse trials. In experiment 2, Coaton t64

TLP@, which qreenhouse was evaluated in cxpenment 112002 (see section 6.3.4), gavc control powdery of mildew that was not sigrúficantly different from that provided by

the Synertrol Horti-Oil@ plus whey powder mixture. Coaton ILp@ may be a replacement

for whey powder if costs are comparative, or if whey powder is not readily available in

some areas.

The experiment conducted at Glenara Wines vineyard was again abandoned due

to the development of severe powdery mildew in the plots where the test materials were

applied. As previous in seasons, the majority of the powdery mildew that established in plots trial was concentrated in the centre of the canopy and the outer leaves were relatively disease-free. The failure of the test matedals to control polvdery mildew

could ha-ve been due to a f,ailure to achievc near iOo?'o coverage, reciuce<ì iight penetration could have impaired the efficacy of the whey component cf the program.

(see sections 7.4 and 5.3.2) or the environmental conditions may have been partie¡larly conducive to the development of powdery milclcw.

The two mixed programs, whey at 45 lL and milk at a 1:10 dilution all reduced the severity of powdery mildew on v. vinifera cv. verdelho to commercially acceptable levels (below 5% infection) at the Temple Bruer Wines vineyard. The degree of control achieved was similar to that achieved with application of sulphur at 0.3%" and might be enhanced if spray coverage could be improved. While the test materials appeared to have no impact on the three quality parameters evaluated, further assessment of juice and wine scnsory athibutes atrtl colour is required. Further assessment of the impact of the test materials on yield is also required before widespread aeceptance of the materials can be expected. There is also a need to assess the test materials on cultivars other than Verdelho and at other locations, particularly if light intensity is important for satisfactory control by whey and milk. 165

Chapter 9 - General discussion

The aim of this research project was to find a biological control or "soft pesticide" for grapevine powdery mildew, to replace sulphur in organic vineyards and

to be used as an alternative to synthetic chemicals in chemically assisted vineyards. The

initial literature search combined with discussions with a wide range of people

associated with organic horticulture revealed a large range of materials and cultural

methods used for the control of powdery mildews on a range of plants and crops.

Materials were chosen for testing based on practicality and availability. As a result of

greenhouse and field experiments, a small number of materials, including milk,

Ecocarb@ and canola oil-based materials, which can be used alone or in mixed

programs for the control of powdery mildew, were selected for evaluation in field

experiments. Other materials, not evaluated in these experiments, such as tea tree oil

and oils extracted from citrus waste, may merit evaluation but are too expensive for

broad scale use.

Warriparinga, a vineyard where no synthetic chemicals had been applied for at

least 10 years and sulphur and copper sprays had been used sporadically, provided a

suitable site for evaluating the test materials. As the vines had been largely abandoned,

loss of production from untreated control vines and those receiving unsuccessful test

materials had no commercial ramifications. Synthetic fungicides could be used for

comparison with the test materials without risking the organic status of the vineyard.

Powdery mildew at Warriparinga was widespread and severe in 2000, indicating that no

effective native control agent was active. The absence of powdery mildew in 2001 may

have been due to a number of factors (see section 4.4). The large population of

Orthotydeid mites present that year may have reduced powdery mildew to undetectable

levels. However, the mite was not present in sufficient numbers to control the disease r66

each year of the project. Like o. lambi, the mite at w'arriparinga was highlv susceptible

to sulphur (Marlow 2002). Sulphur should be removed from the management of

powdery mildew if these mites are to be considered as part of an integrated powdery

mildew control program. The development of alternatives to sulphur is required if natural populations are to establish and persist at sufficiently high numbers to help

reduce the severity of powdery mildew.

Application of a range of biological agents for the control of powdery mildew was investigated in greenhouse experiments; of those evaluated, B. subtilis .¡yas the

most effective' However, the control of powdery mildew by B. subtilis in field

experiments was inconsistent and, while this was attributed partially to incomplete

., wvvvr6tËv ^fal^^ ^^-^^,^-- L1- l' r ur Lrte uarrupl tlrË ulsease perslsteo on areas or the vlnes that received close to

I00% coverage of susceptible tissues. The difference in efficacy of B. subtilis in

greenhottse and field trials may have been related to differences in relative humidity or other environmental fbctors. The mctabolitcs of autociaved cuiiures of B. suhtiiis

reduced the severity of powdery mildew on vines in the greenhouse as effectively as did

Itve errltures" Further evaluation of the cultures and metabolites fion-r oultures of different ages may proviele more consistent control of powdery mildew in the field, particularly if all or alurost all of the susceptibie tissues can be covered with a suitable dose.

Other microbial agents evaluated were no more successful than B. subtilis and. were not considered further. The failure of microorganisnrs to control powdery mildew effectively in the field was possibly due to thc environment iii wi¡ich they were assessed. In cooler regions or those with higher relative humidity, some of the microorganisms may provide superior control of powdery mildew and, if so, could be developed for use in such areas. One rich source of microorganisms, including B. t67 subtilis, is compost teas, which have been shown to control powdery mildew in some conditions. However, the availability of suitable, uniform compost and the time involved in preparation of the teas limit their acceptance as practical options in large vineyards.

Novel compounds that could be used as "soft pesticides" and that were suitable for use in organic viticulture, safe to use, and environmentally benign were also evaluated for their potential to control of powdery mildew. This approach proved successful in the field experiments conducted as part of this research project. Milk, whey and mixtures of botanical oil plus bicarbonate provided control of powdery mildew comparable to that provided by a sulphur-based fungicide in the greenhouse and field experiments.

Milk, whey and botanical oils plus bicarbonate meet many of the initial aims of the project, such as ease of production, storage and application. Milk and whey can be purchased in powder form, are easily stored for extensive periods, easily prepared and can be applied using standard spray equipment. None of these materials presents any health risk to users, and is unlikely to have negative environmental impacts. The only potential environmental impact of these materials is that if oils are applied in hot weather they can contribute to air pollution (Gubler et aI.2002). However, sulphur also contributes to air pollution and, at current recommendations of 5 - 6 kgtha per spray or potentially over 40 kglhalyear, is probably a greater pollution threat. Latge volumes of whey are processed each year as a waste material and, because of its high biological oxygen demand, there are limited options for whey disposal.

The biggest challenge facing viticulturists planning to use these materials in powdery mildew control programs is achieving consistent, high levels of coverage of infected tissues. Milk, whey, oils and bicarbonates appear to act mainly as contact 168

fungicides; if infected plant tissues are not effectively covered the control of powdery

reduced. mildew is The size and density of the vine canopy and the quality and condition of the equipment used to apply the materials have a large bearing on spray

efficacy. For example, the test materials failed to maintain powdery mildew below commercially acceptable levels at Glenara Wines vineyard, at least in part as a result of poor coverage in the large, dense canopy. In vineyards such as at Glenara, the efficacy

of treatments such as milk and whey may be improved by taking measures to reduce the vigour of the vines, to facilitate spray penetration a-nd covcrage. There is a risk ttrraL

reducing canopy and vigour may lead to unacceptable reductions in yield. However, the

cause of the reduced efficacy of the soft pesticides at Glenara needs to be evaluated to

r¡llciÇiv nrrlvúù nfhar fo^fn-a ^--¿-r r.,. uli¡wi i¿vLwi5, siil;ii^',^L as^^ environiüerriai^'^-,:-^---- uondltlons. When pianting new vineyards

where non-systemic materials are to be used for the control of powdery mildew and other diseases, the use of trellis systems that limit the size of the ca¡opy is recommenrled

potential One disadvantage of the above materials is that they may be more expensive than sulphur, increasing the cost of disease management programs and affecting overall profitability. This increased cost is una.¡oidable for products such as

Synertrol Horti-Oil@ and Ecocarb@, and is also the case for milk anrJ whey purchased in powdered form. However, some milk and cheese factories currently pay for disposal of whey stale and milk, and for vineyards located near such factories the only costs associated with their use as controls of powdery milclew could be the labour and fuel reoltirerl tn cnllenf lhcm Â1o^ ;f .,;-^,.^-¡^ r^^^¿^r ,-- r ' ' '--r---^ rÀrov' rr vurvJcrLrù arl^-^ rUU¡l[çU llgAf Oalnes, mllK may be purchased at "farm gate" prices and result in inexpensive disease control programs.

Ecocarb@ is a commercial formulation based on potassium bicarbonate and, as such, is more expensive than the generic potassium bicarbonate available from hardware and r69 grower supply stores. Costs could be reduced if the latter was used in powdery mildew control programs.

Milk and whey provided a level of control of U. necator similar to that of sulphur in greenhouse and field trials and Topas@ at Temple Bruer Wines and

Warriparinga. Other research has found that milk provides control of powdery mildew of zucchini squash similar to synthetic fungicides (Bettiol 1999) and, in glasshouse conditions, also controls powdery mildew in wheat as effectively as sulphur (Drury et al. 2003). Both Bettiol (1999) and Drury et al. (2003) found that the efficacy of milk and whey was related to concentration, which is consistent with the results of greenhouse trials carried out as part of this project (see section 3'3.2).

Although some active components of milk and whey, such as lactoferrin, have been identified, further evaluation of their mode of action is required. A thorough understanding of the mechanisms by which components, such as lactoferrin, interact with U. necator to cause the physical damage seen using SEM will assist in calculating spray timing and concentrations to achieve maximum disease control for cost and effort. For example, if light intensity and duration influence production of free radicals and, therefore, the efficacy of milk in the field, sprays may be more effective if applied in the morning and on bright days than at night or on overcast days.

Investigation of other components of milk is required to assess their ability to control powdery mildew alone and in combination. The efficacy of a material such as lactoferrin may be enhanced if the mechanisms involved in its control of powdery mildew are more clearly understood. A thorough understanding of the mechanisms may

assist in the development of new products for the control of powdery mildew and other plant diseases. For example, if a second protein or molecule is involved in the

interaction of (J. necator and.lactoferrin, mixtures of the two may be more effective 170

than either compound used in isolation. Additionally, a clear understanding of the interaction between lactoferrin and. U. necator may give an indication of the protein's

potential to control other plant diseases. As other components of milk that contribute to the control powdery of mildew are identified, theìr modes of action should be investigated.

While milk and whey have been used successfully to eontrol powdery mildew in two vineyards in these experiments, their efficacy in other environments is largely unknown, The application of milk or r,vhey for control of powdery mildew has been

adopted by a number of growers across Australia and, while they appear to have been

successful in most cases, scientific data have not been gathered to verify the anecdotal

rvvvr!Þ'rañ^r+õ 'I.L^-^:-r'wru rù ^ rrçEu-^-J ¿- '1 tu assess me potentlal of mtlk and whey in a range of environmental conditions, particularly where there is greater rainfall, relative humidity and lower light intensity than in the vineyards used here. The optimisation of timing, concentra-tions and spray inten'als of powdery miidew coiriroi programs that inciucie oii plus bicarbonate mixtures in rotation with milk or whey is also needed in a range of condrtrons.

The effect of different cultivars on the efficacy of the powdery mildew controi

programs and the need for adjustment of rates or timing shoukl be investigated. Most experiments conducted in this project involved the moderately to highly susceptible cultivars' Chardonnay, Verdelho and Viognier. Fewer spray applications or lower concentrations of active ingredients may provide a

Preliminary evaluation of some gape quality parameters indicated that there was no negative impact of the test materials and programs. However, the ultimate

evaluation of the impacts of the milk, whey and programs tested here, is the quality of

the end product, the wine. At this stage, the quality of wine made from grapes sprayed

with milk, whey or the mixed programs does not appear to have been compromised (D.

Bruer, per. com. 2003), but further research, including juice and wine quality

assessment, needs to be carried out. Until there is a clear and concise answer to the

question, "What effect do these disease control measures have on the quality of the

wine?", many growers will not adopt the materials and programs. The question could be

addressed by replicated small batch ferments from grapes sprayed with the test

materials, and evaluation of flavour attributes, colour and other sensory characteristics.

Along with quality, the other major contributor to vineyard income is yield. The

data collected in 2003 at Temple Bruer Wines vineyard at Langhorne Creek showed

that the total yield was greatest from vines treated with sulphur but there \¡/as no

significant difference in acceptable yields for vines treated with sulphur or whey 45 glL

for cv. Verdelho. There was also no significant difference in the proportion of the total

yield considered acceptable for winemaking from vines sprayed with sulphur, milk,

whey or the two test programs (see section 8.3.2). While these results are encouraging,

more data are required to evaluate the effect of the materials on yield in different

seasons, different cultivars and in other grape growing regions.

Difficulties associated with the use of sulphur include phlotoxicity if applied in

hot, humid weather or in slow-drying conditions, and reduced efficacy at low

temperatures (refer section 1.4.3.1). No obvious phytotoxicity was observed for milk,

whey, Synertrol Horti-Oil@ and Ecocarb@ in any of the greenhouse and field

experiments conducted as part of this research. In addition, the degree of control of 172

powdery mildew providecl by these materials did not appear to be temperatr;r.e-

dependent. these If results can be confirmed, the novel control methods would have advantages over sulphur early in the growing season when temperatures are below the

optimum for sulphur or during periods of hot, humid weather later in the season.

One factor in the cost of the spray programs is the time and fuel associated with

the application of the materials. At this stage, the programs involve a 10 - 14 d,ay interval between applications from l5-cm-long shoots to verasion, resulting in six to eight applieations per season. Ak-rng with the financial costs, there are aiso environmental costs associated with spraying, in the form of greenhouse gas production caused by burning fossil fuels, and soil compaction. Further field and greenhouse

experiments are requireri to establish optimum concentrations of the materials applied, water rates, spray timing and spray intervals, to reduce the number of applications

required and, thereby, finzurcial and environmentai costs.

. \Iñ.il^^^^+^Yvrrrrç vuùtù, ^---11¿-- t rr qu¿lty arlo ylelo are all assoctated wtth finaueial sustainability, a major consideration of disease control programs, particularly in organic agriculture, is

environmental sustainability. Sulphur is a biocide and has been shown to be toxic to a number of beneficial organisms, including predatory mites and insects. Honours

projects undertaken by Robert Marlow and Lachian Palmer in association with this project have shown that there are significant differences in arthropod and microbial

populations, respectively, between vines sprayed with sulphur and those sprayed with milk' The impact of the changes in these populations on wine quality and environmental

vsv'ørr¡qu¡'rJcrrcfqinal.ilì+.' ;.ro raÈtvrJ l^*^^1,. .,-t.-^------| urrl\truwrr aIlU wafants Iurtnef tnVeSttgahOn. AISO UnknOWn iS the impact of milk, whey, oil and bicarbonate on the soil microbiota and microfauna and the possible impact of the materials on water in areas of high run-off. t73

Sulphur can have a negative impact on the health and quality of life of vineyard staff and people living in and around areas in which sulphur is used. Milk and whey are food products widely used in the community and, while lactose intolerance affects many in the population, the non-volatile nature of the materials will limit any impacts on human health.

The research conducted in this project has identified materials that meet many of the initial aims. Milk, whey and the mixed programs provided effective control of powdery mildew in the vineyards in Langhorne Creek and were a viable alternative to sulphur and synthetic chemicals, provided adequate coverage of plant tissue could be

achieved. While coverage appeared to be the major factor determining the efficacy of milk and whey in controlling powdery mildew, increased disease pressure and reduced

light intensity in the centre of dense canopies may have also affected effrcacy. Milk and whey are curative treatments for powdery mildew, do not provide an extended period of

protection from new infection and, therefore, must be applied on a regular basis. In

some cases the novel products are more expensive than sulphur, but are safe and easy to

handle, readily available, easily stored, and applied using standard spray equipment.

With further research to optimise spray schedules and to assess quality and

environmental impacts, these novel materials have the potential to become part of

powdery mildew control programs in organic and chemically assisted viticulture. 174

Appendix I - l='ield sites

Map 1 - Glenara Wines and Mountadam Vineyard

s6lt¡ttrilltF ADEIAIIDE MAP I ENVIBONS

I ./

ì?rçÞr

Mountadam Vineyard

S.riltfþH {rEhB

FÛ

= Glenara Wines ll'INShIIhIflJ ts]I¡.ï'lt tÈil€t/'tc nflrunil'rJ $FHTfdlð rHo tË.tutïïJ EIIIL .Fç4fu{,td a H:EUC 1ÏE loËil t !Ë€Af{ 0(Ê\tx3t'ìa \ærá| H¡1f'4 3H:[ tlÌE ìFçá UHEE tÞtÈd¡ il'lt1usï lçF8 SHUfr$ltsï IÐHDÏ +d'rEt srllTrrrJ, ,Þ#,rdu s f EFI ¡rìflT 'f¡¡Fq

I ¡Æ;ä _uFUHlr+l 0 rug+¡ iltFJ Hr¡+¿¡_ïsl,t E J'DlsÈ|ltA s ,+.Ë¡ft!4+ñl Dll Ë¡JËfrtt Ë ryqdrfttlq4q.|{t $¿ UDI lHlH¡¡ilæI $ s¡Êg ruî FEflqI É

Ê 6 Þ ¡FH {4d u.ttlr*ÞJfeÐ ilq Þ rFqNJ¡$ ryqduçuxro¡ lF{s þ JTÀdT4J $ tf+"ífir l¡d U0FAFJ{'JF¡H rù¡fûolo ef¡ lPSl+l tÊ ffiJu¡flÞ ü çù¡Ift|{ m & l0Fa rlllçs Þ tÞ 6 f rË.+n6: tr Ë ËPqlï $ rFq¡g UÉflthsll'II s ¡oeg l¿rF. cJ UÆd+$ ¡,frul#l TTId ¡+d E ìd lDu+l tÍ [çrçS*tû E r,rF l{ & * E IDFá,¡F:l{ Ë EF]ïI ¡r'F-s $ü E tÞl G 6l Ff,{ É+ IE iæ?á F¡++t ü !+ H-lrÊû $ t+ a+Ét þv ¡tDFrv¡¡ 0t 6 I t 9 Il+ry{ _ tl$ÊFrË þ rafl¡8rhsfi lspìl¡Flffill € r6u'f5 4.Edrirf,{ ffiq{fu_rrE IAd u{FÀ¡HoCqì¡S !ùrFt' 1E J$UTINI IOEÐOYIJ tr E Ej L-J f ,lh8lStrtlû¡/

.r+d rcd tl lvcrftf "Ð ;f Lu,¡ t Id'fr.rc EI v

u8u¡rud¡urÀ\ - Z d?IN

çLI 176

Map 3 - Temple Bruer Wines vineyard

+

ü

¡t a{ ,Þc¡.i¡ R E*ndFt f :--C A e Temple Bruer Wines Ptv Ltd l¡ v

rló¡d¿tr .t

'q -5trt ,,tlrãde¡

'%ê4, r'Jr ¿J

fEF IA]/

E¿úlrú

IE¡ìIIHÊE t? HTEBGI.E{ PT On Thb FhF t¡uHG lt5 STNHÁtEV{ F1 B I tot¡BÁR G+ tlH{llFtH KE SNFl.IHG D2 0 4 æruNÊ.l EE I#JI{TB¡f,IIEF FI sJì{v¡l$4Y DI E + Ft'tNtsÊ F5 ¡lËJt{TCOi¡P¡sS B5 TJIEM EEHD Lê L s Fu)q.EÍ E2 MJFÁYFIæE JI ÎEPqHI L2 I ffi.r¡¡Á FE Ml!Aß D{ 'ltEBJltË D+ GC t{4ilmÊF EI m,PailÊl CE ](ñFB/ttË E5 ce ]t/flG¡nrtu 01 II,lRHE FI HCTAFH,Tgæe EI SIIORT TRIPS E1 UIG¿tSERT LE t{,1t8{t1}, E5 r¡t¡¡mHGl EC GI ulG¡tH¡trnl¡¡ J5 t{.lFf, r.flG H5 II'JE.UHSEil L1 ,{9 I.ltFfl(Nt¡E CFEE( HI tfifit¡¡¡{ulJ.E B1 uttlurË4 0+ A.ROUFID 0e t/t4cDtËFlEtD F2 a[D'tt¡lEH ]ç$it tÊ rÀlflttt¡ F2 G6 À{*L¡Fg TUT D+ FÊTT¡.l-(ÉTI t5 $(Ê[cHæ]B Ëc trtd-¡ÊE{ y¡tE Ff,CÉPECTHIIi E+ 'i|¡lKrtill,i B6 ADETAIDE F5 l+ÁFr¡+s EC Mg OBÁY aú \TJHq D5 177

Appendix2 - General methods and materials

extract medium lor Bacíllus subtilís ^2.1Yeast

Growth Media (200 ml)

Vogel's Solution 5'0 ml Sucrose 2'o g

Yeast extract 1'0 g

Distilled HzO 195 ml

Vogel's Solution

Na3 Citrate .2HzO 125 g KHzPO¿ 250 g NH¿NO¡ 100 g

MgSOa. 7 lF'zO 10 g CaCIz.2HzO 5 g

Stir at low heat in775 ml HzO adding each salt individually, adding the CaClz I g at a

time, and then add trace element solution and biotin (10O¡rg/ml) (Vogel 1964).

Trace element solution

Citric acid. HzO 5'0 g

ZnSO¿. 7 ]HzO 5'0 g

FeCNH¿XSO¿)2. 6 HzO 1'0 g

CuSO¿.5 HzO 0'25 g

MnSO¿. HzO 50 mg

Boric acid (anhydrous) 50 mg

Sodium molybdate 50 mg

Mixed in 95ml DD HzO 778

A2.2 Nutri-life 4120@

Distilled Hz O 200 ml (30"C)

4l2O Nurri-life 0.25 g

Sucrose Sigma-Aldrich Co. 0.5 g pty Fish emulsion Garden Maid Ltd 0.5 ml

Chick feed pellets Biotech Organics pty Ltd 1.0 g

Mixed for 25o 48 h at C in 500 ml flask on an orbital mixer then tiitered through I mm gavze. Experiments conducted after the 200012001 vintage that included Nutri-life 4/20

used a modified formulation from Nutri-life as a food source, replacing the sucrose, fish emuision and chick feed pcllets, however, the incubation temperature and time were unchanged.

42.3 MR Formulation

Methionine Sigma-Aldrich Co. 0.15 glL 1 mM Riboflavin Sigma-Aldrich Co. 0.10 g/L26.6 ¡tlll

Laurel sulphate Sigma-Aldrich Co. 1.0 g/L

Copper sulphate Sigma-Aldrich Co. 0.1 g/L 1 mM

(Tzeng and DeVay 1989)

42.4 Wheast

Whey Bonlac Pty Ltd rs gL

Yeast extract Sigma-Aldrich Co. ts s/L a) Temple Bruer Wines PtY Ltd

023747 STRÀTHALBYN Conrnenced: 1861 Last record: 2001 Latitude: -35.2600 S Longítude: 138.8850 E Elevation: 70.0 m State: SA E iTAIi¡ FEB ¡IAR APR !4AY iTI'DI iN'L AUG SEP OCl ¡IOV DEC ÀI\¡N No. %åge :É YrE coûrE) tã Irlean Daily Måx TgrE, (dleg C) z\ri 2't.4 27.4 25-4 2r.8 18.3 15. b 14.8 15.9 18.3 2r.0 23.9 26 -7 2r-3 109.2 78 - D¡eån no. DayE, Matß >= 40.0 deg C X r-.1 0.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.3 2.4 43 .8 99 (, ltean no. DayE, Max >= 35.0 deg C 99 I 5.0 4.4 2 -O 0.2 0.0 0.0 0.0 0.0 0.0 0.2 _t.5 3.1 16 -4 43.8 Dlean no. Days, Max >= 30.0 deg c 10 43 99 9.5 9.4 6.5 2 -3 0.0 0.0 0.0 0.0 0.3 5.6 42.1 .8 F1 HigheEt Ma:< lr€n¡t (dlegr C) 4s.0 43.8 42.9 3'7.4 28.8 26.7 26.8 2 t.4 34-4 37.8 43.4 42.2 4s.0 43.9 99 Þ l+'lË tl( Mean Daily lilin Tq, (deg C) to '?9 taJ 13 . 6 13 .5 72 .r l-0 .0 8.2 6-6 6-7 7.7 8.6 r0.4 72.2 9.5 l-10.8 Dlean no. Dåys, uin =< 2.0 dêg C F ?t 99 0.0 0.0 0 .0 0 .1 1.3 3.8 1.3 0.5 0.0 0.0 13.3 43.8 ltêan no. oays, uin =< 0.0 deg c .1- 99 z 0.0 0.0 0.0 0.0 0.3 1.3 I.I 0.3 0 0.0 0.0 0.0 3.1 43.8 U I-orreEt Mín TqE, (dleg C) 5.0 4.4 3.4 0.4 -2.0 _? q -3.0 1Q -0.8 -0. i- -2.9 4.3 -3.0 43.9 99 a) F ltea¡1 9am Air TgrE (deg c) l-l ) 80 27.1 20.5 19.0 16.4 13.3 TU. I 10-1 Lt.4 L3.1 16.1 18.3 )ñ 15.9 111.8 ?Ê lilean 9an vlet-buIb T€ÍE (dêg c) 15.6 15 .6 14 -1 13 .1 11.1 a, 8-4 a) 1-0.7 t2.2 13 .5 14.8 12 .3 70'7 .4 17 Ê t[êaD. 9arr De!ù Point Tqt (deg C) 90 Þ t_1.3 11.9 11.1 l-0.0 8.9 7.6 6.4 6.1 t.5 8.1 8.8 10.1 9.7 39.7 I Mear¡ 9a¡n Inrnidlity (%) \J Relative 'rt 56 67 63 68 '76 81 79 14 Õ/ b1 56 6't -7 94 Ë ¡lean 9arn $Iind gDê€d (}m/hr) 16.0 14.3 L4.4 13.8 !2.O L2.4 14 -9 ]-7.5 79 -6 2U.¿ 19 -2 18.5 16.1 42.8 97

Mean 3ltrî Air TqrE (dIeS C) 25.3 25.6 23.'7 20 -3 7'1 -0 14.2 13. s 14 -6 16.8 19.3 2L.9 24.7 19.6 106.8 19 \o\ì oo

iIåN FEB IIAR ÀPR MAY i'I'N iII]L AUG SEP ()c1 NOV DEC ANN No. %age Mean 3pm vlet-buIb TeÍE' lasg q¡ 7'7.3 1-1 .5 16.5 14.6 72.8 l-1. r. 10.3 10.8 72.2 13.6 15. 0 16.3 1,4.0 702_4 '75 Mean 3!m Derù Point TsrI, (d€g C) 10.8 r7.2 10.4 9.4 8.8 7 -9 6.1 6.5 1-4 8.0 8.3 o? 8 .'t 3l .8 86 u€an 3pm Relative Etrtnlatíty (%) 42 43 45 52 61 66 64 60 56 50 45 43 52 69.8 92

Dtean 3prn wind Sp€€d (ln/hr) 21 .6 20.0 79 .2 1-7 .1 16.5 1?.0 19.6 20.0 2L.2 2r.2 21 .5 2r.9 19. I 41 .1 94 ll€ar Rainfau (tmr) 20 .2 2r.2 24.L 38.1 54 .3 s8.8 63_7 60.8 53 _ 0 44.'7 28 .6 25.3 493.4 L40 - 0 100 lt€dian (Decile 5) Rairfall (Ím) 13 . 5 L2 -L 75.4 32.5 45 . 9 54-2 6r.5 51 .4 51. 4 42.2 24.8 79.2 490.'7 L39 Decile 9 Rainfall (un) 44 -2 54.1 55.9 16 .3 100.4 98.9 106"3 10s.1 88.4 8r-.4 5'7 .4 56.6 616.0 139 Dêcile 1 Rainfall (Nn) 3.5 1.3 3 -4 9.2 18.9 24.4 28.0 30.0 22.9 13.8 5.9 3.8 369.8 r-39 Mean no. of Râindays 4-8 4-t 6.5 9.9 r3.0 1,4.1, 15.3 16.0 13 .3 11.3 ?o 6. s r23 .2 93 .4 68 Ëighêst Monthly Raínfell (Ím) 220 .5 1,46 .9 tlL.r r4L.2 169 .9 165.9 733 .2 L55 -4 159.3 Lr2.8 98.7 777.2 140.0 100 LoYres! ¡lonthly Råinfa11 (ún) 0.0 0.0 0.0 0-8 r-.1 8.0 10.6 8.5 7r.4 3.6 0.0 0. 0 140 .0 100 High€st Recorded Daily Raín (mûr) 1.42 -2 720 -'7 65 . 0 45 .2 54 .4 49.8 70.0 40.1 32 .4 58 .7 13 .9 68.2 1-42.2 93.6 69

Dl€an no. of Clear Days 9.6 9 .6 8 -4 6.9 5.0 5.2 5.2 5.5 6.7 6.3 6.3 t.ó 87.7 44-r r00 M€an no. of clor¡dy Days 7.3 1.0 10.0 7r.7 13.8 11.5 11.8 11.9 L7.'7 72.r 11.0 10.1 129.9 44 .1 r_00

Last modified 28 May 2001 Weather Data Strathalbyn 2000-2001 YEAR 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 MONTH 10 10 10 10 11 11 11 11 12 12 12 12 Name and Unit Min Air Temp Max Air Temp Precipitation to Av. Relative Min Air Temp Max Air Temp Precipitation to Av. Relative Min Air Ternp Max Ai¡ Ternp Precipiøtion to Av. Relative CC) CC) 9am (mm) Humidi9(%) CC) CC) 9am (mm) Humidity(%) CC) CC) 9am (mm) Humidity(%) Mean 8.6 20.4 2.4 70.9 13 25.7 0.2 69.1 't2.1 26.7 0.1 60.2 Max 31 34 88 39.2 2 93 37.1 2 74 Min30 u 6.7 0 40 5.8 0 44 Total 73.2 4.8 3.6 No. ofObs 31 29 31 31 30 30 30 30 31 31 31 31 I 8.5 16.3 0.4 65 12.2 21 0.2 77 10.9 21.4 0.4 59 2 7.3 17.6 0 71 12.6 21.1 0.8 75 8 31.9 0 46 37.60 75 l0 20.9 0 71 14.9 24.8 0 67 4 6.6 27.5 0 59 9.1 20.1 0 76 10.1 34.3 0 6l 5 10.3 31 0 u 10.1 18.4 0 70 12.8 23.8 0 68 6 13.3 23.1 0 79 7.1 21.7 0 68 10.8 22.6 0 59 7 14 17.6 3 79 6.7 29.8 0 45 13 26.2 0 ô¿ I 6.8 17.1 0 70 20.3 25.6 0 59 12.5 21.7 0 60 I 3.3 17.8 0 70 14.4 21.8 2 81 8.7 31.6 0 62 10 6.4 17.9 0.2 70 12.8 19.8 0 86 't0.9 29.4 0 44 11 3 22.6 0 62 14.1 17 1 93 17.1 28.4 0 60 12 7.1 25j 0 g 14.1 21.1 0.8 86 13.7 24.5 0 66 23.1 0 67 13 9.7 17 1 70 15.1 23.2 0 78 11.9 14 6.8 18.2 0.6 71 12.1 25.9 0 75 10.5 22.2 0 60 't5 10.5 15.6 0 80 12.6 28.9 0 66 7.2 27.6 0 60 16 6.5 20.8 3 67 13.3 28.6 0 70 9.7 37.1 0 50 17 8.5 22.1 0.2 u 14.7 32 0 70 16.6 30.7 0 49 18 13.2 20.9 26 86 15.3 34.9 0 63 16.3 25.7 0.6 74 19 10.1 16.8 U 79 16 30.9 0 74 9.9 31.2 0 68 56 20 10.6 1 86 16.1 22.',| 0 79 1'1.8 36 0 21 6.6 25.8 0.2 73 't4.4 22.1 0 73 '18.9 33 0 56 22 11.3 26.7 0.2 65 10.6 23.1 0 73 16.7 23.5 0.6 67 23 12.8 21.7 0 83 12.4 23.2 0 74 't0.7 24.2 2 65 24 13.4 16.7 0 88 't0 28 0 68 '16.5 25.2 0 68 25 11.8 16.2 3 86 10.8 36 0 53 12.2 24.3 0 62 26 5.8 16.4 0 80 15.1 34.9 0 42 14.1 22.5 0 66 27 9.8 17 0 73 14.9 39.2 0 40 12.8 19.4 0 59 28 7.6 17 0.4 68 22.9 3r.6 0 54 9.8 19.5 0 56 29 3.3 19.8 0 75 9.6 23.8 0 68 5.8 22.8 0 62 oo 305240 61 't0 23.3 0 67 I 28.4 0 62 31 8.9 26.2 0 56 't3.2 32 0 46 oo N)

200F2001 2001 2001 Weather Data Strathalbyn 2001 2001 200'1 2oo1 2001 2001 2OO1 2001 ? YEAR 2001 2OO1 2 333 1 1 222 Av. Relative 1 Min Air Te¡np Max Air Ternp Precipitation to MONTH 1 Max Air Temp Precipiøtion tc' Av. Relative Precipìtation to Av. Relative Min Air Temp (mm) Humidity (%) and Unit Min Air Temp Max Air Temp 9am Name ("C) 9am (mrrr) Humidity (%) cc) cc) 9am (mm) Humidity (%) ('C) 24.8 0.8 ü.5 fc) cc) 0.9 60.8 11.9 60.6 16.6 29.5 15.7 30.9 0.1 37.4 7 80 Mean 41.2 17 81 43.5 2 77 0 30 Max 0 26 6 0 29 10.2 25.2 Min 8.3 24.8 31 2.2 31 3'l 31 Toøl 28 2A 28 30 31 28 0 64 30 31 30 8.6 30.3 No. ofObs 'I',1 .1 39.1 0 40.2 0 29 24.6 0 70 I 14.2 0 30 9.2 48 26 41.2 74 25.2 37.1 0 13.1 25.4 0 2 28.8 0 77 0 6'l 20 0 71 3 11.9 31 81 r 5.9 23.3 16.9 20 0 70 37.1 0 46 13.7 27.7 0 4 18.9 27.1 0 65 0 70 16.3 0 42 5 15.5 23.4 53 14.9 37.4 17.2 36.7 0 40 23.2 0 72 16.2 35.9 0 6 12.4 40 0 61 0 75 20.9 35.7 0 50 7 16.7 24.4 17 47 14 24.7 39.9 30 26 0 72 16.4 36.5 0 8 14.6 23 0 77 0 57 18 0 66 9 16.1 34.4 68 20.7 24.3 16.4 23.8 0.4 '18.6 0 43 20.2 0 60 t0 40.2 0 67 12.4 77 12.6 31.9 0 & 25.3 0 75 13.7 19.9 ll 17.4 29.9 0 63 30.6 68 8.7 20.7 2 l2 14.9 23.3 6 73 0 40 16.3 0 66 14.7 43.5 71 7.8 26.5 IJ 15.5 21.6 0 36.6 0 45 33.5 0 35 t4 21.4 22.1 0.2 67 I 0 68 12.8 0 63 15 19.3 25 68 19.9 27.6 10.9 24.5 0 68 26.5 0 57 11.6 20.2 7 l6 13.1 35.1 0 51 0 68 10.2 0.8 79 t7 13.1 24.1 27 10.2 22.2 16.8 40.3 0 74 23.9 0 68 8.5 23.1 0.4 t8 8.8 39.7 0 26 0 55 25.6 27.7 0 66 l9 12.4 37.6 0 27 9.2 17.5 40.4 80 38.2 0 il 11.'l 15.8 6 20 19.8 27.9 0 67 0 71 19.5 '13 2 78 2l 16.4 27.2 67 18.5 15 23 0 70 32.7 0 il 8.9 25 0 22 15.9 23.5 0 75 0 58 13.9 '13.6 21.1 2 65 23 19.3 39.9 72 13.9 22.4 0 70 38.1 0 65 10.2 20.3 2 24 19.7 28 0.2 71 2 68 16.2 21.2 3 72 25 19.7 36.9 1 74 13.7 71 't6.1 24.3 0 72 19.9 26.3 0.2 70 13.2 19.4 26 13.5 24.8 0 bo 26 0 67 12.6 19.4 0 27 12.9 24.5 0 67 0 60 14.7 18.6 0 67 28 10.9 24.3 6.7 0 66 23.5 0 71 29 11.8 24.8 o 0 65 23.8 0 74 30 14.3 23.8 6.8 0 59 3l 8.3 28.5 Weather Data Strathalbyn 2001-2002 2001 2001 2001 2001 2001 2001 2001 2001 200't 2001 2001 2001 YEAR 12 MONTH 10 10 10 10 11 11 11 11 12 12 12 Av. Relative Min Air Ternp Max Air Ternp Precipitation to Av. Relative Name and Unit Min Air Temp Max Air Temp Precipitation to Av. Relative Min Air Temp Max Air Ternp Precipitation to 9am (mm) Humidity(%) 9am (mm) Humidiry (%) fC) CC) 9am (mm) Humidiry(%) eC) CC) Cc) CC) 68.5 Mean 8.7 18'3 1.3 72.2 10.6 21.9 1.7 70.3 10.9 22.3 1.3 87 Max 25.9 5 u 32.2 l6 93 33.6 I 0 50 Min 3.8 0 52 6.6 0 51 5.8 Total 41.8 50.2 39 30 31 31 31 31 No. of Obs 3'l 31 31 3'1 30 30 30 12 '16.8 4 76 7.7 2. 0 79 5.8 21.9 0 64 t 70 2 5.3 15.9 0.8 74 12.2 19 0 69 13.1 29.3 6 4 87 8.4 16.6 0.4 82 8.8 26.8 0 66 13 15.5 3 4 82 8.3 15.9 2 79 11 29.5 0 61 12.5 20.3 4 72 4.3 18.6 0.2 76 16.7 21.2 0.6 93 7.2 24.2 0.2 s 24.6 6 9.9 14.2 3 73 12.2 17.5 16 75 13.8 I u 82 7 6.5 15.7 4 78 8.2 17.4 0 65 11.5 17.1 5 s 8.5 15.7 4 u 9.9 24.4 0 69 9.7 18.6 5 66 '19.8 0 67 9 3.8 21.9 0.2 71 9.3 18.5 0 70 8.4 0 66 r0 9.2 22.4 0 67 9.5 16.'l 0 72 10.4 18.6 69 6.6 15.3 4 73 11.1 17.1 1 76 il716.52 68 10 16.3 3 76 8.2 16.4 5 86 12.6 20.2 0.2 t2 0 72 17.1 0.6 82 11 18.7 5 78 11.9 21.8 13 I 70 7.6 17.1 2 69 9.5 22.3 0.6 72 11.5 19.4 0 t4 67 20.8 0.2 71 7.4 26.2 0 73 12.4 21 0 ls 6.7 69 8.9 20.3 0 8'l 7.4 31.1 0 66 't2.5 18.9 0 16 68 r7 8.8 l9 0.4 76 14.5 30.2 0 58 7.9 21.1 0 69 7.6 24.7 0 64 10.3 14.8 1 73 't1 18.4 5 18 50 15.5 3 72 o '17.9 0.2 65 9.4 33.6 0 19 8.1 72 20 6.5 20.'l 0 68 9.2 24.2 0 70 15.4 21.3 0 69 2t 4 25.9 0 il I 26.7 0 65 10 22.9 0 12 21.3 0 52 12.9 32.2 0 51 10.5 24.5 0 67 22 66 23.1 3 68 13.1 22.2 12 73 13.2 24 0 23 14.6 't0.1 24 12.4 17.9 0.6 77 14.3 20.8 0 æ 21.4 0 62 13.4 21.7 0.6 64 2s 10.9 17 .6 5 73 13.3 20 0 72 26 8.7 17 0.2 76 11.1 18.4 0.6 78 11 19.4 5 65 69 27 7.6 19.9 2 6'l 12.1 19.8 1 75 11.8 23 0 0 æ 28 9.1 17.9 0 63 10.8 24.1 0.2 68 9.6 28.9 67 10.3 21.2 0 68 9.9 32.6 0 61 oo 29 8.6 18.5 0 UJ 12 '19 0 72 11.8 19.2 0 67 12 21.9 0 57 30 62 31 12.2 19.5 0.2 78 9.6 21.1 0 5oo 'Weather Data Strathalbyn 2001-2002 YEAR 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 MONTH 1 1 1 1 222 2 333 3 Name and Unit Min Air Temp Max Aìr Temp Precipitation t,o Av. Relative Min Air Temp l\{ax Air Ternp Precipitation to Av. Relative Min Air Ternp Max Air Temp Precipitation to Av. Relative CC) CC) 9am (mm) Humidity(%) CC) CC) 9am (mm) Humidity(%) CC) fC) 9am (mm) Humidity (%) Mean 12.8 26.3 0.7 bJ 12.5 25.7 0.1 t!J.J 11.6 24.9 0.7 64.9 Max 39.8 14 86 39.2 1 78 35.8 20 82 Min 6.9 0 29 5.3 0 31 6.9 0 33 Total 20.4 2.2 23.2 No. ofObs 31 30 31 3l 28 28 28 28 31 31 31 31 t 13.4 20.9 0.8 71 13.5 26 0 73 12.7 19.2 2 75 2 11 20.6 1 æ 15.8 19.1 0 71 12.6 21.7 0 73 3 11.4 21.7 0.2 u 12.7 19.7 0.4 59 12.3 20j 0 79 4 11.2 22.9 0 65 11.4 21.3 0 62 8.8 27.7 0 74 s7.6340 53 I 23.3 0 71 10.5 33.9 0.2 59 6 '17.9 29.3 0 51 10.5 29.3 0 67 11.8 25.5 0 67 7 12.2 21 1 65 12 20.4 0 74 12 21.4 0 69 8 12.7 19.6 0.2 71 12.9 18.5 1 78 10.7 20.5 0 61 9 6.9 30.4 0.2 58 13.4 2't.6 0.4 76 6.9 27.6 0 66 l0 12.1 39.8 0 29 12.3 19.9 0.4 64 10.4 34.3 0 41 ll 16.7 28 0 u 5.3 21.6 0 65 14.1 22.4 0 62 t2 10.2 20j 0 59 7.5 29.5 0 64 8.6 21.2 0 66 13 13.9 21 .4 0 63 10.9 36 0 42 9.1 21.8 0 74 t4 14.1 23.4 0 67 14.6 39.2 0 30 8.6 22.2 0 75 r s 11.2 24.6 0 70 22 33.4 0 47 7.7 27.2 0 64 t6 '11.9 24.6 0 66 12.7 22.8 0 62 10.3 31.4 0 38 t7 12.7 23 0 68 9.7 24.9 0 6E 13.6 35.8 0 32 18 9.2 32.6 0 57 't2.2 29.6 0 bb '19.4 30.3 0 52 t9 14.2 39 0 36 't3.2 38.2 0 46 14.2 24.7 0 67 20 21.4 34.5 0 56 15.7 27.7 0 58 13.7 23 0 69 21 15.5 21.2 14 86 11.4 22.6 0 59 '11 22.5 0 74 2215222 76 13.6 21.2 0 66 10.7 27.8 0 63 23 9.3 24.3 0 69 14.9 23.6 0 73 7.2 32.8 0 43 24 11.7 0 63 14.3 30 0 71 13.5 35 0 44 2s 11.9 35.9 0 46 I1.9 34.7 0 61 11.8 2'1.9 0 72 26 15.3 33.8 0 48 12.5 23.3 0 71 12 18.9 20 77 27 19.4 25.3 0.6 73 13 21 0 63 14.1 19.'l 1 81 28 10.5 22.2 0.2 7'l 11.6 20.2 0 65 15 '18.3 0 82 29 14.9 23.4 0.2 78 14.6 19.2 0 75 30 '10.9 24.6 0 74 13 18.9 0 70 31 10.9 24.1 0 71 7.6 26.5 0 64 Weather Data Strathalbyn 2002-2003 YEAR 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 MONTH 10 10 ',to 10 11 11 11 11 1'2 12 12 12 Name and Unit Min Air Temp Max Air Temp Precipitation to Av. Relative Min Air Temp Max Air Ternp Precipitation to Av. Relative Min Air Ternp Max Air Temp Precipitation to Av. Relative CC) CC) 9am (mm) Humidiry(%) fC) CC) 9am (mm) Humidity(%) fC) CC) 9am (mm) Humidity(%) Mean 8.2 21 0.6 66.5 11.2 253 1 62.1 13.1 26.3 0.1 60 Max 32.9 I 94 37.7 21 97 38.1 1 72 Min 3.1 0 34 4.5 0 33 6.1 0 35 Total 17 28.6 2 31 31 31 3l No. of Obs 30 31 30 31 29 29 30 30 1 3.1 25 0 50 4.5 31.4 0 M 7.3 30.'t 0 51 2 9.5 28.4 0 34 18.3 26.6 0.4 52 17.1 30.7 0 44 3 13.2 18.4 0 75 10.6 20.7 0.2 65 8.8 21.3 0 61 4 9.2 ',12.8 4 94 9.1 18.4 0 62 12.2 18.4 0.2 66 r8.9 0.4 71 5 11.2 15.1 1 88 5.6 23.6 0 65 13.7 6 6.3 23.1 0 56 5.8 31 0 40 11.3 18.5 1 56 7 12.9 19.9 0.2 u 14 33.6 0 32 6.1 26.4 0 59 I 7.5 15.'l I 67 9.9 29.3 0 62 10.2 22.9 0 65 I 5.8 15.2 0.2 71 8.4 25.2 0 76 8.4 17.2 0.4 61 10 4 17.3 0 75 13.2 24 0 71 10 18.7 0 57 11 3.4 23.5 0 70 9.2 37.5 0 40 13.1 20 0 65 12 6.9 27.2 0 56 20.5 32.7 0.4 56 13.2 18.9 0 65 13 9.1 17.5 0 65 8.1 19.1 0.2 55 13.3 21.1 0 61 14 9.4 19.1 0 74 6.3 20.5 0 67 7.3 32.5 0 il 15 6.9 25.2 0 u 12.8 23.6 0 6'l 13.7 36.3 0 42 16 8.5 22.4 0 66 7.6 0 34 14.6 33.5 0 58 17 13.'t 203 0 69 17.7 36.8 0 44 17.3 34.1 0 64 18 7.6 31.2 0 62 21.3 0 75 16 28.9 0 72 19 12.7 19.8 0 67 12.3 20.4 0 76 16.6 34.2 0 59 20 7.8 19.2 0 74 13.4 18.9 0.2 78 14.6 29.9 0 67 21 5.6 22 0 67 4.5 26.2 0.2 u 12.6 36.5 0 56 22 28.6 0 59 6.1 25 0 64 16.'1 21.2 0 bb 23 9.3 17.3 71 11.8 37 .7 0 40 11.6 19.9 0 59 24 9.4 18.4 0.4 71 19.6 26.3 0.4 78 12.3 21.1 0 67 25 6.4 18 3 81 14.8 21.5 5 96 13.3 22.6 0 68 26 8.4 16.9 0.2 60 12.2 '17.6 21 79 10.7 25 0 72 27 6.2 18.5 0 73 12 18.4 0.6 79 11.8 26.3 0 65 28 5.4 25.8 0 60 14.2 25.4 0 77 't2.2 38.1 0 35 0 67 25.7 35.4 0 45 oo 29 6.5 32.9 0 42 11.3 22 (J¡ 30 14 19.9 0 71 10.9 19.7 0 63 17.5 33.8 0 64 31 8.1 18.5 0 67 16.5 21.6 0 66 oo o\

\Meather Data Strathalbyn 2002-2003 YEAR 2003 2003 2003 2003 2003 2003 2003 2003 2003 2003 2003 2003 MONTH 1 1 1 1 222 2 333 3 Name and Unit Min Air Temp Max Air Temp Precipitation to Av. Relative Min Air Temp Max Air Temp Precipiøtion to Av. Relative Min Air Ternp Max Air Temp Precipitation to Av. Relative ("C) ("C) 9am (mm) Humidity (%) ("C) fC) 9am (mm) Humidity(%) fC) CC) 9am (mm) Humidity(%) Mean 14.5 29.4 0.4 55.8 14 25 1.6 72.7 11 23.2 0.4 71.6 Max 43.3 I 81 41.3 26 94 36.8 7 83 o Min 0 26 9.6 0 36 7.4 0 33 Total 13 45.8 13.2 No. 31 ofobs 31 30 31 28 28 28 28 31 3'1 31 3l I 12 19.9 0.2 59 12.3 22.3 0 73 10.1 18.2 0.4 66 2 12.7 23.4 o.2 66 10.8 24.3 0 69 9.3 19.5 0.2 71 3 12.2 23.7 0 71 11.9 35.2 0 43 1 1.8 19.1 0 76 4 10.2 23.6 0 66 17.6 41.3 0 36 11.1 19.7 0 75 't0.5 5 34.8 0 54 19 22 0.2 77 8.4 23.2 0 77 6 15.2 38 0 31 14.7 24.8 0 70 9.2 28.1 0 58 7 18.3 æ.2 0 51 14.6 22.1 0 77 11.4 29.2 0 58 8 12.8 21 0 g 13.3 23.4 0 71 12.4 20.7 0 67 9 14.1 22.8 0 56 9.6 24.3 0 71 7.5 21.6 0 68 l0 10.8 26.2 0 67 11.4 22.6 0 69 8.2 23.5 0 81 ll 12.6 35.1 0 47 11.4 28.7 0 68 '12.2 25.7 0 83 l2 14.8 39.2 0 26 11.2 31.7 0 61 12.1 33.9 0 58 l3 ¿o 32.3 0 42 12.7 26.5 0 73 12.9 23.9 0 75 t4 13.8 21.3 0 58 10.9 30.2 0 62 14.2 20 0 68 l5 12.3 22.8 0 65 13.9 21.3 0 72 11.7 19 0 8l t6 12.2 29.9 0 65 15.7 22.3 12 88 8.1 24.8 0.2 73 t7 13.7 38.3 0 45 13.6 23.5 0 71 10.7 34.3 0 45 t8 16.5 25.6 0 71 12.7 24.4 0 60 16 36.8 0 33 t9 13.2 40.4 0 40 15 27.9 0 55 13.5 23.6 7 75 20 23.3 34.3 3 59 16 21.3 26 94 11.9 19.4 4 82 2t 14.4 23 0.2 70 17.3 24 5 89 12 17.4 1 74 22 14.1 24.2 0 71 16.9 22.3 0.4 92 11.8 18.1 0.2 82 o 23 29.5 0 49 16.1 25.9 0.2 u 11.1 19 0 69 24 14.4 40.4 0 29 16.4 26.3 0 86 7.4 25.5 0.2 72 25 20.4 43.3 31 16.2 19 1 88 8.1 23.4 0 76 26 18 21.5 0 81 14.2 22.8 0 u 13.3 23.5 0 78 27 13.5 24.5 0.2 65 14.5 21.9 0 77 12.8 22.6 0 77 28 13.3 31.6 0 55 13.4 18.5 1 75 12.3 20.9 0 82 29 r 6.9 38.7 0 50 9.9 21.3 0 83 ô 30 14.6 23.6 70 11.6 20.5 0 76 3l 12.6 21.5 0.2 61 7.5 22.6 0 79 b) Glenara Wines Pty Ltd

Averages for LENSTilOOD RESEARCH CENTRE

2001 O238OI LENSWOOD RESEÄRCH CE\IIIRE Conrnenced: 1967 Last record: Latitude: -34.949'7 S Longitude: 138.8058 E Elevation: 452-0 m State: SA MAR APR MAY iII'N JI'L ÀI'G SEP OCT !K'V DEC À¡¡N No. %age JÀII FEB Yrs cwt llean Daily Mâx T4t (dleg c) 23 18.3 96 25.2 25 -7 22.9 18.9 ]-4.8 12 -7 Lt -4 t2.5 ]-4.7 I'7.8 20.8 -3 30.8 lfean no. Days, Ètax >= ¿0.0 deg C 0.L 9'l 0.1 0.0 0.0 0 .0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 30.9 lleån no. Days, lfarß >= 35.0 deg C 2-O 1.8 0.5 0.0 0-0 0.0 0.0 0.0 0.0 0.0 0.2 0.5 4.9 30.9 9't DIear¡ no. DayE, Ma¡ß >= 30-0 deg C 7.5 8.0 3.8 0.3 0-0 0.0 0.0 0.0 0.0 0.4 2.3 5.0 27 .4 30.9 97 EighêEt lita¡ß TsrE (deg C) 4L-3 39.6 39.3 33.9 30.5 20.6 23.9 29.9 33.9 38.4 11 a 41.3 32.1 r00 ![ean Daily uin Tsq, (Cleg C) 1) -L-L .b 9.4 9'7 12.9 13 .5 12 -3 10 .6 8.6 6.7 6.0 6.3 8.4 10.0 30.9 ueen no. Day6, Dfin =< 2.0 deg C 0.0 0.0 0.0 0.0 0.1 0.5 0.8 0.7 0.4 o.2 0.0 0.0 z.ó 30.9 91 Mean no. Days, Min =< 0.0 deg C 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.2 30.9 9'l r.ovreEÈ uin Tqr (dteg c) 4.2 5.8 4.4 2.3 1.5 -l-.8 0.6 -0.1_ 0.'l 2.4 5.1 -1.8 32 .1 100

¡[eån 9a¡ß Air TenE) (dleg c) oo 1b. b 13 .1 30.8 96 t7 .6 77 .9 16.5 l-4.3 LT.2 8.9 10.8 L2.9 t4.5 uean 9arn wet-trulb TgrE (deg C) oo 1) '7 10.3 30.8 qÁ 13 .6 13 .7 l-3 .0 11.3 a^ /.o 6.8 8.4 11.1 Dlean garr Dew Poínt Tdtr) (deg c) 10.0 10.1 9.8 8.4 6.1 5.1 5.0 5.6 6.5 /.b 8.9 7.5 30.8 96 garn Hur¡Édlity (%) Dfean Relêtive '72 65 65 68 7r 79 83 óz 79 t3 68 6t 64 30.8 96 llean 9arl wind speed (lon/hr) 7L.4 9.6 10.3 r0.7 10.8 13.0 14 -3 1¿ aì 13 .9 74 -7 12.3 11.5 1-2.2 29 .8 96 oo\ì

!trean Rainfatl (ftr) 100 33.0 28 -3 43.6 76.6 115.1 I32.8 160.0 148.8 779.4 82-7 45.0 43.4 1'028-6 33.6 oo oo Median (Decíle 5) Rainfall (¡run) _ 24.4 L4-9 33-1 67,6 104.8 130.3 161.0 l Aa ) 70'7 .4 ?tr o 40.3 3 /.5 t-018.0 33 Decilê 9 Rainfall (mn) _ 87 .2 '73 .2 90 . 9 I49 .'t 244 .8 205. B ))1 0 )41 a 202.3 1¿q a 90.6 ÔE t 1303.4 33 Decile 1 Rainfall (m¡r) _ 7.0 1,.4 2.9 10.6 34.9 46 .4 64.3 61.5 55.6 22.1 9.1 13.0 '7 68 .4 33 llean Bo. of Raindays 6.9 5.1 8.1 72.4 16.5 71 .6 10 a 10 0 ),6-2 13.8 10.3 ao 156.0 33 .6 100 HigheEt Monthly Rainfall (rrrr) 1-03.2 7'74.8 L27 .1 290.2 268.4 343.0 356.6 311.0 aoa ) r91.6 134. 0 164.8 33.6 100 LordesÈ Monthly Rainfall (Ím) 3.6 0 .2 0.2 3.2 29 .6 41_.6 5r_¿ aa. ) 41.I 7.6 3.0 7.8 33.6 l-00 Eighest Recorded Daily Reín (ÍEn) 50.8 723.2 79-A 96.5 '7'7.4 tó-./, 61 -4 104.0 /b. ð 54.6 4A .6 59 .4 723.2 33 .0 98 Mean Daily Sunshine (h¡s) 9.8 9.4 7.8 6.3 4.1 20 4-r 5_1 6.1 7.6 9.2 6.9 28.8 99 Mean Daily EvaDoration (Íün) 6.3 6.0 4 -4 2.'t 1.6 1.1 1.1 r.6 2.5 ). t 4.8 5-7 3.5 30.8 99

I¿st modìfied 28May 2001 Weather Data Mt Barker 2000-2001 YEAR 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 12 t2 MONTH l0 l0 l0 10 t1 1l 11 il 12 12 Max Ternp Precipitation to Av. Relative Name and Unit Min Air Temp Max Air Ternp Precipitation to Av. Relative Min Air Temp Max Air Ternp hecipitation to Av. Relative Min Air Ternp Air fC) Cc) 9am (mm) Humidity (%) ('C) fC) 9am (mm) Humidiry (%) ('C) ("C) 9am (mm) Humidity(%) 0.6 70.s 0.3 60.2 Mean 7 .5 19.5 3.1 70.2 t2.l 25.2 tt.2 27.2 76 Max 29.5 35.4 93 36.6 6 96 38.1 5.2 Min 0.9 0 26 6 0 38 5.8 0 39 10.2 Total 97 '2 18.8 3l 31 No. ofObs 31 31 3l 31 30 30 30 30 31 31 I 6.6 15 7.6 68 l2 18.8 0.6 79 8.4 223 0 @ 2s13.70 85 I 1.5 19.9 5 85 7.5 29.5 0 50 3 2.6 zl.t 0 77 9.4 20.1 0 75 15 28.8 0 59 62 4 6.6 25.9 0 5l 8.6 2l 0 79 10.4 32.2 0 0 ól 5 l0 .3 29.5 0 26 8.4 l9 0 72 lt.6 28 6 ll.l 24.5 0 74 6 22.3 0 69 9 24.3 0 59 62 7 t2 17 8.8 76 8.6 27.4 0 46 l1.9 28.3 0 62 I 5.2 17.2 0 72 18.7 23.5 0.2 48 10.5 22.4 0 9 1.8 16.6 0 74 12.3 21.3 6 87 9.8 30.4 0 59 l0 7.4 18.2 0 76 I 1.6 r 8-9 0.2 94 10.6 33.4 0 39 0 55 l l 0.9 20.9 0 63 l, s 15.5 t.4 96 16 32.8 12 8.1 22.2 0 40 12.6 18.6 I 9l 11.6 27.4 0 68 13 8.4 14.8 4 68 l3 23.t 0.2 84 9.5 24.3 0 69 t4 5.1 14.7 2.4 80 I 1.6 26.7 0 80 8.5 23.3 0 57 15 9,6 16.2 8.2 82 13.8 28.5 0 67 8.r 28J 0 59 16 4.5 20.2 0 73 12.7 28.5 0 72 9 34,7 0 54 t'7 11 20.5 0 52 13.5 30.1 0 7l 15.7 29 0 39 2 73 18 I 1.9 17 .9 35.4 93 t4.6 34.6 0 60 14.6 29.4 t9 7.9 16.7 24.8 74 15.9 32.8 0 68 9.8 33.5 0 47 20 8.5 18.5 1.4 84 t4 19.I 4.2 82 r2.3 38.1 0 56 't) 21 7.2 25.4 0 68 12.3 23 0 19 32.3 0 52 22 l0 24.6 0 55 t0.2 25.4 0 68 16. r 2l,l 5.2 74 23 13 21.6 0 79 ll 26.4 0 69 t0.2 22.2 1.6 67 1.2 76 I 1.9 t7.6 0.2 89 I t.4 30 0 7t 14.3 22.4 24 1) 25 10.3 l7.4 3.6 89 9.5 34 0 54 9.s 22.3 0 26 4.5 15.9 0.2 8l t4.7 36. l 0 49 I r.4 20.9 0 69 27 7.6 16.8 0 80 l6 36.6 0 38 l0.l t9.2 0.2 60 28 4.7 t7.s 0.6 60 19.8 27 0 50 7.6 20.1 0 64 68 9.9 25.t 0 67 5.8 24.4 0 59 æ 29 3.6 20.5 0 \o 30 4.9 2r.9 0 66 7.5 22.6 0 70 9290 63 31 10.7 24.1 0 52 13.5 28 0 56 \o

Weather Data Mt Barker 2000-2001 YEAR 2001 2001 2001 2001 200t 2001 2001 200t 2001 2001 200t 200t MONTHIIII2 222 3 333 Name and unit Name and Min Air Temp Max Air precipitation unit Temp to Av. Relative Min Air Ternp Max Air Temp Precipitation to Av. Relative l\{in Air Ternp Max Air Temp Precipitation to ('c) 9am (mm) (%) cc) Humidity ('c) cc) 9am (mm) Humidity(%) fc) fc) 9am (mm) Mean 14.9 3 1.9 0.2 56 t5-2 29.6 0.4 6l .8 10.5 24.2 I.9 67.8 Max 41.8 3 78 38.5 6.8 90 35.2 2t.4 87 Min 7.6 0 30 9.1 0 2t 4.6 0 34 Total 5.4 I r.8 s8.8 No. ofObs 3t 3t 3l 3l 28 28 28 28 3l 3l 3l 31 I t3 37 0 4t 9.t 35.3 0 4l 9.2 30.7 0 55 ) 25 35.3 0 36 23.7 38 0 )1 10.5 25.5 0 7l 3 10.3 33 l 0 64 19.5 30.8 0 6l r0.9 29.5 0.2 78 4 18.9 35.2 0 30 15.4 19.6 0 90 13. I 24.5 0 70 5 t4.5 24.7 0 65 t3.7 28 0.2 67 12.5 30.6 0 72 6 25.6 il.3 0 67 16.5 35.7 0 60 t4.2 35 0 46 7 t4.5 26 0 73 l9;7 37.7 0 6l 15.3 35.2 0 39 8 t4.3 28.7 0 72 23.4 38.2 2 40 t4 345 0 50 9 13.9 35.7 0 59 t7.3 20.6 0.2 85 13.7 35. I 0 44 t0 16.2 38. I 0 42 t4.l 25.7 0.8 69 20.2 22.2 0 65 t4.9 ,-0.4 ll 32.7 0 47 1.2 33. r 0 69 2r.8 0 66 t2 t2 32.t 0 66 t 5.5 29.8 0 7t 9.8 20.5 0 66 l3 41.8 l6 0 42 l5 24.1 6.8 78 6.1 22 0.6 62 t4 22.6 39.7 0 32 t3.7 21.9 0 76 5.9 26.6 0 66 l5 17.8 22.9 0 69 Ú.2 22.5 0.4 72 9 3l 0 34 l6 28.5 lt5 0 56 9.3 26.3 0 68 t7.l 22 1.8 82 r'7 I 1.6 26.5 0 65 t0.7 32.6 0 57 9.4 18.8 t7.6 70 l8 8.9 28 0 6l r6.9 38.5 0 28 9.6 2l.4 0 82 l9 t2.3 36.7 0 58 25.9 38.1 0 2t r0.7 25.4 0 78 20 18.4 35. I 0 34 16.6 37.5 0 33 8.8 24.4 0 64 2t r 5.l 30.9 0 66 19.3 24.9 0 53 7.4 13.7 21.4 87 22 165 39.9 0 47 t2.6 26.3 0 6l r0.5 l9 3.2 85 71 20.7 40.9 0 35 9.9 28.5 0 77 7.3 23 0 78 24 22.4 37.8 0 38 13.3 24.6 0 76 t t.7 t7.9 3.4 82 25 20.4 35.6 3 69 t4.3 27.9 0.6 10.5 t7.6 0.6 80 26 18.6 26.5 2.4 78 t4.7 21.5 0.8 70 u.5 18.4 9.2 84 27 t0.4 24.4 0 72 1 3.5 28 0 70 10.7 l8 0.4 7l 28 7.6 o 26 65 r0.9 26.2 0 7l r0.l t9.2 0.4 69 29 10.7 tl 68 4.6 18.ó 0 7t 30 I 1.9 25.2 0 64 4.8 23 0 70 3t 9.1 29-9 0 57 5.4 25.9 0 ó8 Weather Data Mt Barker 2001'2002 2001 2001 YEAR 2001 200t 2001 200t 2001 2001 2001 2001 2001 2001 MONTH l0 10 l0 l0 11 ll ll ll 12 12 t2 t2 Precipitation to Av. Relative Min Air Ternp Max Air TenP Precipitation to Av. Relative Name and Unit Min Air Temp Max Air Temp Precipitation to Av. Relative Min Air Ternp Max Air Ternp 9am (mm) Humidity(%) CC) CC) 9am (mm) Humidity (%) ('C) ('C) 9am (mm) Humidity(%) CC) CC) I .7 2t.9 I 70.9 Mean 7.5 ló.9 3'l 75.2 9.4 21.2 72.8 9.7 7 95 23.4 10.4 9l 29.9 13 94 31.ó Max 38 Min 2] 0 40 4.1 0 45 4.2 30.8 Toral 97.6 51.4 30 31 31 3l 3l No. 31 31 3l 3l 30 30 30 ofObs 0 69 l0 15.2 6-2 87 9.8 23.5 0 78 4.2 22.6 I 7 63 15 2.2 78 10.3 19.5 0 70 12.6 26.7 2 7.3 95 3 8 16.3 l0 9l 7.9 23.8 0 7l tl.'l t3.7 6.4 0 10.2 21.4 4.4 84 4 6.3 15. I 5.6 83 12.6 28.1 61 23.s 0 69 5 2.9 l7 0 8l 16 l9.l r.2 94 7.4 23 s 8ó 6 8 11.4 6 82 10.4 17.4 l0 82 13.6 16'4 t.4 89 7 4.6 15 5.4 85 5.7 17.8 0 68 10.4 0 18,6 2-2 70 8 7 16.5 3.ó 85 8.5 22.5 64 7.4 20 0 69 9 2.7 20.5 0.2 6'l 7.2 18.3 0 74 6.5 0.2 17.5 0.2 7t l0 9.8 20.1 0 46 8.4 16 71 8.4 Ló l0 16.7 0.4 82 I I 6.3 t4.6 7 -2 74 4.1 15.5 8l 2 22 0 68 12 8.7 13.9 2.6 80 ó.5 15.6 90 9.8 1.6 23.8 0 70 13 6.8 16.2 2.6 85 9.3 t9.2 8l 10.5 20 0 78 t4 4.3 15.8 4 7'7 6.5 23.5 0 67 10.3 0 66 15 3.8 19.6 0 68 6.4 25.9 0 67 l0.l 22.2 0 20.4 0 73 16 7.5 22 0 80 I 29.9 65 10.7 0 22.9 0 65 t7 8.9 16.8 0.8 83 13 28.5 45 7.5 0 6l 18 9.1 14.8 7.8 80 l0 l5.l 8 8s 8.6 26.2 0 52 19 5.5 16 0.8 72 6.5 18.3 0 64 8.ó 31.6 0 't8 20 3 18.2 0 62 8.6 24.1 0 72 t3.7 22.1 22.7 0 73 21 5.3 23.4 0 40 10.5 26.2 0 69 9.4 0 7l 22 15.5 19.5 0 45 t2.3 28.7 0 49 8.6 25.4 13 22.5 0 74 23 l3.5 21.5 3.ó 73 ll.6 19.9 80 I2:l 0 59 l0.l 15.2 5.2 90 12.2 18.9 5.4 74 11.6 19.4 24 75 25 9 15.8 l0'4 79 11.5 17.2 1.2 8l ll 18.3 3 4.4 7.8 19.6 0.8 70 26 8.5 14.5 7 -2 89 10.5 l8.l 84 0 7l 27 4 l7.8 4.4 '71 9.3 18.4 2.2 83 9.3 20.9 0 63 28 8.8 16 0 75 8.3 23.4 0 66 9 27.'1 0 38 \o 29 5.6 17.2 0 77 8.5 23.5 0 75 9.8 30.9 0.ó 9.2 20.5 0 66 30 10.5 I ó.s 0 75 l0.l 18.8 75 0 8l 31 10.6 17.5 l'8 73 9.7 l9.l \o ì\) Weather Data Mt Barker 200l-2002 YEAR 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 MONTH I I I I 222 2 3 3 3 3 Name and Unit Min Air Temp Max Air Temp Precipitarion to Av. Relative Min Air Temp Miax Air Temp hecipitation to Av. Relative Min Air Ternp Max Air Temp Precipitation to Av. Reìative (mm) ("C) ('C) 9am Humidity (%) ("C) fC) 9am (mm) Humidity(%) ('c) fc) 9am (mm) Humidity (%o) Mean I 1.4 25.7 Cr.9 66.3 r0.4 25.6 0.2 67 t0.4 25.2 0.5 68.9 Max 37.1 12.2 89 36.5 3 88 347 6 ot

Min 5.4 0 3l 5.5 0 33 6 0 _12 Total 2E.8 6.4 t4.4 No- ofObs 3l 3t 3l 3l 28 28 28 28 3l 30 3l 3l I n.2 t7.2 2 78 9.2 3l .l 0.2 79 10.9 20-9 0-4 i7 2 8.6 t7.9 8.2 82 l3.l 17.6 0 8l 9.7 24 0 i4 3 7.2 20.2 0.4 70 8.8 20.1 0.8 64 l 1.8 20.4 0.8 85 4 7.5 24.1 0 69 8.4 22 0 66 I 1.9 28.4 0 i3 5 7.5 30.4 0 58 7.9 25.7 0 73 9.2 - 32.3 0 66 6 l ó.5 23.4 0 52 8.3 28.4 0 66 il.5 30 0 58 7 I 1.8 17.9 3.2 73 9.8 2t.6 0 77 n.2 23 0 76 8 ll 2t 0 74 l0 t7 4.2 88 7.t 2t.2 0 70 9 5.4 29 0 63 t0.7 21.5 3 82 6.2 28.1 0 7t l0 ll5 37.1 n 3l 9.8 18.l 2 80 8.6 3l .9 0 5l lt ts.2 22.2 0 6l 5.5 22.4 0 68 l4.t )2 0 72 t2 8.5 2t.l 0 66 7.t 29 0 66 6 23.2 0 7l l3 10.9 ))a 0 t5 9.3 33.8 0 52 9.9 22.9 0 75 t4 t2.t 25.5 0 65 13.3 36.5 0 33 9.6 23.7 0 76 l5 10.4 27 0 6'7 t9 28.1 0 39 7.1 27.9 0 72 16 t0.5 27.2 0 63 8.6 23.9 0 67 ll.l 29.9 0 42 t7 t 1.5 25.7 0 66 7.2 27.1 n 74 t2 34.7 0 4t r8 8.3 32.6 0 59 lt 30.5 0 66 18.2 31. I 0 32 t9 t4 36.9 0 34 t2.7 35.2 0 45 l3.l 24 0 7l 20 20.6 32.6 0 49 13.8 2t.3 0 65 9.6 )2 n 80 2l t4.5 18.8 t2.2 89 9.4 2t.2 0 60 8.5 24.4 0 78 22 t37 22.8 1.4 83 10.5 22.1 0 70 10.9 26.7 0 70 23 6,8 24.9 0 68 12.5 26.5 0-2 77 '7 3l.l 0 50 24 t0.2 29.7 0 67 t2.2 3l .5 0 66 I 1.3 33.3 0 4{t 25 l0;l 34.1 0 58 10.5 33.4 0 63 t2.6 20.6 0 n 26 t4.5 32.7 0 58 10.3 26.9 0 70 9.5 18.5 6 83 27 l8 22.8 0.6 88 l 1.9 21.8 tt 66 I 1.5 19.3 3.8 85 28 l0 3 2l .8 0.2 7t t0.2 22.r 0 72 t2.s 17.8 r.8 92 29 t3.3 24.4 0.2 78 t2 l9.s 0.6 u 30 t r.8 27.8 0.4 75 l0 20.2 0.8 7l 3l 10,6 26.5 0 68 7.3 0.2 65 Weather Data Mt Barker 2002-2003 YEAR 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 MONTH 10 10 10 10 1'l 11 11 11 12 12 12 12 Ternp Precipitation to Av. Relative Name and Unit Min Air Temp Max Air Temp Precipitation to Av. Relative Min Air Temp Max Air Ternp Precipitation to Av. Relative Min Air Ternp Max Air (%) CC) ('C) 9am (mm) Humidity(%) CC) CC) 9am (mm) Humidity(%) ec) CC) 9am (mm) Humidity 67.7 '11.1 26.8 0.7 63.3 Mean 7 .1 19.5 1 .'l 72.5 9.3 25 1.7 Max 29.4 8'2 97 35 35.4 99 40 7.6 85 Min 1.7 0 25 1.8 0 23 5.1 0 43 Total 33.2 51.8 22 31 31 3l No. of Obs 31 31 31 31 30 30 30 30 31 I 3.9 22.7 0 38 5.5 28.4 0 62 5.7 28.3 0.2 64 2 10.1 24.9 0 25 14.9 24 0.4 47 17.1 24.6 0 58 1 5.8 20.2 0 71 3 10 12.5 1 95 9.6 '18.4 74 4 7 12 7.2 97 5.6 17.5 0.6 75 9.6 l6 0.4 69 5 9.2 14 1.6 92 1.8 22.4 0 71 10.6 17 7.4 85 6 7.3 20.5 0 66 3.4 29.5 0 53 8.3 '17.8 5.6 69 7 10.3 17.6 0 u 12.7 30.5 0 23 5.1 24 0 66 I 5.6 13 8.2 73 8.3 27.4 0 59 8.1 24.1 0 62 I 1.7 14 0.6 u 6.2 29.3 0 60 5.6 16.4 0.6 82 10 1.9 18.1 0 74 11 26.7 0 71 7.2 18.8 0.2 71 11 3.1 22.3 0 71 8.7 34.9 0 57 10.4 22 0 71 12 6.1 24.4 0 49 18.8 '19.4 2 85 11 22.2 0 69 13 8.4 14 0.4 82 5.'l 21.1 3.4 68 9.4 23.7 0 68 60 14 8.3 16.6 2.4 91 3.7 21.9 0 77 6.1 31.5 0 15 7.5 23 0.6 72 9.1 25.2 0.2 67 11.6 36.6 0 43 't6 11.5 23.2 0 45 6 31.4 0 56 13.9 38.8 0 44 17 10.6 23.9 0.2 81 15.1 34.4 0 30 19.3 40 0 48 18 7.7 28.5 0 57 11.2 25.5 0 70 14.1 32.6 0 66 19 10 18.4 0 78 I 25.4 0 77 14.5 37.6 0 57 20 5.4 19.5 0 79 11.4 17 .6 0.6 83 13.7 34.6 0 48 21 5.6 23 0 75 5.2 27 0.4 65 12.7 38.5 0 45 22 7.2 25.4 0 61 5.7 27 0 67 14 22.2 0 74 23 6.6 14.4 3.8 86 9.7 35 0 43 5.8 21.4 0 62 89 9.9 22.5 0 65 24 7.8 17.4 1 82 17.8 25 3 25 5.6 14.4 5.4 92 13.6 22.2 4.6 99 11 25.1 0 64 26 6.3 16.9 0.2 73 10.8 '16.7 35.4 90 10.9 27.6 0 67 27 5 19.3 0 83 10.4 17.2 0.2 82 9 29.8 0 67 28 6.4 23.6 0 73 11.6 25.7 0 79 10.2 36.5 0 47 29 7.4 29.4 0 39 9.8 22.9 0 82 23.2 32.6 0 45 (,\o 30 12.'l 18.1 0.6 80 8.4 2'1.3 0 71 17.6 31.2 0 74 31 5.2 20.7 0 72 13.5 17.6 7.6 u \o 5

Data Mt Barker 2002-2003 2003 2003 Weather 2003 2003 2OO3 2003 2003 2003 2003 2003 3 YEAR 2003 2003 2 1 333 1 1 222 Precipitation to Av. Relative MONTH 1 to Av. Relative Min Air Ternp Max Air Temp to Av. Relative Min Air Temp Max Air Ternp Precipitation Name and Unit Min Air Temp Max Air Ternp hecipitation 9am (mm) Humidig (%) 9am (mm) Humidity(%) CC) ("C) 9am (rnm) Humidity (%) fC) CC) 74.8 CC) fC) 75.9 23.6 0.5 59.6 12.3 27 1.7 9.2 Mean 12.5 29.8 0.6 6 94 39.1 25.8 98 34.7 42.7 9.4 83 32 Max 44 0 31 7.6 0 4.2 Min 8.7 0 17 46.2 18.8 31 Total 28 31 31 31 28 28 28 31 No. ofObs 31 31 31 1.8 78 23.6 0 80 8 18.2 o4 17.5 7.2 74 9.7 80 I 73 '17 o 75 9 27.7 0 6'1 '1 2 8.9 24.4 0.2 0 75 36.9 0 59 9.5 21 24.6 0 78 7.6 77 3 11.9 44 22.5 0 63 16.6 39.1 0 9.2 4 8.7 26.1 0 0 78 23.5 1-4 82 8.4 25.1 33.6 0 58 16.6 69 5 11.3 67 28J 0.2 41 13 29.5 0 6.7 6 12.6 36.4 0 0 59 24.7 0 76 8.2 30.2 36.1 0 39 12.1 76 7 15.3 71 21.7 0 62 11.4 26 0 11.5 8 9.8 22.5 0 0 75 8.1 29.5 0 74 6.9 23 9 10.1 24.4 0 æ 0.2 82 10 25.4 0.2 74 7.5 24.9 10.1 28.2 0 70 0.2 82 l0 oo 0 71 10.5 27.9 0 58 3l 10.2 u 31 0.2 72 ll '10 29.5 0 60 11.1 .9 l2 13.1 36.8 0 38 0 77 10 29.7 0 69 10.9 271 26.4 29.7 0 46 0 66 l3 0 65 11.3 2',1.7 0 69 9.8 33.7 t4 10.2 22.5 17 0 83 12.9 29 0 73 11.2 .6 l5 10.5 24.5 0 60 0.2 76 20.3 6.2 97 8.2 27.7 0.2 68 14.6 48 l6 10.9 31 77 33.1 0 45 12.1 25.4 1.6 10 t7 12.2 36.6 0 0 32 25.5 0 62 14.2 34.7 30.8 0 72 11.1 82 l8 12.3 65 17 5.8 50 13.4 24 0 11.7 l9 13.1 40 0 6 78 15.8 20 25,,8 98 7.9 18 2',1.9 36.2 0.6 54 1.2 83 20 23.8 9.2 s3 g-4 16.1 25 0 66 16.1 94 2l 13.1 95 17 0.4 67 15.6 20.6 0.ô 8.9 22 11.6 27.5 0 0.2 78 14.4 29.5 0 88 9.8 19.1 32 0 59 0.2 80 23 9.4 0.2 86 4.2 24.9 0 42 15.1 31.1 24 11.7 40 0 68 14.4 24 0.6 88 6.8 25.7 42.7 0 31 0 76 25 16.7 0 82 11.1 25.5 0 79 12.1 28 26 16.1 22.8 0 79 't 1.8 26.5 0 81 10.9 24.5 26.9 0.6 69 0 79 27 11.2 o"4 71 12.3 20.8 0 61 11.6 18.6 83 28 11.1 31.6 8.4 22.5 0.2 0 31 74 29 16.8 38 8.9 22.3 0 9.4 B3 78 30 l3 20.2 6.8 23 0.2 0.6 73 31 9.1 21.5 c) Mountadam Vineyards Pty Ltd

Averages for MOUNT CRAWFORD FOREST HEADQUATERS

023763 MOUNT CRAV/FORD FOREST HEADQUATERS Commenced: 1954 Lastrecord:2001 Latitude:-34.7139 S Longitude: 138.9453 E Elevat ion: 395.0 m State: SA

üå¡¡ FEB MÀR àPR MÀY iII'N i'I'L ÀuG SEP Oel rfov DEc À!¡\I No. %agê Yra cq,

![ean Daily Uarß Teqt (dlegf C) 21 -1- 21 .5 24.3 20 -7 16 . 0 13.0 I¿.¿ L3 .1 15.5 18.9 27.9 24 -9 19.8 26.1 94 MeaD no. Days' lilatß >= 40.0 deg C 0.6 0.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 1.U 1< O OÃ Mean no. Days, Dtax >= 35.0 deg C 3.8 3.4 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.3 I.7 1-0.2 26 -9 95 D¡ean no. Days, Ma:< >= 30.0 deg C 9.8 70.4 5.5 1.1 0.0 0.0 0.0 0.0 0.0 0.5 3.0 1.L 21 F 26.9 95 Eighest Max Tû8, (dfeg c) 20 n 42.5 42.5 40.6 35 . 0 26 .5 22.0 23.5 25.0 29.3 33 .0 40.0 42.5 28.3 r-00 uean Daily r[in Teqr (¿leg c) '1 1 It.2 LL.2 9 -7 7 .0 4.9 3.6 3.1 3.4 4.3 6.1 9.5 6.9 26 .6 94 ![ean no. DayE, Min =< 2.0 deg C qq zó- I 94 0 .1 0 .1 1.0 4.7 8.3 r-0.3 11.0 8.1 5.2 2 -1- 0.8 67.1 [!ean no. Days, Min =< 0.0 d€g c 1^ 94 0.0 0 .0 0.1 1.1 3.6 5.6 6.6 5.5 4.3 0.6 0.0 28.8 26.1 LoleêEt Min Tqt (deg c) 2.O 1.0 -2.2 -3.5 -5.0 -7.0 -'1 .0 -4 .0 2q -2.0 0.1_ -7.0 28.3 99

Èlean 9a¡n Air TerE) (dlegr C) r-8.9 18.6 I't .2 1,4.1 10.6 't1 a) 8.6 l-l-.1 L3.'t 75 .4 L1 .1 13.7 26 -6 94 ltean 9a¡n wet-bu1b Ts¡qr (deg c) 1a 14.2 1,4.0 13 .5 11 . 8 7.7 6.3 9.0 10.5 71 .'7 13 .1 r0.7 2r.5 /o Mean 9a¡r Derù Point Tq, (deg C) 10.0 10.0 10.0 9.0 /.b 5.9 5.0 ç1 6.6 7.r 8.0 9.0 ?q 2L.5 lo gaI¡r Relatiwe nu¡nidity (%) ![êan 1) 59 67 66 72 84 88 88 85 '76 64 60 2\.5 76

\o ![ean 9aÍi wind Speed (lol/hr) (,r 23 .0 22 -3 20.8 79 .7 t-9 .3 18.8 20 .3 27.9 22.2 23 .r 22.2 22 .3 2L -4 25 -1 91'

ltean 3I[ß Àir Te¡q, (deg C) 25.8 26 -r 23.7 19.9 r-5.3 72.0 11.3 L2.7 14.2 't7.7 20.6 23.4 19-3 23-4 83 \o o\ Mean 3tn lifet-bulb Tetrqr (dleg C) ao Itr I 16.3 r-6.6 l-5.5 13.6 11-.5 9.4 8.8 10.5 7:2.2 13.8 13 .3 1/.b 62 Mean 3I[l Dew point TsrE) (deg C) 8.2 8.1 8.9 7 .7 1.(l 6.2 Ê.^ '72 7.9 t.a 19.0 6i M€a¡¡ 3pm Relatív€ llu¡nidity (%) 36 36 44 49 65 121 73 t0 61 55 45 41- 52 t] .9 64 Mean 3I!î Wind Spe€d (¡m/hr) 23 .2 22.3 21 -9 20 .1 20 .4 19.t| )) ¿. 24 -U a1 1 23 _4 a2 0 ))a ))a 81 Mean ttainfau (Ím) 24.5 23 .7 28.2 53 .3 88.9 o? cr LLs .4 103 .4 82.9 o).4 5ó. I 35 .4 755.7 41 .9 10c Median (Dscile 5) Rainfall (¡run) _ L9.2 l-1.8 21,.1 46.4 15.6 94.1) 107.5 l-01.6 15.1 55. s ?? o 28 -0 140 .2 47 D€cile 9 Råínfall (Íñr) _ 54.4 6'7.2 68.9 109.8 191.1 2\5 -4 1-81 .2 165.5 139.5 71:-.5 67 .6 64 .4 1035.0 47 Pecile 1 Rainfall (rnn) _ 3 .6 0.6 r.'7 '7 -2 27 .2 24.C 49 .6 39.8 40.9 1? .1 7r .4 1.6 481, -9 47 llean no. of Ral.ndays 10 a 5.7 4.8 6 -4 9 .9 L4.4 16. C 18.s 72.7 8.8 1.9 l-39.3 a1 a. 99 Highêst Uonthly Rainfall (Íû) 11 .2 733 .2 ]-02 .3 759 -'7 247 .4 248 .4 26L.5 208.0 rt¿t- t 702.2 16ó-¿ 4I.9 100 Lotrêst uonÈhly Rainfall. (rur) 0.3 0.0 0.0 2.5 17.0 14.2 23.5 22.4 4.2 0.3 2.3 47 .9 100 Eighêst Record€d DaíIy Raln (rllrn) 40 . 1 702 .9 65 .2 40 .1, sA.7 53 .3 45 .6 '77.4 35.6 54.5 40.1 49. 0 L02.9 3L.2 vb Iiteaa no. of Clear Daya ¿? 73.7 74.7 1.2.O 8.2 4.7 3.6 4.3 4.6 7.7 l-0.4 93. r_ 28.3 100 ÈIeaB Eo. of Cloudy Days q¿ s.9 5.1 6.3 7.6 9.6 10.8 10.9 a? 1.9 /.fi t-9 98.2 28.3 100 Ìtean Daily Evaporation (mn) 6.3 6.0 4.5 2-6 1.5 1.0 1.0 L.4 2.r ¿1 Rtr 3.3 24.L 98 t¿st modified 28 May 2001 2000 2000 2000 YEAR 2000 2000 2000 2000 2000 2000 2000 2000 2000 12 12 12 MONTH 10 10 10 10 11 11 11 11 12 Relative Min Air Temp Max Air TemP Precipiøtion Av. Relative Name and Min Air Temp Max Air Temp Precipitation to Av. Relative Min Air Ternp Max Air Ternp Precipitation to Av. to 9am (mm) Humidity(%) Unit ('C) fC) 9am(mm) Humidity(%) ('C) ('C) 9am(mm) Humidity(%) fc) cc) 26.2 0.4 58.5 Mean 8.7 16.9 2.4 77.5 12.8 23.2 0.6 73.2 12.5 37.6 12 90 Max 23.3 24 100 34.8 15 100 29 6.2 0 23 Min 4.7 0 23 5.9 0 12.8 Total 75.2 16.8 30 30 30 29 31 tlumber of Ob: 30 30 31 31 29 28 29 15.5 0.4 92 8.1 20.9 0 66 1 6 12.1 I 81 10.4 29.3 0 41 2 5.8 11.5 0 97 10.4 16.4 0.6 91 8.8 17 28.1 0 59 3 7.2 18.9 0 83 10.3 18.1 0.2 88 31.5 0 45 4 I 22.8 0.2 55 8.6 18.1 0 85 10.7 20.1 29.5 0 50 5 18.1 0 23 8.3 17.2 0.2 83 24.7 0 59 b 22.9 0 71 5.9 19.6 0 71 8.1 11.2 28.3 0 62 7 't0.8 16.9 1 91 't1 25.6 39 10 21.9 0 66 I 6.5 15 2 82 16.8 21.3 0 67 9.2 29 0 il I 5.6 13.7 0.2 77 12.7 19.8 0 93 0 22 't0 7.8 15.7 0 86 11.7 19 0 98 16.8 v 100 47 11 4.7 r8.8 0 63 11.7 17.5 15 11.2 26.3 0 72 12 10.4 l9 0 54 11.9 18.7 0 97 11 24.8 0 75 13 7.8 12.1 6 u 1 1.8 21.5 0 87 8.8 23 0 66 14 5 12.5 2 98 11.1 24.6 0.2 83 28.8 0 51 15 8.5 14.3 12 91 14.8 25.4 0 72 9.4 34 0 32 't6 6.3 17.4 0.2 78 15.3 26.1 0 78 14 22 30.2 0 36 17 11.1 18.7 0 68 16.5 0 69 13.7 30.5 61 18 11 13.2 24 100 18.9 30.9 0 55 32.3 0 19 6.5 14.1 18 90 20.3 30.8 0 64 16.5 v 37.6 0 44 20 7.5 17 0.2 93 12.1 20.7 0 90 12.4 25 30.4 0 69 21 9.7 22.6 0.2 72 10.8 22.4 0 83 19.5 12 85 22 16.7 23.3 0 46 8.5 24.7 0 74 14.1 10.6 20.2 0.4 80 23 13.7 19.3 0 86 1'1.5 26.7 0.2 72 20.5 0.2 90 24 1',l.5 16.1 0 96 12.1 29 0 59 13.5 20.6 0.2 79 25 9.6 16.6 0.2 92 16.4 32.8 0 32 10.2 19.8 0 82 26 6.5 13.5 0 92 21.3 34.8 0 29 11.8 17.4 0 68 27 8.2 14.8 0 u 20.7 0 u 9.5 17.6 0 71 28 5.3 14.7 0 76 28.8 0 66 8.4 \o 23.2 0 6'l { 29 4.7 17.4 0 78 10.4 23.6 0 70 6.2 10.7 27.2 0 49 30 6.8 19 0 59 10.2 20.4 0 77 25.8 0 36 3'l 13.6 21.9 0 56 16.9 \o oo YEAR 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 MONTI{ 111 1 222 2 333 3 Name and Min Air Temp Max Air Temp Precipitation to Av. Relative Min Air Temp l\4ax Air Temp Precipitation to Av. Relative Min Air Temp Max Air Temp Precipitation Av. Relative Unit ('C) (mm) CC) 9am Humidity(%) ("C) CC) 9am (mm) Humidity (%) ("C) fC) to 9am (mm) Humidity(%) Mean 17.1 31.7 0 50.3 17.3 30.4 0.2 58.1 12.5 23.9 1.1 65.8 Max 41.1 0 83 38 2 93 33.2 14 94 Min l0 0 20 9.2 0 to 6.8 0 21 Total 0 4.2 34.6 No. ofObs 30 31 30 31 22 26 25 28 28 28 31 31 1 r 9.3 35.4 0 22 17.4 34 0 24 10.8 29.8 0 60 2 25.6 35.2 0 37 25.5 36.3 0 28 r0.8 25.6 0 68 3 16.6 32.9 0 32 23.9 v.4 0 56 12.6 30 0 u 4 22.5 33.4 0 44 15.2 25.1 0 93 1l.9 23.8 0 74 5 13.2 24.2 0 76 13.4 27.4 z 70 13.8 30.7 0 65 6 10.5 26.1 6'l 19.1 u.2 0 5l 20.3 33.2 0 32 7 13.1 25.6 0 76 23.4 37.8 0 53 21.9 32.7 0 26 13.3 I 28.4 0 oz 26.8 38 0 32 20.6 32.5 0 30 9 17.5 u.5 0 42 28.8 0 86 21.7 33 0 22 22.2 l0 37.5 0 30 12.8 25.3 0 73 21.6 27.8 0 65 't1 21.7 't 35.8 0 34 1.8 32 0 56 9.9 21.9 0 72 12 15.5 32.1 0 34 20.5 27.2 0.2 76 9.8 20.1 0 66 13 21.5 41.1 0 20 83 9.2 21.3 0.2 65 14 39.1 0 29 81 8.8 24.6 0 58 't5 18.7 24.6 0 70 21.8 0 78 15.1 29.5 0 21 16 11.9 27.6 0 50 9.2 25.5 0 65 6 75 17 11.8 28.8 0 59 14 31.2 0 39 16.7 0 79 10.8 l8 28.5 0 52 22.8 36.9 0 24 10.6 20.1 0.2 85 15.6 l9 34.6 0 40 37.3 0 22 10.1 0.4 71 20 25.3 36.2 0 36 24.4 36.3 16 15.2 24.5 0 æ 21 15.6 36.7 0 60 20.9 28.7 0 58 14 90 22 23.1 38.7 0 32 r 0.8 27.9 0 57 9.9 't8.4 4 85 23 23.6 40.8 0 23 29.3 0 59 9.2 22.9 0 71 24 27.8 37 0 54 12.1 26.1 0 79 10.8 19.9 2 85 25 22.1 u.7 0 66 13.2 28.2 2 58 7.8 15.9 0.8 89 26 '18.6 17.8 25.6 0 78 29.5 0 ô¿ 11.7 16.3 6 94 27 't2.8 22 0 82 l3 25.7 0 76 11 16.7 0.8 86 28 10 24.4 0 69 11.9 25.4 0 75 9.9 16.9 0.2 78 29 11 27.8 0 72 6.8 17.1 0 75 30 11.4 24.3 0 71 7.5 2t.8 0 72 31 11.1 28.3 0 45 11.5 25.1 0 53 d) Warriparinga

Averages for ADELAIDE (WAITE INSTITUTE)

023031 ADELAIDE (WAITE INSTITUTE) Conrnenced: 1925 Lasts record 2000 Latitude: -34.969'7 S Longitude: 138-6331 E Elewation; 115.0 m State: SA e"age üÀIi¡FEB!,TARÀPRMAYi'I'Ni'T'I.ÀUGSEPOCTNOVDEC À¡ù¡[ No. Yrs coûrE

!{ean Daily Max TerE (deg C) 27 .1 21 -6 25.6 2t.4 !'7 .8 15.0 14 .1 L5 -2 [t.5 20.2 23-¿ 25.e 20.9 54.5 88 trtearr no. Days, Max >= 40.0 dêg c 98 0.8 0.6 0 .1- 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 t.'7 20.5 uean no. Daya, Max >= 35.0 deg C no 1? O 20.5 98 4 -7 4.2 1.3 0.1 0 .0 0.0 0.0 0.0 0.0 0.0 2-8 Dlean no. DayE, Mar< >= 30.0 deg C 1a 7L.7 10. I 6.3 L.'t 0 .0 0.0 0.0 0.0 0.0 4.6 8.5 44 -2 20 -5 98 Highest Matr TerE (deg C) 43.6 43.1- 47.4 36.6 28.8 27.9 25.5 26 -3 21 2 33 .4 40 -7 47.2 43.6 20.5 98

¡lear¡ Daily ltin 1gl8, (dteg c) 88 76 .2 16 .5 75 .4 L2 .9 r0.7 8.5 7.7 8.1 9.¿ 10.9 12.8 L4.7 11.9 s4.5 ¡ilean no. DayE, Min =< 2.0 deg C 0.0 0.0 0.0 0 . 0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 20.5 98 ltean no. Days, Min =< 0.0 deg C 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 20.5 98 (deg C) Loweat ltin Tql oa 8.0 10.1 7-8 5.4 5.0 3.3 2.6 2.9 3.8 4.3 5.6 8.4 2.6 20.5

¡il€an 9a¡n Àir TerrE (deg C) 22.0 27.7 20 -4 1,7 .1 14.L 11".4 L0.4 11.4 13 .6 16 .0 18.4 20 .6 tb.4 54 .4 88 uêan 9an rùêt-h¡lb TdE, (deg c) o? on 15.5 15.1 14.8 13.0 -Lt. r 8.4 L0.2 11.8 13 .3 14.6 72.2 54 .4 88 Meaa 9arll Delr Point TeÍE (deg C) 70.4 10.6 10.6 9.0 8.0 Áo 6.L RO 6.4 t.r 8.0 o1 8.1 20.3 96 Mean 9a¡tt Rêlative Humidity (%) 49 52 53 6L 68 15 /b 77 64 59 54 50 bt 54 .4 88

Mean 9am wind sDeed (lcn/hr) r0 .2 70 -2 10.0 9 .2 9.0 '7 .9 10_5 10.3 11 .3 L2.r 72.8 13.0 t_0.8 2.3 96 \o uean RainfaU (rEß) \o q)) to R 23 .8 23 .5 24.0 52.O 76.4 /b.b 81 .6 Il -4 63 .5 36.7 62]-.3 75.1 100 Median (Ilecile 5) Rainfall (m) 19.'7 74.r 77.4 43.8 66.7 69 .4 8r-.9 7r-6 63 .6 ,o < 32.0 21 .3 611.5 14 N)

Decile 9 Rainfall (tl[n) '135 _ 45-'7 63.3 59.1 77'7.6 741 .5 138.4 -2 120. 0 L02.4 ot o 70.0 5t,r / tó.3 14 Decile 1 Rainfall (¡¡n¡r) _ 2.2 0_s 1.€ 10.4 26.6 28 .6 42 .0 30.8 2 t.4 15.6 L0 .4 5.,9 503.1 74 ltean no. of Raindays 5.2 4.7 5.8 10.5 r4.8 L6.4 18.4 L8.2 14.5 12 .6 oo 7.2 13'7.2 Áo 1 0l HigbesÈ Monthly Rainfall (mn) 92.2 ]-01 -2 l-05.6 189.5 71 4.9 119.0 183 .1 162 .6 r/2.¿ 155.0 i20 -2 02 tr '75.r L0) Lor'rest Uonthly Rainfall (¡r¡n) 1a ) 0.0 0.0 0.0 r.4 1_6 30.0 1l-.5 9.0 2 -4 2.r 2.4 75.7 1,0) Eighest Recorded Daíly Rain (Íflr) s6.9 81.8 41 .6 5t .7 45.0 48. 5 44.1 44 .4 36.0 51 .0 65.0 Jt.4 81 .8 69.2 92 Mean Daily Sunshine (hrs) '7.5 '1 A oÊ 9.5 9.1 5.s 4.0 J.J 4.4 5.4 7.0 '7.9 6.3 20.4 91 ¡tteaa Daily Evaporatíon (rmn) 1.4 7.1, 5.3 3-4 2.7 1.15 r.6 2.2 t1 4.3 5.6 6. t 4.2 15. B 75 t¿st modified 28 May 2001 Weather Data KentTown 2000-2001 YEAR 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 MONTH l0 10 l0 l0 ll ll ll ll t2 l2 t2 t2 Relative Min Air Temp Max Air TernP Precipitation Av. Relative Name and Min Air Temp Max Air Temp Precipitation to Av. Relative Min Air Ternp Max Air Ternp Precipitation to Av. (%) ('c) to 9am (mm) Humidity(%) Unit ('C) CC) 9am (mm) Humidity(%) cc) ('C) 9am (mm) Humidity fc) 28.3 0.4 46.9 Mean I t.8 21.2 2.7 61.5 t7.l 27.6 0.3 s3.3 16.1 66 Max 32.4 28 88 39.3 8 72 40 8.6 24 Min60 30 tt.2 0 24 I 1.8 0 Total 82'4 9.6 13.4 30 3l 3l 3l 3l No. 31 31 3t 30 30 30 ofObs 31 s6 lO.t 16.2 5.6 57 r4.2 20.8 0.8 69 l r.8 23 o I 0 30 0 a) t4.8 2t.5 0.8 68 13.4 3l 2 to 16.4 46 3 6.2 22.2 0 72 12.8 2l .8 o 63 20.4 27.9 0 38 4 12.3 28.8 0 36 t2 22.5 n 65 14.9 33.4 0 0 47 5 t7.8 32.4 0 30 tt.2 20.9 0 59 17.5 30.3 0 4t 6 15.2 23.5 0 68 t2.3 23.9 0 4l 13.3 27.8 28.9 0 45 7 13.9 18 13.2 75 16. I 30.4 0 25 16.4 0 50 8 8.7 17.8 1.2 62 22.3 25.6 0 60 t4.3 25.2 0 4l 9 6.7 l8.t 0 64 16.5 24.1 8 65 t3 32.4 0 29 10 t2.6 18.9 0 63 t6.2 25.5 0 69 17.5 35 47 ll 6 22.4 0.4 52 15.4 22 0 72 20.4 3t.6 0 0 60 t2 12.7 24.2 0 46 ló 22.6 0 72 t5.I 25 0 58 13 t2.2 16.1 1.8 59 l6 25.5 0 60 t4.5 25 48 t4 9.8 r7.l 3.2 72 166 28.8 0 55 12.6 25.2 0 38 15 12.3 l8.l 5.4 73 17.4 29.6 0 5l 13.8 31.8 0 24 16 9.4 22.2 0 60 18.6 30.3 0 53 r 9.5 36.2 0 49 t7 16.2 22.8 0 56 22.3 3l .9 0 49 18.9 29.8 0 0 38 17.8 29.8 1.6 55 I 8 l4.l 17 .l 28 88 ))) 35.3 10.8 17.3 23.6 67 2t.4 33.6 0 50 16.8 33.4 0 35 19 n 20 10.8 21 0 77 16.5 23.9 0 67 t6.6 40 32 58 21 9.8 26.2 0 6l 15.5 25.5 0 56 24.5 32 0 22 15 27.5 0 53 13.6 29.2 0 50 t7.2 22.5 8.ó 66 62 23 ló.6 25.3 0 7l 15.6 3l .6 0 48 r4.ó 24 3 0.2 6l 24 14.6 20 0 79 17.2 32.7 0 4t t6.4 23.6 25 12.2 20.6 0 80 17.7 35.4 0 32 t3;l 22.8 0 62 7)< 0 ól 26 9.2 t7.4 0 76 22.9 37.7 0 26 t5.7 27 l2.l 18.7 0 59 27 -5 39.3 0 24 14.6 20.5 0 53 12.5 2t.4 0 52 28 8.9 19.2 0 53 26.t 28.9 0 47 t\) 29 10.7 21.2 0 55 13.8 24.8 0 60 t2.2 25.1 0 46 36 30 l3.l 24.t 0 33 12.9 23.1 0 64 16.5 29.7 0 22.5 30.8 0 25 3l l'1 .3 27 0 38 N) N) Weather Data KentTown 2000-2001 YEAR 2001 2001 200r 200 I 2001 2001 200 l 200 I 2C0t 2001 2001 2001 MONTH I I a1 I I 2 2 33 3 3 Name Min Air Ternp and Max Air Temp Precipitation to Av. Relative Min Air Ternp lvfax Air Temp Precipitation to Av. Relative Min Air Temp Max Air Temp Precipitation Av. Relatrve Unit ('C) (.C) 9am (mm) Humidiry(%) ("c) (mm) fc) 9am Humidity(%) cc) fc) to 9am (mm) Humidity (9zo) Mean 0.6 42-9 20.3 33.7 19.9 3l .6 0.4 47.2 14.4 zs.s l.ó 54.2 Max 43-3 19.6 7l 40 8.6 75 36.4 15.6 75 Min l4.l 0 l9 13.8 0 t7 9.2 0 2I Total 19.6 t04 49 No. ofObs 31 31 3l 3l 28 28 28 28 3l 3l 3l 3l l 19.8 39.1 0 l9 20.7 37.9 0 t7 t4.7 29.2 0 52 2 28.3 35.8 0 4l 28. l 39.8 0 26 t3.4 28.2 0 5l 3 15.2 36 0 43 23.3 36.5 0 57 15.3 29.4 0 54 4 22.6 36 0 38 20.5 27.7 0 60 t6. I 26.7 0 53 5 16.9 27.4 0 57 18.8 3t.2 0 47 t7.6 33.7 0 44 6t4928.80 53 20.5 36.5 0 43 24 36.4 0 26 7 16.6 29.3 0 57 28 39.7 0 44 192 34.3 0 29 8 t8 317 0 45 29.9 40 0 34 204 34.3 0 27 9 2t3 35.9 0 33 23.2 25.2 0 65 20.6 36.4 0 2t t0 24 38.3 0 35 t5.2 28.8 0.8 56 2t.8 26.7 0 53 I I 20.8 37.t 0 30 t5.6 33.8 0 50 t3.7 24.6 0 \) 12 t9.3 35 0 27 2t.4 28.2 0.4 75 l4.l 2t.9 0 52 13 25.2 43.3 0 20 17.3 24.3 8.6 67 l2.s 22.1 0.8 52 t4 26.5 41. 0 36 t7.l 23.4 0 58 l l.8 26.4 0 42 15 2t.9 25.3 0 55 t4.7 24.4 0 57 14.I 32.3 0 24 16 17.4 31 5 0 38 13.8 28.8 0 46 18.8 2t.9 2 63 17 I 6.8 3t.7 0 43 16.7 34.8 0 26 13.3 20.6 13.8 64 18 15 32.t 0 4t 25.t 39.7 0 23 I 1.5 22.2 0 73 t9 18.4 36.s 0 39 28.3 38.7 0 l9 t2.l 24.4 0 66 20 25.3 37 0 33 22.8 38.9 0 2t 13.2 26.4 0 6l 'r7 0 21 2t.6 36.9 0 48 22.6 0 46 il.5 t 5.8 t5.6 74 22 23.t 39.3 0 40 t4.6 too 0 46 13.7 20.8 3.8 73 23 26.2 41 0 30 14.3 28 0 50 t0 24.9 0 ól 24 27 I 38.9 0 45 l6.l 27.9 0 60 13.8 20.7 3.4 67 25 26-5 38 0 6l 18.9 32.s 0.6 52 I r.7 r 9.5 4 70 26 2t 25.t 19.6 7t t9.9 30 0 52 13.5 20.6 5.2 75 27 15.8 24.t 0 6l t5.2 24.8 0 64 13.9 t9.6 0.2 68 28 l4.l 24.6 0 55 15.4 25.6 0 59 12.7 20.3 0.2 60 29 l5.l 28.8 0 57 9.8 20.4 0 56 30 16.3 27.s 0 47 9.2 23.2 0 59 3l 18 31.9 0 3t 9.5 25.8 0 56 Weather Data KentTown 2001-2002 YEAR 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 MONTH l0 l0 l0 l0 ll ll ll ll t2 12 12 t2 Av. Relative Name and Unit Min Air Ternp Max Air Temp Precipitation to Av. Relative Min Air Ternp Max Air Ternp Precipitation to Av. Relative Min Air Ternp Max Air Ternp Precipitation to fC) CC) 9am (mm) Humidity (%) fC) CC) 9am (mm) Humidity(%) CC) CC) 9am (mm) Humidity(%) Mean I1.8 21.2 2.7 61.s t7.l 27.6 0.3 53.3 r ó.1 28.3 0.4 46,9 Max 32.4 28 88 39.3 I 72 40 8.ó 66 Min60 30 |.2 0 24 11.8 0 24 Total 82.4 9.6 t3.4 3l 3l No. ofObs 31 31 3l 3t 30 30 30 30 31 31 I l0.l ló.2 s.6 57 14.2 20.8 0.8 69 il.8 23 0 5ó 2 r0 16.4 0 17 14.8 2t.5 0.8 68 13.4 31 0 30 3 6.2 22.2 0 72 r2.8 2t.8 0 63 20.4 27.9 0 46 4 12.3 28.8 0 36 t2 22.s 0 65 t4.9 33.4 0 38 5 17.8 32.4 0 30 Û.2 20.9 0 59 t7.5 30.3 0 47 6 15.2 23.5 0 68 t2.3 23.9 0 4l 13.3 27.8 0 4l 7 l3.9 18 13.2 75 l6.l 30.4 0 25 16.4 28.9 0 45 0 60 0 50 8 8 .7 17.8 1.2 62 22.3 25.6 14.3 25.2 41 9 6.7 l8.l 0 64 I ó.5 24.1 8 65 13 32.4 0 l0 12.6 18.9 0 63 r6.2 2s.5 0 69 17.5 35 0 29 't2 il 6 22.4 0.4 52 ts.4 22 0 20.4 31.6 0 47 12 12.7 24.2 0 46 l6 22.6 0 72 ls.t 25 0 ó0 13 12.2 16.l 18 59 l6 25.5 0 60 14.5 25 0 58 t4 9.8 l7.t 3.2 72 16.6 28.8 0 55 t2.6 25.2 0 48 15 12.3 18.I 5.4 73 17.4 29.6 0 5l 13.8 31.8 0 38 16 9.4 22.2 0 ó0 l8.6 30.3 0 53 l9.s 36.2 0 24 17 16.2 22.8 0 56 ))7 31.9 0 49 18.9 29.8 0 49 1aa 1.6 55 I 8 l4.l 17 .l 28 88 35.3 0 38 17.8 29.8 19 10.8 17.3 23.6 67 21.4 33.6 0 50 r 6.8 33.4 0 35 20 10.8 2t 0 77 16.5 23.9 0 67 16.6 40 0 32 2t 9.8 26.2 0 6l 15.5 25.5 0 56 24.5 32 0 58 22 15 27.5 0 53 13.6 )o) 0 50 17.2 22.5 8.6 66 23 16.ó 25.3 0 7l 15.6 3t.6 0 48 14.6 24 3 62 24 t4.6 20 0 79 17.2 32;l 0 4l 16.4 23.6 0.2 63 25 12.2 20.6 0 80 l'7.7 35.4 0 32 t3.7 22.8 0 62 26 9.2 t7.4 0 76 22.9 37.7 0 26 r5.7 22.5 0 6l 2'7 l2.r 18.7 0 59 27.5 39.3 0 24 t4.ó 20.5 0 53 26.r 28.9 0 47 21.4 0 52 28 8.9 19.2 0 53 t2.5 ì.') 0 55 13.8 24.8 0 60 12.2 25.1 0 46 O 29 10.7 2t.2 u) 30 l3.l 24.1 0 33 12.9 23.1 0 64 16.5 29.7 0 3ó 31 17.3 27 0 38 22.s 30.8 0 25 ì.J À Weather Data KentTown 2001-2002

YEAR 2001 2001 2001 200 I 2001 200 I 2001 200 I 2001 2001 200t 200 I MONTH I I I I 2 2 2 2 3 3 3 3 Name and Unit Min Air Temp Max Air Temp Precipitation to Av. Relative Min Air Temp Max Air Temp Precipitation to Av. Relative Min Air Temp Max Air Temp Precipitation to Av. Relative fC) CC) 9am (mm) Humidiry (%) cc) ('c) 9am (mm) Humidity(%) cc) cc) 9am (mm) Humidity (0/6) Mean 20.3 33.1 0.6 429 t9.9 3l .6 0-4 47.2 14.4 25.5 1.6 54.2 Max 43.3 19.6 7T 40 8.6 75 36.4 15.6 '15 Min l4.l 0 l9 13.8 0 t7 9.2 0 2t Total 19.6 10.4 49 No. ofObs 31 31 3l 3t 28 28 28 28 3l 3l 3l 3l r 19.3 39 I 0 l9 20.7 37.9 0 l7 14.7 29.2 0 52 2 28.3 3s8 0 4l 28.t 39.8 0 26 t3.4 28.2 0 5I 3 15.2 36 0 43 23.3 36.5 0 57 15.3 29.4 0 54 4 22.6 36 0 38 20.5 27.7 0 60 l6.l 26.7 0 53 5 16.9 27 .4 0 57 18.8 3t -2 0 47 t7.6 33.7 0 44 6 14.9 28.8 0 53 20.s 36.s 0 43 24 36.4 0 26 7 16.6 29.3 0 57 28 39.7 0 44 19.2 34.3 n 29 8 t8 31.7 0 45 29.9 40 0 34 20.4 34.3 0 27 9 21.3 35.9 0 33 23.2 25.2 0 65 20.6 36.4 0 2t l0 24 38.3 0 35 t5.2 28.8 0.8 56 2t.8 26.7 0 53 1r 20.8 37.1 0 30 15.6 33.8 0 50 t3.7 24.6 0 52 12 19.3 35 0 2'7 21.4 28.2 t).4 75 t4.1 21.9 0 I 13 25.2 43.3 0 20 17.3 24.3 8.ó 67 12.5 22.1 0.8 52 t4 26.5 4t 0 36 17.t 23.4 0 58 I 1.8 26.4 0 42 15 2t.9 25.3 0 55 t4.7 24.4 0 57 14. I 32.3 0 24 16 17.4 3t.5 0 38 l3.8 28.8 0 46 18.8 21.9 2 63 t7 16.8 3t .7 0 43 t6.7 34.8 0 26 13.3 20.6 13.8 & 18 15 32.1 0 4l 25.1 39.7 0 23 I 1.5 22.2 0 73 19 18.4 36.5 0 39 28.3 38.7 0 l9 t2.l 24.4 0 66 20 2s.3 37 0 33 22.8 38.9 0 2l 13.2 26.4 0 ól 2t 21.6 3ó.9 0 48 22.6 27.9 0 46 I 1.5 t5.8 15.6 '74 22 23.1 39.3 0 40 t4.6 29.9 0 46 13.7 20.8 3.8 73 23 26.2 41 0 30 14.3 28 0 50 l0 24.9 0 6l 24 27.1 38.9 0 45 l6.l 27.9 0 60 13.8 20.7 3.4 67 25 26.s 38 0 6l 18.9 32.5 0.6 52 I t.7 19.5 4 70 26 2t 25.1 19.6 7l 19.9 30 fì 52 13.5 20.6 52 15 27 15.8 24.t 0 6l t5.2 24.8 0 64 13.9 19.ó 0.2 68 28 t4.t 24.6 0 55 15.4 25.6 0 59 12.7 20.3 0.2 60 29 l5.l 28.8 0 57 9.8 20.4 0 56 30 ló.3 27.5 0 47 9.2 23.2 0 59 3l 18 31.9 0 3l 9.5 25.8 0 56 All weather data is from the Australian Bureau of Meteorology 205

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