UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The effects of seed-storage lipid emulsion sprays on the interactions between plants and their fungal… Young, Roderick James 1994

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1994-0247.pdf [ 2.78MB ]
Metadata
JSON: 831-1.0087345.json
JSON-LD: 831-1.0087345-ld.json
RDF/XML (Pretty): 831-1.0087345-rdf.xml
RDF/JSON: 831-1.0087345-rdf.json
Turtle: 831-1.0087345-turtle.txt
N-Triples: 831-1.0087345-rdf-ntriples.txt
Original Record: 831-1.0087345-source.json
Full Text
831-1.0087345-fulltext.txt
Citation
831-1.0087345.ris

Full Text

THE EFFECTS OF SEED-STORAGE LIPID EMULSION SPRAYSON THE INTERAcTIONS BETWEEN PLANTSAND THEIR FUNGAL PATHOGENSbyRODERICK JAMES YOUNGBSc, The University of British Columbia, 1991A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Botany)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAApril 1994© Roderick James Young, 1994In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.__________________________________Department of E oçpjThe University of British ColumbiaVancouver, CanadaDate PrPPJL_ 2C, 1qqL/DE-6 (2/88)I. ABSTRACTEmulsions of the seed-storage lipids jojoba wax and canola oil were testedfor phytoprotective activity against two powdery mildews and two rot-causing fungi, andfor phytotoxicity on two host plants, grape ( Vitis vinifera) and cucumber (Cucumissativus). Against the powdery mildews tested (Erysiphe cichoracearum and Uncinulanecato,), greenhouse cucumber plants and field grape plants treated with jojoba waxemulsions showed 75-100% reductions in powdery mildew disease severity, ascompared to water sprayed plants. The canola oil emulsion reduced cucumber powderymildew disease severity by 63-69%. Scanning electron microscope analyses of E.cichoracearum conidia on cucumber leaves sprayed with the seed-storage lipidemulsions showed normal germination structures with inhibited appressorium formation.The jojoba wax emulsions did have slight phytotoxic effects observable on both thecucumber and grape plants, especially during periods of higher temperature. However,these effects were not observed to reduce the yield or survival of the grape plantssprayed in field trials.Jojoba wax and canola oil emulsions did not seem to have an effect againstthe infection processes of rot-causing fungi (Botrytis cinerea and Didymelia bryoniae).Instead, the lack of differences between the growth of B. cinerea colonies on potatodextrose agar (PDA) treated with the seed-storage lipid emulsions or the emulsifyingsurfactant Triton X-100 alone indicated that the presence of the surfactant wascorrelated with reduced colony growth. Colony growth on Triton X-1 00-treated PDA wasreduced by 87-92% as compared to the water control-treated PDA. In addition, fieldgrape plants treated with emulsions containing Triton X-1 00 showed a 68-87% reductionIIin B. cinerea-caused disease incidence. While similar reductions in D. bryoniae growthwere observed in vitro for PDA treated with emulsions containing Triton X-100, thesurtactant or the lipid emulsions did not reduce D. bryoniae-caused disease incidence inthe in vivo trials on greenhouse cucumbers plants. Applied treatments may have beenwashed away by exuding sap.IIIII. TABLE OF CONTENTSI. ABSTRACT iiII. TABLE OF CONTENTS ivIll. LIST OF TABLES viiiIV. LIST OF FIGURES xV. ACKNOWLEDGMENTS xi1. INTRODUCTION 11.1. Fungal Infection Processes 11.1.1. Activation 21.1.2. Germination 31.1.3. Recognition 31.1.4. Penetration 41.2. Previous Studies of Lipid Emulsion Use on Plants and TheirFungal Pathogens 61.3. Systems Chosen for Study B1.3.1. Lipids Emulsions to be Applied to the Plants 81.3.2. Plant-Fungal Systems Chosen for Study 91.3.2.1. The Powdery Mildews 91.3.2.2. The Rot-Causing Fungi 121.4. Study Outline 142. MATERIALS AND METHODS 152.1. Preliminary Studies 152.1 .1. Determination of Phytotoxic Lipid EmulsionConcentrations 152.1.2. Effect of Exogenous Lipid Emulsions on the Growth ofDidymeila bryoniae and Botrytis cinerea In Vitro 162.2. Comparison of Exogenous Lipid Emulsions as Phytoprotectantsand as Antitranspirants of Cucumber Plants 182.2.1. Effects of Exogenous Lipid Emulsions on Powdery Mildewof Cucumber Plants 192.2.1 .1. Scanning Electron Microscope Studies of theEffects of Exogenous Lipid Emulsions on Erysiphecichoracearum Infection of Cucumber Leaves 202.2.2. Effects of Exogenous Lipid Emulsions on Black Rot andGrowth of Cucumber Plants 212.3. Effect of Seed-storage Lipid Emulsions as Phytoprotectants andas Antitranspirants of Grape Plants 222.3.1. Plant Material, Husbandry, and Spraying 222.3.2. Effects of Exogenous Lipid Emulsions on Powdery Mildewand Bunch Rot of Grape Plants 252.3.3. Effects of Exogenous Lipid Emulsions on Grape PlantGrowth, Yield and Survival 263. RESULTS 303.1. Effects of Exogenous Lipid Emulsions as Phytoprotectants ofGrape and Cucumber Plants 303.1 .1. Effects of Exogenous Lipid Emulsions on Powdery Mildewof Cucumber Plants 30IvTABLE OF CONTENTS (Continued)3.1.1 .1. Scanning Electron Microscope Studies of theEffects of Exogenous Lipid Emulsions on Erysiphecichoracearum Infection of Cucumber Leaves 313.1.2. Effects of Exogenous Lipid Emulsions on Powdery Mildewof Grape Plants 343.1.3. Effect of Exogenous Lipid Emulsions on the Growth ofDidymella bryoniae and Botrytis cinerea In Wtro 353.1.4. Effects of Exogenous Lipid Emulsions on Black Rot ofCucumber Plants 373.1.5. Effects of Exogenous Lipid Emulsions on Bunch Rot ofGrape Plants 373.2. Effect of Exogenous Lipid Emulsions as Antitranspirants onCucumber and Grape Plants 383.2.1. Determination of Phytotoxic Lipid EmulsionConcentrations 383.2.2. Effects of Exogenous Lipid Emulsions on CucumberGrowth 413.2.3. Effects of Exogenous Lipid Emulsions on Grape PlantGrowth, Yield and Survival 423.2.3.1. Growth and Survival 423.2.3.2. Yield 484. DISCUSSION 504.1. Effects of Seed-Storage Lipid Emulsions as Phytoprotectantsagainst Powdery Mildews 504.1 .1. Efficacy of Lipid Emulsions against Powdery Mildews 504.1.2. Mechanism of lipid Emulsions against Powdery Mildews 534.2. Effects of Seed-Storage Lipid Emulsions as Phytoprotectantsagainst Rot-Causing Fungi 584.3. Overall Discussion 595. LITERATURE CITED 626. APPENDICES 70Appendix 1. Number of E. cichoracearum colonies per six-week old“Chicago Pickling11 cucumber leaf (mean for three samples perplant) both two weeks after application of exogenous lipidemulsions 70Appendix 2. Percent of experimental jojoba wax emulsion-treated field-grown ‘Auxerrois’ grape plants in a particular growth stage asrecorded throughout the growing season in 1991 71Appendix 3. Stomatal resistance (S moH rn-2) measurements from thecentre of experimental jojoba wax emulsion-treated field-grown‘Auxerrois’ grape leaves on the south and outer side of grapeplant canopies as recorded throughout the growing season in1991 72Appendix 4.1. Number of leaves per shoot of experimental jojoba waxemulsion-treated field-grown ‘Auxerrois’ grape plants throughoutthe growing season in 1992 72vTABLE OF CONTENTS (Continued)Appendix 4.2. Number of internodes per shoot of experimental jojobawax emulsion-treated field-grown ‘Auxerrois’ grape plantsthroughout the growing season in 1992 73Appendix 4.3. Length of the fifth internode (cm) on shoots ofexperimental jojoba wax emulsion-treated field-grown Auxerroisgrape plants throughout the growing season in 1992 73Appendix 4.4. Length of the 10th internode (cm) on shoots ofexperimental jojoba wax emulsion-treated field-grown ‘Auxerrois’grape plants throughout the growing season in 1992 74Appendix 4.5. Mean area of the 10th leaf (cm2) on shoots ofexperimental jojoba wax emulsion-treated field-grown ‘Auxerrois’grape plants throughout the growing season in 1992 74Appendix 4.6. First cluster rachis length (cm) on shoots of experimentaljojoba wax emulsion-treated field-grown ‘Auxerrois’ grape plantsthroughout the growing season in 1992 75Appendix 4.7. Berry volume in mm3 from berries sampled at the bottomof first clusters on shoots of experimental jojoba wax emulsion-treated field-grown ‘Auxerrois’ grape plants throughout thegrowing season in 1992 75Appendix 4.8. Amount of chlorophyll in mg/cm2for experimental jojobawax emulsion-treated field-grown ‘Auxerroi& grape leaf samplestaken from the south and outside of grape canopies at the 1.5 mlevel throughout growing season in 1992 76Appendix 5.1. Number of leaves per shoot of experimental seedstorage lipid emulsion-treated field-grown ‘R iesling’ grape plantsthroughout the growing season in 1992 76Appendix 5.2. Number of internodes per shoot of experimental seedstorage lipid emulsion-treated field-grown ‘Riesling’ grape plantsthroughout the growing season in 1992 77Appendix 5.3. Length of the fifth internode (cm) on shoots ofexperimental seed-storage lipid emulsion-treated field-grown‘Riesling’ grape plants throughout the growing season in 1992 77Appendix 5.4. Length of the 10th internode (cm) on shoots ofexperimental seed-storage lipid emulsion-treated field-grown‘Riesling’ grape plants throughout the growing season in 1992 78Appendix 5.5. Area of the 10th leaf (cm2) on shoots of experimentalseed-storage lipid emulsion-treated field-grown ‘Riesling’ grapeplants throughout the growing season in 1992 78Appendix 5.6. First cluster rachis length (cm) on shoots of experimentalseed-storage lipid emulsion-treated field-grown ‘Riesling’ grapeplants throughout the growing season in 1992 79Appendix 5.7. Berry volume (mm3)from berries sampled at the bottomof first clusters on shoots of experimental seed-storage lipidemulsion-treated field-grown ‘Riesling’ grape plants throughoutthe growing season in 1992 79VITABLE OF CONTENTS (Continued)Appendix 5.8. Amount of chlorophyll in mg/cm2for experimental seedstorage lipid emulsion-treated field-grown ‘Riesling1 grape leafsamples taken from the south and outside of grape canopies atthe 1.5 m level throughout growing season in 1992 80Appendix 6. Weight of harvested grape clusters sampled fromexperimental seed-storage lipid emulsion-treated field-grown‘Auxerrois’ grape plants in 1992 80vi’III. LIST OF TABLESTable 1. Experimental seed-storage lipid emulsions applied to 1% w/vsucrose-enriched potato dextrose agar to evaluate the relative efficacyof the emulsions on the growth of D. btyoniae and B. cinerea 17Table 2. Experimental seed-storage lipid emulsions applied to “ChicagoPickling” cucumber plants in greenhouse trials for determining theeffect of the emulsions on powdery mildew disease severity 19Table 3. Experimental seed-storage lipid emulsions and schedule of theirapplication to Auxerrois grape plants at TV and ‘Riesling’ plants atACRS, applied to determine the effects of the various treatments inreducing powdery mildew disease severity and/or incidence 24Table 4. Number of E. cichoracearum colonies per leaf area 17 days afterinoculation on greenhouse-grown “Chicago Pickling” cucumber plantstreated with experimental exogenous lipid emulsions 31Table 5. Number of E. cichoracearum infection structures observed by SEMon “Chicago Pickling” cucumber leaves air-dried 48 hours afterspraying with experimental exogenous lipid emulsions, and subsequentinoculation with E. cichoracearum 32Table 6. Mean number of U. necator colonies per experimental seed-storagelipid emulsion-treated ‘Auxerrois’ grape leaf in the preharvest, andmean number of experimental jojoba wax emulsion-treated ‘Auxerrois’grape clusters at harvest showing symptoms of powdery mildewdisease 35Table 7. Difference in B. cinerea and D. bryoniae colony area between 1 and4 days after inoculation on experimental exogenous lipid emulsiontreated 1% sucrose-enriched PDA 36Table 8. Percentage of black rot-diseased inoculated petioles on six-week-old“Chicago Pickling” cucumber plants 17 days after application ofexperimental exogenous lipid emulsion treatments, and inoculationwith D. biyoniae 37Table 9. Mean number of experimental seed-storage lipid emulsion treated‘Auxerrois’ and ‘Riesling’ grape clusters harvested per plant showingbunch rot disease incidence 38Table 10. Amount of experimental jojoba wax emulsion-treated ‘Auxerrois’grape leaf damage calculated using greenhouse plants which weresprayed at 7 weeks and sampled at 10 weeks 39Table 11. Leaf area of six-week-old greenhouse-grown “Chicago Pickling”cucumber plants, treated 17 days previously with experimentalexogenous lipid emulsions 42Table 12. Percentage of experimental jojoba wax emulsion-treated field-grown ‘Auxerrois’ grape plants observed in a growth stage, andstomatal resistance measurements of grape leaves 45Table 13. Stomatal resistance at mid-season of field-grown ‘Auxerrois’ grapeleaves treated with experimental jojoba wax emulsions 46Table 14. Wet weight of experimental seed-storage lipid emulsion-treated‘Auxerrois’ and ‘Riesling’ grape plant cane prunings per plant beforeand after the growing season for field trials 48VIIILIST OF TABLES (Continued)Table 15. Cluster weight and berry weight and composition measurementssampled from grapes harvested from experimental jojoba waxemulsions-treated field-grown ‘Auxerrois’ plants in 1991 49Table 16. Yield, and berry composition of grapes harvested from experimentalseed-storage lipid emulsions-treated field-grown ‘Riesling’ grape plants 49ixIV. LIST OF FIGURESFigure 1. Diagram of interrelationships between data and the method used todetermine the effects of the interrelationships on a single variable, sothat the effects may be accounted for in subsequent ANOVA 28Figure 2. Examples of E. cichoracearum infection structures observed bySEM on “Chicago Pickling” cucumber leaves air-dried 48 hours afterspraying wfth experimental exogenous lipid emulsions, and subsequentinoculation with E. cichoracearum 33Figure 3. Photographs of experimental jojoba wax emulsion- and Triton® X100 surfactant treated-’Auxerrois’ leaf damage using greenhouseplants which were sprayed at 7 weeks and sampled at 10 weeks 40Figure 4. Comparison of the phytotoxic effects of the commercial control andthe 1.0% “all-season” jojoba wax sprays on ‘Auxerrois’ grape plants 44Figure 5. Growing degree days for 1991, 1992 and previous 80-year mean,for the Central Okanagan Region, in which are located both TV andACRS 45Figure 6. Residuals from stepwise multiple regression analysis showing jojobawax emulsion treatment effect on ‘Auxerrois’ grape leaf chlorophyll atTVin 1992 47xV. ACKNOWLEDGMENTSI acknowledge with gratitude the financial assistance and thoughtful advice giventhroughout the study of my supervisors, Dr. Edith Camm, Dr. Robert Copeman, and Dr.Andy Reynolds, without which I would have found this project difficult. I also thank mywife, Pamela, for her capable technical and research assistance in 1992 in both the fieldand laboratory sections of the study, and Glen McKay for his help in 1991 and 1992.Additionally, the financial assistance of the Agricultural Research and DevelopmentCorporation of British Columbia, and the scholarships given by the Science Council ofBritish Columbia were invaluable, and much appreciated. Finally, I thank God for Hisgrace, without which the project would have had no meaning.XI1. INTRODUCTIONEvery year, diseases caused by fungal plant pathogens destroy approximately12% of crops produced worldwide, resulting in an estimated loss of $9.1 billion in theUnited States alone [Agrios 1988]. While many chemical and biological controls offungal pathogens exist, recent research has shown the potential for using antitranspirantlipid emulsions as plant prophylactics against fungal pathogens [Quarles 1991].However, commercially available antitranspirants like Folicote, WiltPruf, Sunspray andothers with known prophylactic effects are too costly for application in situations largerthan small-scale nurseries [Das and Raghavendra 1979]. Alternatively, the emulsifiedstorage lipids of some seeds, such as canola oil and jojoba wax, are inexpensive andpresent a low hazard to labourers and consumers [Ackman 1990, National ResearchCouncil (US) 1985]. In this study, the effects of jojoba wax and canola oil emulsions onthe infection processes and diseases caused by certain powdery mildews and rot-causing fungal plant pathogens were studied in comparison with the effects of Folicoteand WiltPruf. These comparisons were done (1) to determine whether seed-storage lipidemulsions have potential as plant prophylactic substances and (2) to explore how lipidemulsions applied to plant epicuticles affect fungal plant pathogen infection processes.1.1. Funqal Infection ProcessesThere are four general processes which the fungal spore must undergo beforesuccessfully entering into plant tissues: activation from the dormant state in which itarrives on the plant [Sussman and Douthit 1973], germination with the production of thegerm tube [Van Etten et a! 1983], recognition during which information regarding apenetration site is received from the epicuticle [Kunoh 1984, Kunoh et a! 1991], and1penetration during which a specialized hypha called a penetration peg is forced throughthe epicuticle and the underlying cuticle into plant tissues [Emmett and Parberry 1975].While some fungal pathogens can (and do) enter into the plant via injuries and naturalopenings such as stomates [Agrios 1988], many fungal pathogens gain access to theirhosts by penetration of unwounded tissue [Koller 19911. The simplest manner ofpenetration is via stomates, but stomates take up on average only 1 % of the entire leafarea on most plants [Gay and Pearce 1984]. Therefore, most pathogenic fungi mustovercome the epicuticle-cuticle-cell wall barrier before entering plant tissues. Any or allof the processes summarized above may be affected by applications of exogenous lipidemulsions. The following describes in greater depth background information on thefungal infection process.1.1.1. ActivationFungal spores are in a quiescent state before the activation of physiologicalprocesses necessary for germination [Van Etten et a! 1983, Sussman and Douthit1973]. The major reason spores remain dormant is their state of desiccation, whichmaintains the endoplasmic reticulum in vesicle form, thus compartmentalizing enzymesand separating mRNA from translational compounds [Sussman and Douthit 1973]. Inaddition, compartmentalization of respiratory substrates lowers respiration and anabolicprocesses [Sussman and Douthit 1973]. Desiccation is an exogenous constraint;particular species may be additionally constrained by constitutive constraints, such asthe slow decay or leaching of some inhibitory cellular component feeding back to thegenome which prevents activation until the concentration of the component has aninsignificant inhibitory effect [Macko 1981]. Activation occurs when sufficient amounts ofwater and heat activate the spore’s metabolism to allow for growth [Van Etten et a!1983, Sussman 1966]. Activation of spores from a particular species may also depend2on the elimination of constitutive constraints [Sussman 1966], or the presence of exudedsugars on the epicuticular surface as a result of diffusion through underlying anticlinalcell walls, such as at leaf veins [Carvers and Thomas 1990, O’Connell et al 1985].1.1.2. GerminationActivation starts the process of germination, an irreversible process ofmorphological change from the dormant form of the organism to the active form [VanEtten et a! 1983, Kennedy 1990, Sussman and Douthit 19731. During germination thespore develops a germ tube which breaks through the spore cell wall using combinedphysical pressure and enzymatic degradation; the developing germ tube forms aprotective chitin cell wall after extension [Gooday and Trinci 1980]. During the growth ofthe germ tube and the penetration process, a sheath of mucilage is produced by thespore which surrounds the entire organism [Nicholson 1984]. This mucilage acts toattach the growing spore to the plant surface, to seal the penetration site, and to protectthe growing fungus against limiting environmental conditions - functions which are vitalfor successful germination [Kunoh et all 991, Nicholson and Epstein 1991, Nicholson1990, Koller eta! 1982].1.1.3. RecognitionAppropriate penetration sites are recognized on the basis of the local chemicaland topographical cues from the epicuticle, although particular species may use onemore than the other in site location [Emmett and Parberry 1975]. Chemical, orchemotropic, recognition may refer to the recognition of particular epicuticularcomponents and component chain lengths [Hoch and Staples 1991]. This type ofrecognition was observed in Colletotrichum gloeosporioides Penz., where appressorium3formation on avocado (Persea americana Miller var Haas) fruit was shown to occurpreferentially in the presence of very long chain primary alcohols isolated from theavocado fruit epicuticle [Podila et al. 1993]. Chemical recognition also may refer to therecognition of sites where a greater rate of exosmosis occurs relative to other localareas of the plant surface, as a result of diffusion through underlying anticlinal cell walls.This has been suggested due to the high correlation of fungal penetration sites with celljunctions, such as leaf veins [Carvers and Thomas 1990, Emmett and Parberry 1975,O’Connell et al 1985, Hoch and Staples 19911. The other type of recognition which usescues from the topography is referred to as the tactile, or thigmotropic recognition of thedistribution of particular wax bodies or structures in the epicuticle [Emmett and Parberry1975, Hoch and Staples 1991, Staples and Hoch 1987]. Upon successful recognition ofa penetration site, the fungal germ tube begins to form the structures needed forpenetration.1.1.4. PenetrationAfter recognition, the fungal germ tubes produce appressoria, which are lobatestructures at the ends of the germ tubes. These adhere to the plant surface and producea penetration peg that moves through the epicuticle, the actual surface of the plant,down through the cuticle and cell wall and eventually into the plant epidermis [Kunoh1984]. Growth of the peg is similar to the germ tube, except the peg stays narrow indiameter while passing through the epicuticle-cuticle-cell wall barrier [Mims et all 989].This barrier consists of the epicuticle, a biopolyester of unsubstituted and monoestericlong-chain hydrocarbons, 22-50 carbons long; the cuticle, an underlying supportingstructure of interesterified hydroxy- and epoxy- fatty acids and tn- and di-acylglycerides,16-18 carbons long; and the cell wall, composed mainly of cellulose [Gay and Pearce41984, Kollatukudy 1976,1984]. The appressorium provides both mechanical anchoringand barrier-weakening enzymes which enable growth of the penetration peg.Cuticle-weakening enzymes are provided in the same mucilage whichsurrounded the growing germ tube. This mucilage, which also surrounds theappressorium and penetration peg, contains in some species positively-chargedglycoproteins [Tunlid eta! 1991, Epstein et a! 1987]. These glycoproteins have beenshown to be cutinases and serine-esterases, proteins with high specificity for hydrolysisof cuticle, and particularly ester bonds [Koller 1991, Deising et al 1992, Pascholati et a!1992] which the fungal spore produces in response to the presence of cuticularmonomers [Kollatukudy 1985]. Hence, in addition to protecting the spore during growth,mucilage initiates the breakdown of the cuticle by stimulating the production ofcutinases, which degrade the cuticle in advance of the penetration peg. However, thelonger the chain length of the compound, the greater the resistance to degradation[Kollatukudy 1985]. As a result, the mucilage-borne enzymes alone are not usuallysufficient to breach the epicuticle [Kollatukudy 1985, Kollatukudy eta!. 1987]. Therefore,in addition to the degradation of the cuticle by the mucilage enzymes, mechanicalpressure exerted on the growing peg by the appressorium anchored to the epicuticle bywater tension and the mucilage [Howard et al 1991] can force the penetration pegthrough the epicuticle, and expose the cuticle to further chemical degradation.Exogenous lipid emulsions applied to plant epicuticles may alter infection processes bychanging conditions needed for activation and/or germination, by interfering withrecognition, and/or by inhibiting or promoting penetration.51.2. Previous Studies of Lipid Emulsion Use on Plants and Their FungalPathoaensLipids were studied and used as antitranspirants before their consideration asplant prophylactic substances against fungal pathogens. In general, antitranspirantsreduce drought-induced water stress and improve water use efficiency [Quarles 19911.For instance, when water-stressed peach trees were sprayed with WiltPruf (10% 3—pinene (monoterpene) polymer emulsion, Nursery Specialty Products, Greenwich, NT),the trees showed a 30% reduction in water use over 90 days with no loss of growth[Steinburg et a! 19901. However, polymer antitranspirants provide no advantage inirrigated crops, and phytotoxic effects can retard plant growth and reduce plant yields[Quarles 19911. Tomato and cotton plants sprayed with antitranspirants have shown asmuch as a 4°C increase in leaf temperature, as transpiration was diminished [Gale andHagan 1966]. Also, photosynthesis decreases, as gas exchange and water vapourmovement across the cuticle are inhibited [Quarles 1991, Kastori et al 1991].Surfactants (such as the Triton X-series) used to emulsify lipids can induce theformation of ethylene and other symptoms associated with a wounding response[Lownds and Bukovac 1989]. Triton X-100 increased cuticular permeability up to 15times when applied at 0.2% on pear arid orange leaves [Riederer and Schonherr 19901.Only in areas which have little water or poor soil does the increased water use efficiencyoutweigh the detrimental effects of the antitranspirant sprays [Kamp 1985]. However,lipid emulsions applied to plants as antitranspirants have been shown in several casesto reduce fungal disease [Quarles 1991]. In these cases, the protection against fungiand subsequent increases in yields overcome the detrimental physiological effects ofthe antitranspirant substances.6Folicote, an paraffin wax emulsion (Crystal Soap and Chemical Co., Langsdale,PA), has shown some use as a plant prophylactic against fungal pathogens. Ziv andFredericksen [1983, 1987] demonstrated that Folicote (10% active ingredient) reduceddisease incidence by 66-78% for Erysiphe graminis DC. ex Mercat (wheat powderymildew), Puccinia recondita Rob. ex Desm. (wheat rust), Puccinia polyspora Underw.(corn rust), or Excerohilum turcium Pass. Leonard and Suggs. (sorghum leaf blight), ascompared to plants sprayed with the fungicide benomyl (Benlate, du Pont Chemical,Wilmington, Delaware). Also, the incidence of disease caused by Botrytis cinerea Pers.ex Fr. on bean, tomato, pepper and cucumber leaves treated with a 3OgIL solution ofFolicote was reduced 60% 8 days after inoculation [Elad et al, 1990]. Formation ofappressoria by E. graminis was shown in another study [Zekaria-Oren et al 19911 to bereduced 20% after application of Folicote (2% active ingredient). At this concentrationFolicote did not form a continuous coat on the leaf surface when observed by SEM.Thus, previous work has shown that Folicote forms a concentration-dependent film overthe plant epicuticle which apparently interfered with fungal recognition processes.Germination was not prevented by the paraffin wax emulsion, but appressoria formationand subsequent penetration were inhibited.While the mechanism of action of the triacylglyceridic oils from canola, sunflower,safflower and soybean seeds is not yet known, applied emulsions of these oils havebeen reported to have plant prophylactic activity [Northover etal 1993]. Applied at 0.5%as emulsions in 0.012% Agral surfactant, they reduced U. necator (grape powderymildew)-caused disease incidence on grape plants by at least 86% [Northover et a!.1993]. In situations where a large amount of inoculum was present, the efficacy of theoil emulsions was reduced, however. This may be due to insufficient coverage on thehost plant, an effect similar to the concentration-dependent coverage afforded byFolicote.7WiltPruf is also a useful plant prophylactic against fungal pathogens. WiltPruftreated wheat plants showed a 99% reduction in the incidence of Erysiphe graminis f.sp. tritici DC. (powdery mildew)-caused disease as compared to an untreated control[Ziv and Fredericksen 1983]. On cucumber plants a 55% reduction in disease incidencecaused by Erysiphe cichoracearum DC. (powdery mildew) was observed when 2OgILwas used; WiltPruf was observed to inhibit germination [Elad et a!, 1989]. Against B.cinerea, WiltPruf at 2OgIL reduced disease incidence by —50% on bean, tomato, pepper,and cucumber leaves and fruits [Elad et a!, 1990]. The high degree of control affordedby these lipid emulsions indicates possible practical usage for these substances asplant prophylactics.1.3. Systems Chosen for Study1.3.1. Lipids Emulsions to be Applied to the PlantsThe four lipids that were selected for testing in this study included twocommercial preparations, Folicote and WiltPruf, and two seed-storage lipids, canola oiland jojoba wax. Both Folicote (paraffin wax) and WiltPruf (f—pinene) consist of lipidsthat are similar to compounds which are abundant in the cuticle, but not the epicuticle.The first seed-storage lipid, canola oil, also has similarities to cuticular components. It isthe storage lipid of rapeseed (Brassica campestris L.), which consists 93% oftriacyiglycerides with oleic fatty acids [Ackman 1990]. The second seed-storage lipid,jojoba wax, was selected for its similarity to epicuticular lipids. It is a product of the fruitof the jojoba bush (Simmondsia chinensis L.) composed 98% of esters of cismonounsaturated w9 C18 to C26 alcohols and cis-monounsaturateci w9 C16 to C24 fattyacids [National Research Council (US) 1985, Desert King Jojoba Corp. 1990]. The8majority (—90%) of the waxes are between 38 and 44 carbons long [Johnson andHinman 1980], and thus have similarities to the esters in plant epicuticles. The twoseed-storage lipids were emulsified using either Triton X-100, which is I -(p-tertoctylphenyl)-w.-hydroxy-poly(oxyethylene) nonionic surfactant (Union Carbide, Montreal,Que.), or ISOL-RB, a polyglycerol nonionic surfactant (Leo-Chem lnc, Oakville, Ont.).The fact that the exogenous lipids have at least some similarity to epicuticle/cuticle lipidsmay be useful in suggesting the role that the epicuticle/cuticle lipids have in interferingwith fungal infection processes.1.3.2. Piant-Fungal Systems Chosen for StudyIn order to compare the effects of jojoba wax and canola oil with Folicote andWiltPruf on interactions during infection processes, four plant-fungal systems werechosen. Cucumber plants (Cucumis sativus L.) and two pathogens, Erysiphecichoracearum DC. (powdery mildew) and Didymella bryoniae Auersw. (black rotfungus) were selected. Grape plants (Vitis vinfera L.) and two pathogens, Uncinulanecator Schw. (powdery mildew) and Botrytis cinerea Pers. ex Fr. (bunch rot fungus)were also selected. The obligate powdery mildews and the nonobligate rot-causing fungiwere chosen for their dissimilar interactions with the epicuticle-cuticle, so as todetermine whether the exogenous lipid emulsions would have an effect on particularfungal infection processes.1.3.2.1. The Powdery MildewsBoth the powdery mildews (U. necator and E. cichoracearum) are common andconspicuous obligate pathogens of their respective hosts forming spots or patches ofwhite to grayish powdery growth on the upper surface of leaves and on shoots, and9covering young plant organs [Sitterly 1978, Pearson and Gadoury 1987, Jarvis andNuttall 1979, Bulit and Lafon 1978]. Both types of fungi penetrate only green, livingepidermal cells, invariably forming an appressorium over and entering through anticlinalcell walls and forming haustoria in the cell lumen [Bulit and Lafon 1978, Sitterly 1978,Aist and Bushnell 1991, Pearson 1988]. The resultant infection rarely kills the host, but,in utilizing photosynthate and nutrients via endohaustoria in epidermal cells, they canreduce the yield of the host by 20-40%.Research on infection mechanisms exists for at least two species of powderymildew. U. necator (powdery mildew of grape) has been observed to germinate and toproduce functional appressoria on any surface that presents a relative humidity of99.8% in the absence of free moisture [Blaich et al 1989, Heintz 1986]. Appressoriaformed regardless of whether these conditions were maintained by the use of differentosmotica in or an amyl acetate polymer over an artificial agar substrate, or by the use ofdried grape epidermal cell calluses in appropriate conditions of high humidity [Blaich etall 989]. Germination did occur at lower relative humidities and in the presence of freewater, but no appressoria were developed. Therefore, it seems unlikely that theepicuticle-cuticle layer (or a exogenous lipid emulsion layer) might interfere with U.necator spore activation or germination. Also, the presence or absence of cuticle or flatsurface or even normal tissue topography (thigmotropic recognition cues) were notimportant for appressorium formation. Moreover, under the strict environmentalconditions needed for appressorium formation, Heintz [1986] showed that penetrationpegs were able to penetrate artificial membranes impervious to enzymatic degradation.This result indicates the importance of the strict environmental conditions needed forappressoria formation, and the unimportance of chemotropic recognition cues. Waard[1971] suggested that a condition of high relative humidity in the absence of freemoisture necessary for conidial infection processes could be provided by the10transpirational gradient of grape leaves. Thus, the epicuticle-cuticle barrier (andtherefore perhaps the lipid emulsions) may act to modulate the transpirational gradientand thus interfere with the formation of appressoria by U. necator on the epicuticlesurface. The epicuticle-cuticle layer may provide resistance to penetration, but thelayer’s first action would seem to be interference with appressorial formation, sinceappressoria need to form before penetration is possible.Erysiphe gramirils f. sp. avenae (powdery mildew of rye) also requires particularconditions for appressorium formation. Appressorium formation and penetration occursnaturally on E. graminis-inoculated ryegrass (Lollum spp.), but not in vitro as seen in amicroscopic survey of E. graminis on agar [Carver and lngerson 1987]. This indicatesthe requirement for some particular surface or condition modulated by the surface toinitiate appressorium formation. However, appressorium development has beenobserved on the upper surface of ryegrass leaves with or without the epicuticle-cuticleremoved [Carver eta! 1990, Carver and Thomas 1990]. Since the cell wall morphologyof the upper surface observed with epicuticle removed was different from theepicuticular wax morphology [Carver and Thomas 1990], recognition by the spore onthe surface was not by thigmotropic or chemotropic cues, as these would have beenaltered by removal of the epicuticle-cuticle from the surface. The main effect of theepicuticle may have been interference with environmental conditions necessary forfungal infection by some factor intrinsic to the wax [Carver et al 1990]. Again, theepicuticle-cuticle layer may provide resistance to penetration, but the layer’s first actionwould seem to be interference with appressorium formation, since appressoria need toform before penetration.For both species of powdery mildew, it appears that the epicuticle-cuticle barrierprimarily interferes with particular environmental conditions needed for appressorium11formation. If all powdery mildew fungi behave similarly to the two described, then theepicuticle-cuticle barrier (and perhaps the applied exogenous lipids) of other host plantsmay interfere with appressorium formation in a similar manner.1.3.2.2. The Rot-Causing FungiB. cinerea is a nonobligate pathogen which establishes appressoria at thejunction of the berry and the pedicel, and then grows to cover grape berries with a grayfruiting layer [Jarvis 19771. These infections damage the grape epidermis, leading touncontrolled evaporation and eventual desiccation of the berry [Bulit and Dubois 1988].B. cinerea can penetrate intact epicuticle-cuticle barriers by the use of appressoria likethe powdery mildews [McKeen 1974], even penetrating nonhiving substances likeparaffin and gold film [Brown and Harvey 1927]. Serine-esterases have been detectedduring the penetration process [Shishiyama et a! 1970] and they seem to aid thepenetration peg, as penetration sites in the epicuticle-cuticle have sharp edges, ratherthan the indented appearance expected of a purely physical penetration [McKeen 1974].However, the fungus has been observed only rarely to form appressoria over living,turgid cells, with penetration most often occurring into senescent tissues [Brown andHarvey 1927, Buhit and Dubois 1988, Dugan and Blake 1989]. Direct penetration intohealthy tissues also seems unlikely because of the observation that cutinases producedby B. cinerea on tomato with no external sugar source degraded only 3-5% of theepicuticle-cuticle thickness in 18 hours [Salinas et a! 1986]. Most penetration attemptshave been observed in areas of the epicuticle which are thin due to injury and necrosis[Marois et al 1986] or to microfissures particularly near the pedicel [Blaich et all 984] orin peristomatal areas of grape berries where grape berry sugars are in greatestabundance [Jarvis 1977]. The fungus is often observed microscopically to move towardsan abundance of exuded sugars [Jarvis 1977, Dugan and Blake 1989, Padgett and12Morrison 1990]. Therefore, the epicuticle-cuticle probably interferes with access toexudates required for both the germination and growth of B. cinerea spores.Black rot of cucumber, caused by D. bryoniae, is a nonobligate pathogen ofcucumber [Jarvis and Nuttall 1979]. After penetration, D. bryoniae occupies cucumbertissues, causing wilting, desiccation, and canker formation [Jarvis and Nuttall 1979,Leski 1984]. Entry into plant tissues has been shown to be primarily via injured material[Svedelius 1990, de Neergaard 1989, Bergstrom et al 1982, Van Steekelenburg 1985,Svedelius and Unestam 1978], as opposed to direct penetration using appressoria. D.bryoniae pycnospores germinated in distilled water, tap water, or a supernatant ofmacerated cucumber or squash; the degree of germination differed only in that the germtubes in the macerated cucurbit extract exhibited greater branching and more vigorousgrowth [Chiu and Walker 1949, Svedelius and Unestam 19781. This stimulating effect ofcucurbit extracts on the infection process of D. bryoniae pycnospores has been shownin areas of hydathode guttation [Svedelius 1990], and at necrotic areas and near veins[Svedelius and Unestam 1978]. However, while artificial nutrient supplements increasedthe number of lesions produced by a conidial suspension as opposed to the number oflesions produced by an unsupplemented conidial suspension, mycelial suspensionsproduced an equal number of lesions regardless of the presence or absence ofadditional nutrients [Svedelius 1990]. This observation indicates that the exuciates atdamaged or necrotic sites on the plant likely promote germination, and have only asmall effect in chemotropic recognition of these damaged sites by the germ tube; ifchemotropic recognition had been important, then one would expect the supplementedmycelia to cause more lesions. Therefore, similar to the proposed exudate barrier of theepicuticle-cuticle for B. cinerea, the epicuticle-cuticle barrier may interfere with thegermination of D. bryoniae by limiting access of the pycnospores to the germinationpromoting plant exudates.131.4. Study OutlineThe literature survey suggests that epicuticle-cuticle barriers interfere withthe environmental conditions required for the induction of appressorium formationneeded for infection by powdery mildews, and with the exudation of exuded sugarsneeded for infection by rot-causing fungi. Seed-storage lipid emulsions may have asimilar effect, as the emulsions are lipidic like the epicuticle-cuticle. The objectives ofthis study were the following: (1) to determine whether seed-storage lipid emulsionshave potential as plant prophylactic substances and (2) to explore how lipid emulsionsapplied to plant epicuticles affect fungal plant pathogen infection processes. Seedstorage lipids were compared with commercial lipids with known modes of action in invitro, greenhouse and field trials.142. MATERIALS AND METHODSFor all experiments (except where indicated), data were analyzed forsignificant differences between treated groups by ANOVA, followed by Duncan’smultiple range tests where applicable, except where other tests are indicated. For alltests, a=0.05. ANOVA and Duncan’s multiple range tests were completed using thecomputer program PROC GLM provided in the SAS statistics package [SAS Institute1985].2.1. Preliminary Studies2.1.1. Determination of Phytotoxic Lipid Emulsion ConcentrationsIn order to determine the lipid and surfactant concentrations that were likely toprovide the most interference with fungal processes and the least damage to the subjectplants, a greenhouse study was done at Agriculture Canada Research Station (ACRS)at Summerland, B.C. One hundred ‘Auxerrois’ grape cuttings from one parent plantwere rooted and potted in 10 cm pots in topsoil and peat. At the start of the experiment,each four-week-old plant was pruned for the strongest shoot, which was tied to a 1 mstake pushed into the pot. The plants were hedged to the top of this stake when theywere seven weeks old and every two weeks thereafter; adventitious shoots wereremoved as they became apparent. All plants were watered regularly, receivingapproximately 100 mL of water per weekday, and were maintained at 25°C and 80%R.H. throughout the experiment.15Four blocks of plants consisted of six treatment representing six concentrationsof jojoba wax - 0%, 0.1%, 1.0%, 2.0%, 5.0% and 10.0% - in water with 1.0% Triton X207 (Rohm and Haas, Philadelphia, PA) surfactant. Four plants were unsprayed andwere used as a control for the effects of the surfactant. All plants were sprayed at sevenweeks with 1.0 L of treatment sprays using a 12 L Sanex backpack sprayer. Both sprayswere applied to run-off, which means the entire aerial surface of the plants was covered,and the spray was dripping off leaf margins. While Triton X-207 was used in this test, itwas due to its availability that it was used in lieu of Triton X-100, which was used in allother experiments. Triton X-1 00 is generally more available, and is extensively used inthe agricultural industry as a spreader/sticker [Vielvoye 1984]. A preliminary experimentalso showed that it was able to emulsify jojoba wax to the same degree (subjectivelydetermined) as Triton X-207.The effect of the sprays on the plants was determined once at 10 weeks (21 daysafter spraying) by measuring the area of the primary leaf from the sixth node of eachtreated plant with a Licor 3000 Aerometer (Li-Cor Inc., Lincoln, Nebraska). Damage tothe leaf was estimated by cutting necrotic material out of the leaf, and determining leafarea remaining; the difference was the leaf damage. In addition to these measurements,subjective observations were made throughout the experiment, and photographs weretaken six weeks after spray application. This experiment was done once.2.1.2. Effect of Exogenous Lipid Emulsions on the Growth of Didymellabryoniae and Botrytis cinerea In WtroThis experiment was designed to determine the effect of jojoba wax and canolaoil when the lipids were placed in direct contact with B. cinerea and D. bryoniae. It alsotested the effects of the surfactants Triton X-1 00 and ISOL-RB on the fungi, so that this16effect could be accounted for in subsequent studies of the lipids’ effects. The trialsconsisted of 520 agar petri dishes separated into 13 treatments, as listed in Table 1.Table 1. Experimental seed-storage lipid emulsions applied to 1 % wlv sucrose-enrichedpotato dextrose agar to evaluate the relative efficacy of the emulsions on the growth ofD. bryoniae and B. cinereaControl Treatments Lipid-Containing TreatmentsUntreated 1.0% jojoba wax + 0.5% Triton X-100Water only 2.0% jojoba wax + 0.5% Triton X-1 000.5% benomyl/1 .0% Captan mixture 1.0% canola oil + 0.5% Triton X-100(B. cinerea commercial control) 2.0% canola oil + 0.5% Triton X-1 001.0% iprodione 1 .0% jojoba wax + 0.5% ISOL-RB(0. bryoniae commercial control) 2.0% jojoba wax + 0.5% ISOL-RB0.5% Triton X-100 WiltPruf (10% active ingredient)0.5% ISQL-RB Folicote (4% active ingredient)The sucrose-enriched potato dextrose agar petri dishes were made using amixture containing 40 g of potato dextrose agar (Difco Laboratories, Detroit, Mich.), 10 gof sucrose and 1.0 L of tap water, which was sterilized at 1500 kPa in a BarnsteadAutomatic Autoclave (Barnstead Still and Autoclave, Co, Boston, Mass.). The resultingsolution was poured into 80, 10 cm-diameter petri dishes per litre. The treatments wereprepared under sterile conditions in an Ultraclean laminar flow hood (Agnew-HigginsInc., Garden Grove, CA) using sterile water. For each treatment a 300 pL aliquot of thetreatment solution was spread onto each petri dish allocated to the treatment using aLabLine petri dish rotary autoplater (LabLine Instruments Inc., Melrose Park, Ill.) and abent glass rod. The 300 pL treatment volume was used because a preliminaryexperiment showed that 300 pL aliquots allowed differentiation between the surlactantand the lipid effect.After a 24 hour drying period, the petri dishes for each treatment were separatedinto four blocks of 130 petri dishes, one for each of the three B. cinerea isolates (from17Interior Spruce, from Western Hemlock, and from Lodgepole Pine, each supplied by B.Dennis, Pacific Forestry Centre, Victoria, B.C.) and one for a D. bryoniae isolate isolatedfrom a black rot-diseased greenhouse cucumber plant. The petri dishes for each isolatewere inoculated in three equidistant spots with 10 p1 of the respective spore suspension(10 x 1 0 spore mL-1), and then were incubated at approximately 20°C. The inoculationspot boundaries were marked with a fine black pen after 24 hours, and the diameters ofthe original spot and the final colony were measured after 4 days. As there was noindependence between spots on the individual petri dishes, means for the spotdiameters for each plate were used in the subsequent analysis. Also, data on colonydiameters from the petri dishes for the separate B. cinerea isolates were pooled, sincethere was no significant difference for the treatment effects on the isolates. Thisexperiment was completed twice.2.2. Comoarlson of Exogenous Lipid Emulsions as Phytoprotectants and asAntitranspirants of Cucumber PlantsAll plants for the cucumber plant trials were “Chicago Pickling” cucumbers startedfrom seeds (Sunset Seed Co, Ltd., Armstrong, B.C.) and grown in a greenhouse duringOctober and November in 10 cm pots using a medium of 20 kg topsoil, 1 kg peat, and170g of 14-14-14 Osmocote fertilizer (Grace Sierra Horticultural Products, Malipatus,Calif.). The plants were staked and tied as necessary, watered daily, and maintainedbetween 15 and 30°C at —60% R.H. If the plants were on a mist bench, the temperaturewas maintained at —22°C and >95% R.H. with free moisture.182.2.1. Effects of Exogenous Lipid Emulsions on Powdery Mildew of CucumberPlantsTrials were done on cucumber plants in greenhouse situations at USC in 1993 inorder to determine the effects of exogenous lipids on E. cichoracearum . Eleven, 6-week-old plants were used per treatment for both trials (Table 2). Treatments wereapplied using a 12 L Solo backpack sprayer.Table 2. Experimental seed-storage lipid emulsions applied to “Chicago Pickling”cucumber plants in greenhouse trials for determining the effect of the emulsions onpowdery mildew disease severityControl treatments Experimental treatments0.5% Triton X-100 1.0% Jojoba wax + 0.5% Triton X-1 000.5% ISOL-RB 1.0% Jojoba wax + 0.5% ISOL-RB1.0% wlv sulfur1 1.0% Canola oil + 0.5% Triton X-100Water only WiltPruf (10% active ingredient)Folicote (4% active ingredient)1. Kumulus D (sulfur) manufactured by BASE, GermanyThe cucumber plants were inoculated by taking sporulating powdery mildew-diseased leaves from seed-grown cucumbers and various squash, and shaking theseleaves over the cucumbers plants, one day after they were sprayed. The number ofspores was determined for the second trial by placing 20 microscope slides with 4 cm ofdouble-sided Scotch tape (3M Canada, London, Ont.) on the greenhouse table next toand inside the block of cucumber plants. Spores adhering to the tape were countedunder a microscope at 10 microscope fields per slide using a 40x objective.Approximately 2.0 x iO spores cm2 were being placed on the cucumber leaves.Estimates varied from 2.4 x iO spores cm2 on the outer leaves to 1.7 x iO sporescnv2 on the inner leaves. Since powdery mildew requires humidity for germination, the19plants were kept on an operating mist bench for 4 days with precautions to ensure thatno free moisture could accumulate on the foliage.The powdery mildew disease severity on the cucumber plants was measured 17days after inoculation by counting the number of colonies per third, fourth and fifthleaves, then measuring leaf area (Licor 3000 Aerometer). These measurements wereaveraged per plant for subsequent analyses. Estimations of the colony sizes on theleaves were noted. A preliminary trial done in 1992 with the same design was done, butis not discussed, since the results were not as clear as the 1993 trials. This experimentwas done twice in 1993.2.2.1 .1. Scanning Electron Microscope Studies of the Effects of Exogenous LipidEmulsions on Ervsiphe cichoracearum on Cucumber LeavesIn order to more closely visualize the effects of the applied lipids on the E.cichoracearum infection signs, nine 6-week-old cucumber plants, one for each of thetreatments listed in Table 2, were separated from the other cucumber plants. Theseplants were then inoculated by the same method as the other cucumber plants, and twoleaves were removed from the plants after 48 hours. For each leaf from each treatedplant, a 1 cm diameter cork borer was used to remove a leaf disk immediately adjacentto the main leaf vein disk from the centre of the leaf. These disks were immediatelyaffixed using paper glue (UHU Stick, Eberhard Faber, Oakville, Ont.) to 1 cm-diameterscanning electron micrograph (SEM) plugs. The leaf-covered disks were left to air dryfor 10 days.After the drying period, the dried disks were gold coated in an argon environmentusing a Nanotech II SEMPrep Sputter Coater. The disks were then observed under a20Cambridge 250 Scanning Electron Microscope (Cambridge Scientific Instruments, Inc.).Micrographs were taken using Polaroid Landmark 55 film. Spores observed on the driedcucumber leaf disks were similar in proportion and shape compared to micrographs ofE. cichoracearum spores on disks of air-dried cucumber leaves displayed by Samuels eta!. [1991].The micrographs were studied to determine what differences, if any, might existin spore germination on leaves treated with different exogenous lipid emulsions. Thenumber of germinated spores and the number of spores with appressoria in the each ofthe 38 SEM sample fields (300 pm wide) observed per leaf disk were counted. Thisexperiment was done once.2.22. Effects of Exogenous Lipid Emulsions on Black Rot and Growth ofCucumber PlantsIn 1993 in the same greenhouse as the trials testing the effects of exogenouslipids on powdery mildew, trials testing the effect of exogenous lipids on black rotdisease incidence on cucumber plants were done. Eleven, 6-week-old plants were usedper treatment for both trials (Table 2). The same treatment sprays that were used in thepowdery mildew trial were used, except that 1.0% w/v iprodione (Rovral, May and BakerLtd., Essex, England) replaced sulfur as the commercial control. The same treatmentswere used because a preliminary trial had shown Folicote and Triton X-1 00 might beeffective in reducing black rot disease incidence. Since D. bryoniae is a woundpathogen, cucumber plants were prepared by slicing the petioles on the third, fourth,and fifth nodes in half. After a 3 hour drying period, the treatments were applied using a12 L Solo backpack sprayer; the sprays were allowed to dry for 3 hours. Then 10 iL of aD. bryoniae spore suspension (5 x 1 0 spores mL-1) was dropped onto the spray21covered injured petioles. The plants were then placed on a mist bench for 4 days, thenremoved to the greenhouse. The effects of the treatments were determined by countingthe number of diseased inoculation points per plant 17 days after inoculation. Thisexperiment was done twice.The leaf area measurements taken for all the leaves that were sampled forpowdery mildew colony presence gave an indication for growth. In order to determinethe effect of the lipid emulsion applications on growth, the random effect of the numberof powdery mildew colonies was accounted for in the subsequent ANOVA.2.3. Effect of Seed-storage Lipid Emulsions as Phytoprotectants and asAntitranspirants of Grace PlantsIn order to test how jojoba wax and canola oil would perform as prophylactics infield situations, three trials were completed at two sites - Torrs Vineyard (TV) inKelowna, B.C., and the Agriculture Canada Research Station (ACRS) at Summerland,B.C. Trials were done at TV for two years (1991 and 1992) and at ACRS during 1992only.2.3.1. Plant Material, Husbandry, and SprayingThe trial at ACRS used ‘Riesling’ grape plants 3 years old spaced at 3 m x 2 m(row x vine) in five blocks. At TV, in Kelowna, B.C., 4-year-old ‘Auxerrois’ plants spacedat 2.4 m x 1.5 m were used in six blocks. Vines were trained to 0.6 m-high bilateralcordons and pruned to 16 shoots m1 as 2-node upward-oriented spurs. Shoots weretrained upward to form a vertical canopy. Each block was divided into treatments, witheach treatment represented by a “post-length” of grapes, meaning the grape plants22between the two posts which supported the trellis wires for those plants. Treatmentswere randomized with respect to their position within the blocks to reduce the in-blockvariation. Treatments and spray application dates for the trials are listed in Table 3. Thetreatment names “bloom”, “pre-veraison”, and ‘post-veraison” refer to general period ofgrape plant growth as adapted from Eichhorn-Lorenz growth stages [Pearson andGoheen 1988]. In both field sites, rows of plants were oriented north-south.23Table 3. Experimental seed-storage lipid emulsions and schedule of their application to‘Auxerrois’ grape plants at TV and ‘Riesling’ plants at ACRS, applied to determine theeffects of the various treatments in reducing powdery mildew disease severity and/orincidenceTV 1991 May Jun Jun Jul Jul Jul Aug AugTreatment 22 4 22 3 14 28 10 25Water control + + + + + + + +Commercial control2 + + + + + + + +1.0% Jojoba “All-season”3 + ÷ + + + + + +1.0% Jojoba “Bloom” + + +1.0% Jojoba “Pre-veraison” + + +1.0% Jojoba “Post-veraison” + + + +TV 1992 May May Jun Jun Jul Jul AugTreatment 12 23 6 23 6 21 54Water control + + + + + + +0.2% Triton X-100 control + + + ÷ + + +1.0% Jojoba “All-season” + + + + + +0.2% Jojoba “All-season” + + + + + + +1.0% Jojoba “Pre-veraison” + + +0.2% Jojoba “Pre-veraison” + + +ACRS 1992 5 Jun Jul Jul Jul Aug AugTreatment 18 3 17 31 12 6 21Water control + + + + + +Commercial control + + + + ÷1.0%Jojoba + + + + +1.0%Canola + + + + +0.5% Canola + + + + +1Treatment names refer to types of controls (ACRS and TV), concentrations of sprays(ACRS and TV), or the general period of spray application (TV). ‘÷‘ indicates a particulartreatment was sprayed on a certain day.2. Commercial spray was 5% sulfur (Kumulus S, BASF, Germany) sprayed biweeklyfrom 10 to 15 cm new growth to 30 days prior to harvest, and at TV, 1 % Captan(Standard Oil Development Co.) or 1% iprodione (Rovral, May and Baker Ltd., Essex,England) (I) at bloom, (ii) at 80% capfall, (iii) at bunch closure, (iv) at veraison and (v) 10to 14 days later if needed. Captanhiprodione combinations were not sprayed at ACRS.3. Sprays included the lipid at the concentration indicated in water with 0.2% Triton X100 surfactant for emulsification.• Due to overabundance of powdery mildew, the commercial control was used afterAugust 5 for all plants.5. All plants were sprayed with 5% sulfur until two weeks before June 18.6. Due to farmer error, plants were sprayed with 5% sulfur on August 12.24With respect to husbandry, all plants were watered, pruned, suckered, and tiedaccording to grape industry standards [Vielvoye 1984]. ACRS grapes were watered by amicrojet system, whereas grape plants at TV were watered by overhead sprinklers. Bothvineyards were hedged in midsummer, and both were cluster-thinned to two clusters pershoot. Treatments were applied in the 1992 field trial at ACRS by a 12 L Sanexbackpack sprayer. All other sprays were applied using a tractor mounted 220 L sprayer.Sprays were applied to run-off; that is, the entire aerial surface of the plants werecovered, and the spray was dripping off leaf margins.2.3.2. Effects of Exogenous Lipid Emulsions on Powdery Mildew and BunchRot of Grape PlantsIn 1991, grape plants at TV were inoculated with U. necator on August 11 byhanging powdery mildew-diseased grape clusters collected from ACRS from the 1.5 mwire of the grape trellis and irrigating the vineyard for two hours, and then removing thediseased grape clusters 24 hours later. Sufficient overwintering inoculum was present in1992 at TV to provide adequate natural inoculation. In 1992, infected clusters from TVwere used in two attempts in late July to inoculate vines at ACRS, but this failed toinitiate disease, and further attempts were not made. At TV in 1991 and at ACRS in1992, plants were inoculated with B. cinerea two and three weeks before harvestrespectively. Berry clusters were sprayed to run-off with a conidia suspension ofapproximately 2.5 x spores mL1 in a 2% sucrose solution, Inoculation of TV in 1992was not attempted due to an U. necator epiphytotic, which had already resulted in thedesiccation of the berry clusters.To assess the degree of powdery mildew disease incidence, U. necator coloniesfor two leaves per vine at the 1.5 m level were counted at TV in 1991 and 1992. In25addition, the number of mildewed grape berry clusters was counted at harvest in 1991.The number of clusters with bunch rot-diseased areas greater than the size of a singlegrape were counted at TV in 1991 and at ACRS in 1992.23.3. Effects of Exogenous Lipid Emulsions on Grape Plant Growth, Yield andSurvivalIn 1991 at TV, transpiration was measured in the centre of leaves on the southand outside of the grape plant canopies at the 1.5 m level 4-6 days after each sprayusing a Licor 1600 porometer (Li-Cor Inc., Lincoln, Nebraska). At TV in 1992, stomatalresistance was measured at 9:30am and at 12:30 am for leaves selected as in 1991 onthree dates in mid-season (June 7, 17, July 3). This was done to determine differentstomatal aperture sizes at the beginning of the day, so that stomatal resistancedifferences in mid-day, when the plants normally experienced some water stress (Dr. AReynolds, pers. comm.), could be determined controlling for the original aperture sizes.No transpiration measurements were taken at ACRS.Growth was estimated at TV in 1991 by comparing the grape plants to anadapted Eichhorn-Lorenz growth stage outline [Pearson and Goheen 1 988J. At TV andACRS in 1992, measurements were made throughout the growing season from twoshoots on each plant, one nearest to and one furthest from the trunk on the south side.Fifth and tenth internodes were marked with spray paint, and every tenth leaf wasidentified with a twist tie. On these shoots, weekly measurements were made of thenumber of leaves and internodes per shoot, of the length of the fifth and tenthinternodes, the tenth leaf width, basal cluster length, and the average berry width. Leafarea was estimated by assuming the leaf width represented the diameter of a circle, thearea of which was then calculated. Berry volume was estimated by calculating the26volume of a sphere with berry width as the diameter. Also, leaves were sampled weeklyfor chlorophyll determination. Chlorophyll was extracted in 5 mL of N-Ndimethylformamide from 2 cm2 leaf portions next to the main vein, using the method oflnskeep and Bloom [1985].In order to isolate the effects of treatment on each measurement, without thecomplicating effects of other interdependent measurements on the same plant, a type ofpath analysis (described by Li [1975]) was done. In the first of two steps, the effects ona particular variable of other measured variables were removed, and then, in the secondstep, the effects of the treatments alone on the particular variable were studied. In thefirst step, each of the dependent measured variables (number of leaves, number ofinternodes, fifth and tenth internode length, tenth leaf area, leaf chlorophyll, rachislength, berry volume, and the amount of powdery mildew where applicable) was isolatedas single dependent variable, with the remaining dependent variables held asindependent. Using PROC REG [SAS Institute 1985], the subset of the variablesconsidered independent for the analysis which made a contribution to the correlationcoefficient was selected by forward stepwise selection. The residuals from this modelwere then taken into the second step of the analysis, where they were subjected to anANOVA to determine whether the various treatments had effects on the subject variablewith respect to its predicted value. The steps in the analysis are diagrammed in Figure1. This kind of analysis may be considered to create a regression representing theamount of variation in a given dependent (e.g. berry volume) contributed by each of theother variables (e.g. leaf chlorophyll, leaf number, rachis length, etc.). The regression isthus a calculated mean for the dependent variable which takes into account thecontributions of the variables held as independent variables; that is, the other dependentvariables. The regression does not take into account variation caused by treatment orblock effects. Therefore, a subsequent ANOVA of the residuals of the regression, the27IndependentMeasurementsCorrelations between X maydirectly or indirectly affect Y.Residuals are Y after effects ofXj are accounted for.DependentMeasurementsResidualsAffected by:TreatmentsReplicationsFigure 1. Diagram of interrelationships between data and the method used to determinethe effects of the interrelationships on a single variable, so that those effects may beaccounted for in subsequent ANOVA. Adapted from Li [1975].“distance” between the observations and the calculated mean for the dependentvariable, allows study of the variation in the dependent variable not complicated by theeffects of the other dependent variables held as independent variables in theregression. This variation will be due to the treatment and block effects. This two-stepprocess permits the separation of true treatment effects.Grape clusters were harvested October 11 at TV in 1991, September 20 at TV in1992, and October 16 at ACRS in 1992. During harvest, the number and weight ofgrape berry clusters were determined for each plant. Except for the TV 1992 trial, fromwhich the grapes were too desiccated by the U. necator epiphytotic to analyze further,random berry samples (100 berries per treatment) from each treatment wereimmediately taken to ACRS and stored at -40°C. At a later time, the berries were28weighed (per hundred berries), juiced, and the concentration of soluble solids (in °Brix)for the berries was determined using an Abbé refractometer (AC Instruments, Buffalo,NY). Then, pH was determined for the berries with a Fisher 825MP pH meter (FisherInstruments, Vancouver, B.C.). The total titratable acids were determined for 50gextracts of whole berries using the extraction and measurement method described byMattick [1983].Both titratable acidity and pH were measured because they are not necessarilycorrelated quantities. The main berry acids (tartaric acid and malic acid) exist partiallyas potassium salts at normal berry pH [Coombe 1992]. After veraison, catabolism ofthese acids, primarily malate, occurs as respiration increases, but tartarate remainsmainly in salt form, degrading at a minimal rate in comparison to the malate [Winkler eta! 1974, Coombe 1992, Gutierrez-Granda and Morrison 1992]. Malate degradationcontributes directly to increased pH, while the titratable acidity measurement donewhich includes tartarate does not necessarily.The effect of the lipid sprays on plant survival was determined in all trials beforeand after the growing season by weighing cane prunings (the winter before, and thenafter) on site using a dairy scale accurate to 10 g. The difference between the beforeand after prunings indicated changes in the degree of survival to the next growing year.293. RESULTS31. Effects of ExoQenous Lipid Emulsions as Phytoprotectants of Grape andCucumber Plants3.11. Effects of Exogenous Lipid Emulsions on Powdery Mildew of CucumberPlantsWhen the seed-storage lipid emulsions were tested in comparison to commerciallipid emulsions against E. cichoraceaum, only plants sprayed with jojoba wax/Triton X100 consistently showed as few E. cichoracearum colonies per leaf area as thosetreated with the commercial control in 1993 (Table 4). However, the trend was that thejojoba wax/ISOL-RB, WiltPruf, and canola oil/Triton X-100-treated leaves had fewercolonies per leaf area than leaves sprayed with other treatments. Fewer colonies wereobserved on the leaves treated with the Triton X-1 00 control than the water control-,ISOL-RB control- or the Foilcote-treated leaves. These latter three treatments showedno significant difference in the number of E. cichoracearum colonies per leaf area. Whilethe separation between plants sprayed with the various treatments was not as clear asthe 1993 trials, similar trends were observed for trials with an identical design done in1992 (Appendix 1).30Table 4. Number of E. cichoracearum colonies per leaf area 17 days after inoculation ongreenhouse-grown “Chicago Pickling” cucumber plants treated with experimentalexogenous lipid emulsionsNumber of E. cichoracearum colonies per cm2 leafTreatment Trial 1 Trial 2Water control 0.8 A 1.3 A0.5% ISOL-RB control 0.7 A 1.3 A4.0%Folicote 0.7 A 1.2 A0.5% Triton X-100 control 0.4 B 0.7 B1.0%Canola/0.5% Triton X-100 0.3 BC 0.4 C10% WiltPruf 0.2 CD 0.1 D1.0% Jojobal0.5% ISOL-RB 0.1 D 0.3 CD1.0% Jojobal0.5% Triton X-100 0.1 D 0.1 DCommercial control 0.0 D 0.0 D1. For both trials, n=1 1. Means represent averages of all plants of a particular treatmiiifwith counts of three leaves (3rd, 4th, and 5th from base) per plant. Column meanssharing the same letter are not significantly different according to Duncan’s multiplerange test, p.<0.05.3.1.1.1. Scanning Electron Microscope Studies of the Effects of Exogenous UpidEmulsions on Powdery Mildew of Cucumber PlantsThe scanning electron micrographs taken of cucumber leaves 48 hours afterinoculation revealed different infection structure morphology on leaves treated withdifferent exogenous lipid emulsions. Germination occurred on all leaves except thosetreated with WlltPruf (Table 5). Only the water and surfactant control-treated leaves hadgerm tubes which actually produced appressoria (Table 5). After 48 hours, while thecommercial control- and Folicote-treated leaves had germinated spores with a fewappressoria formed, all leaves sprayed with the jojoba wax or canola oil emulsionsshowed germinated spores without appressorium formation.31Table 5. Number of E. cichoracearum infection structures observed by SEM on“Chicago Pickling” cucumber leaves air-dried 48 hours after spraying with experimentalexogenous lipid emulsions, and subsequent inoculation with E cichoracearumTreatment % Germinated 1 % Germinated withappressoriaWater control 50.0 100.00.5% Triton X-1 00 control 60.5 60.90.5% ISOL-RB control 44.7 100.0Commercial control 55.3 4.8Folicote (4% active ingredient) 42.1 6.31.0% Canola oil! 0.5% Triton X-1 00 60.5 0.01.0% Jojoba waxl0.5% Triton X-1 00 39.5 0.01.0% Jojoba wax!0.5% ISOL-RB 39.5 0.0WiltPruf (10% active ingredient) 0.0 0.01. n=38.When observed with the SEM, the leaves sprayed with ISOL-RB were similarto the water control-sprayed leaves; therefore, in the micrographs, only a sample fromthe water control-treated leaves is shown (Figure 2A) Similarly, Folicote-, canolaoil/Triton X-1 00- and jojoba wax/Triton X-1 00 emulsion-treated leaves were not differentfrom one another, so only a sample from the jojoba wax emulsion-treated leaves isshown (Figure 2D). Spores germinated on jojoba wax/ISOL-RB leaves were similar tothe water control-sprayed leaves, except that no appressoria were formed. Spores onWiltPruf-treated leaves did not germinate or produce any germination structures. Thewater- and ISOL-RB-treated leaves had spores with long, branched germ tubes with anappressorium at the end of each germ tube branch. However, the Triton X-1 00 controltreated leaves showed extremely short germ tube growth with large, elaborateappressorial structures (Figure 2C). Like the spores on the Triton X-1 00 control-treatedleaves, the leaves treated with the commercial control (Figure 2B) or any of the lipidTriton X-100 combinations except WiltPruf showed extremely short germ tubes;however, there was no appressorium development at the end of the germ tubes.32Figure 2. Examples of E. cichoracearum infection structures observed by SEM on“Chicago Pickling’ cucumber leaves air-dried 48 hours after spraying with experimentalexogenous lipid emulsions, and subsequent inoculation with E. cichoracearum. (A)Water control-treated leaves. (B) Commercial control-treated leaf. (C) Triton X-100control-treated leaf. (D) Jojoba wax emulsion-treated leaves.333.1.2. Effects of Exogenous Lipid Emulsions on Powdery Mildew of GrapePlantsOn ‘Auxerrois’ grape plants at TV, the pre-harvest measurement of September27, 1991 showed no U. necator colonies on the 1.0% “all-season” and the commercialcontrol-treated leaves (Table 6). The 1.0% “pre-veraison”- and 1.0% “post-veraisontreated leaves were also not significantly different from the commercial control. In 1992,a September 7 pre-harvest colony count showed fewer colonies on the 1.0% “allseason”-sprayed leaves as compared to all other treatments. In addition, the Triton X100 control, both the 1.0% and 0.2% “pre-veraison”, and the 0.2% “all-season”-treatedleaves showed fewer U. necator colonies than those treated with the water control(Table 6). At harvest in 1991, there was no difference in the number of powdery mildew-diseased clusters in the 1.0% “all-season”, 1.0% “pre-veraison”, 1.0% “post-veraison”,and commercial control-treated plants.Harvest data for 1992 is not displayed for TV because overwintering E.cichoracearum inoculum from 1991 initiated a substantial epiphytotic before the jojobawax emulsion sprays were applied. By the harvest time, the berries on the grapeclusters were diseased with powdery mildew to the point where all the berries weredesiccated, and many had fallen off the clusters. Because of the U. necator epiphytotic,no colony counts after August 7 were collected either (Table 6). However, leaves whichwere observed to be healthy (uninfected) early in the season which received at leastone spray of the jojoba wax emulsion remained healthy throughout the season.34Table 6. Mean number of U. necator colonies per experimental seed-storage lipidemulsion-treated ‘Auxerrois’ grape leaf in the preharvest, and mean number ofexperimental jojoba wax emulsion-treated ‘Auxerrois’ grape clusters at harvest showingsymptoms of powdery mildew diseaseNumber ofNumber of colonieslleaf diseased clustersTreatment Sept. 271991 Aug. 71992 Oct. 111991Triton X-100 control - 26.9 B -1.0% “Bloom” 69.2 A - 4.8 AWater control 63.3 A 65.2 A 4.3 AB1.0% “Pre-veraison” 40.8 AB 25.8 B 1.8 BC0.2% “Pre-veraison” - 27.4 B -0.2% “All-season” - 18.9 B -1.0% “All-season” 0.0 B 3.2 C 0.4 C1.0% “Post-veraison” 20.0 B 1.2 CCommercial 0.0 B - 0.4 C1. For 1991 samples, n=24; for 1992, n=16 for the Triton X-100 control, and both “Preveraison” treatments, n=24 for both “All-season” treatments, and n=64 for the watercontrol. Leaf means represent averages of all plants of a particular treatment, withcounts of two leaves per plant. Column means sharing the same letter are notsignificantly different according to Duncan’s multiple range test, p<0.05.3.1.3. Effect of Exogenous Lipid Emulsions on the Growth of Didymellabryoniae and Botrytis cinerea In VitroThe petri dishes spread with the commercial control, WiltPruf, and treatmentswhich contained Triton X-1 00 showed retarded B. cinerea and D. bryoniae colonygrowth (Table 7). The untreated petri dishes and those spread with the water control, alltreatments containing ISOL-RB, and Folicote (with the exception of the first D. bryoniaetrial) showed the greatest colony growth. The trend was that the petri dishes spread withthe treatments which contained Triton X-1 00 showed the least colony growth for bothspecies of fungi, consistently second only to that presented in the commercial control-treated petri dishes (Table 7). In addition, the lipid components of the Triton X-1 00-35containing emulsions did not seem to make a consistent difference amongst the petridishes in which Triton X-1 00-containing treatments were spread. The trend, however,was that the petri dishes which were spread with an emulsion which included a lipidcomponent showed more growth than the petri dishes spread with Triton X-1 00 alone.Table 7. Difference in B. cinerea and D. bryoniae colony area between 1 and 4 daysafter inoculation on experimental exogenous lipid emulsion treated 1 % sucrose-enrichedPDADifference in B. cinerea Difference in 0.. biyoniaecolony area (mm2) colony area (mm2)Treatment Trial 1 Trial 2 Trial 1 Trial 2Untreated control 1500.0 A2 1500.0 A 1500.0 A 1500.0 AWater control 1500.0 A 1500.0 A 1397.2 A 1500.0 A0.5% ISOL-RB control 1500.0 A 1500.0 A 1460.0 A 1500.0 A1.0% Jojobal0.5% ISOL-RB 1500.0 A 1500.0 A 1500.0 A 1500.0 A1 .0%CanolaIO.5% ISOL-RB 1500.0 A 1500.0 A 1500.0 A 1500.0 A4.0% Folicote 1500.0 A 1500.0 A 1120.0 B 1500.0 A10% WiltPruf 1000.7 B 865.7 B 719.8 D 583.6 B1 .0%Canola/0.5% Triton X-100 525.7 C 119.6 D 846.9 C 246.8 D2.0%CanolaIO.5% Triton X-100 222.1 D 201.3 C 489.8 E 506.4 B1.0% Jojobal0.5% Triton X-100 118.3 E 159.3 CD 156.6 F 358.4 C2.0% JojobalO.5% Triton X-100 233.0 D 100.6 D 445.0 E 236.0 D0.5% Triton X-100 control 195.1 DE 125.5 D 226.1 F 3866 CCommercial control 0.0 F 0.0 E 20.0 G 21.0 E1. Maximum colony diameter before observable colony interference was 44 mm; that is,1500 mm2.2. For B. cinerea trials, n=30; for D. bryoniae trials, n=10. Each means representsaverages of all petri dishes of a particular treatment with three colonies per dish.Column means sharing the same letter are not significantly different according toDuncan’s multiple range test, p<0.05.363.1.4. Effects of Exogenous Lipid Emulsions on Black Rot of Cucumber PlantsWhen the exogenous lipids were tested against D. bryoniae, plants sprayedwith any treatment except the Folicote spray in the first trial and the commercial controlin both trials did not differ in the percentage of diseased, inoculated, injured petioles ascompared to the water control (Table 8).Table 8. Percentage of black rot-diseased inoculated petioles on six-week-old “ChicagoPickling” cucumber plants 17 days after application of experimental exogenous lipidemulsion treatments, and inoculation with D. bryoniaePercentage of black rot-diseasedpetioles per plantTreatment Trial 1 Trial 21.0% Jojoba/0.5% Triton X-1 00 67 A1 48 A1 .0%Canola/0.5% Triton X-1 00 68 A 50 A10% WiltPruf 57 AB 47 A0.5% ISOL-RB control 57 AB 50 A1.0% Jojobal0.5% ISOL-RB 50 AB 50 AWater control 47 AB 50 A0.5% Triton X-100 control 33 BC 43 A4.0%Folicote 17 CD 33 ACommercial control 0 D - 3 B1. For both trials, n=1 1. Means represent averages of all plants of a particular treatment,with three petioles per plant. Column means sharing the same letter are not significantlydifferent according to Duncan’s multiple range test, p.<0.05.3.1.5. Effects of Exogenous Lipid Emulsions on Bunch Rot of Grape PlantsAt TV in 1991, there was no difference in the number of bunch rot-diseased‘Auxerrois’ grape clusters between treated plants sampled 14 days after inoculation,except that the 1.0% “all-season” and “post-veraison”-treated plants were significantlydifferent from the 1.0% “pre-veraison”-treated plants (Table 9). However, there were37fewer bunch rot-diseased ‘Riesling’ grape clusters from plants treated with any of thethree lipid sprays at ACRS in 1992 than were observed on plants treated with either thecommercial or water control (Table 9). No assays of bunch rot disease incidence for the‘Auxerrois’ plants at TV in 1992 are indicated because the U. necator epiphytoticdamaged the grape berries beyond the point where such damage could be accountedfor in subsequent analyses.Table 9. Mean number of experimental seed-storage lipid emulsion treated-’Auxerrois’and ‘Riesling’ grape clusters harvested per plant showing bunch rot disease incidenceTreatment Number of Treatment Number of‘Auxerrois’ grape ‘Riesling’ grapeclusters with clusters with bunchbunch rot 1991 rot 199?Water control 1.7 AB1 Water control 19.1 ACommercial control 1.2 AB Commercial control 11.9 A1.0% “All-season” 0.8 B 1.0% jojoba wax 3.3 B1.0% “Bloom” 1.2 AB 1.0% canola oil 4.2 B1.0% “Pre-veraison” 3.1 A 0.5% canola oil 6.0 B1.0% “Post-veraison” 0.4 B1. For ‘Auxerrois’ grape clusters, n=24; for ‘Riesling’ grape clusters, n=12. Columnmeans sharing the same letter, or where letter is not indicated, are not significantlydifferent according to Duncan’s multiple range test, p<0.05.3.2. Effect of Exogenous LipLd Emulsions as Antitranspirants on Cucumber andGrace Plants3.2.1. Determination of Phytotoxic Lipid Emulsion ConcentrationsThe primary goal of the greenhouse study was to determine the maximumconcentration of jojoba wax that could be applied to grape plants without observabledetrimental effect. The area of leaves showing necrosis increased for plants sprayed38with concentrations higher than 1.0% (Table 10, Figure 3D, E, F). In fact, plants sprayedwith 10% jojoba wax had no leaves when the treatments were photographed; no pictureis included in Figure 3. The plants sprayed with the 1.0% concentration were notdifferent from the water controls. As a result, 1.0% was used as an upper limit toconcentrations of both jojoba wax and canola oil in subsequent trials.Table 10. Amount of experimental jojoba wax emulsion-treated ‘Auxerrois’ grape leafdamage calculated using greenhouse plants which were sprayed at 7 weeks andsampled at 10 weeksLeaf area (cm2)Treatments Total Undamaged Damaged(Necrotic + (Necrotic)Undamaged)Water control - 0.0Triton X-207 control 96.4 94.6 1.80.1% Jojoba wax/i .0% Triton X-207 88.8 87.2 1.71.0% Jojoba wax/i .0% Triton X-207 84.8 79.4 5.42.0% Jojoba wax/i .0% Triton X-207 83.0 65.6 17.45.0% Jojoba wax/i .0% Triton X-207 65.7 43.3 22.510.0% Jojoba wax/i .0% Triton X-207 64.2 40.1 24.21. For all plants of a particular treatment, n=24.39A/BwDFigure 3. Photographs of experimental jojoba wax emulsion- and Triton X-1 00 surtactanttreated-’Auxerrois’ leaf damage using greenhouse plants which were sprayed at 7weeks and sampled at 10 weeks. (A) Water control-treated plant. Remainder of plantstreated with 1.0% Triton X-207 and jojoba wax at (B) 0.0%, (C) 0.1%, (D) 1.0%, (E)2.0%, (F) 5.0%.C•1VE F40In addition to information about appropriate concentrations of jojoba wax, thisportion of the study also provided data on the effect of the Triton X-series surfactants.Jojoba wax could not be dispersed into solution without the aid of a surfactant.However, all plants sprayed, even those receiving only the Triton X-207 surfactant(Figure 3B, C, D, E), appeared a lighter green than those receiving the water controls(Figure 3A), and developed translucent spots in leaf depressions. In order to reducethese effects, 0.2% Triton X-1 00 surfactant, the lowest concentration observed whichcould emulsify jojoba wax, was utilized in subsequent in vivo trials.3.2.2. Effects of Exogenous Lipid Emulsions on Cucumber GrowthThere were no significant differences in cucumber leaf size between plantssprayed with different treatment (Table 11). However, there were other signs of damagepresent. While the canola oil/Triton X-1 00 and water control-treated plants showed noleaf damage, all other treated plants showed some leaf margin necrosis, with thegreatest damage observed in the commercial control and WiltPruf treated plants. Thiswas similar to the damage seen in the grape greenhouse study (Figure 3D, E, F) foremulsions containing jojoba wax concentrations over 1.0%.41Table 11. Leaf area of six-week-old greenhouse-grown “Chicago Pickling” cucumberplants, treated 17 days previously with experimental exogenous lipid emulsionsLeaf area (cm2)Treatment Trial 1 Trial 20.5% Jojoba waxl0.05% Triton X-1 00 97.61 114.70.5% Canola oill0.05% Triton X-100 95.4 132.50.5% Jojoba wax/O.5% ISOL-RB 95.5 141.94% Folicote 96.9 101.90.5% ISOL-RB 108.4 123.10.05% Triton X-100 107.4 105.3Commercial 103.3 95.8Water 119.6 123.610% WiltPruf 100.4 100.91 For both trials, n=1 1. Means represent averages for all plants of a particulartreatment, with three leaf areas (3d, 4th, and 5th leaves from base) per plant.3.2.3. Effects of Exogenous Lipid Emulsions on Grape Plant Growth, Yield andSurvival3.2.3.1. Growth and SurvivalAll grape plants sprayed with lipid emulsions (those containing jojoba wax in allthree trials and canola oil at ACRS in 1992) exhibited some lightening of leaf colour andslight chlorosis. This seemed to be correlated with the number of spray applications.Plants sprayed with the “all-season” (TV 1991), 1.0% “all-season” and 0.2% Triton X100 control (TV 1992), and 1.0% jojoba wax and 1.0% canola oil (ACRS 1992)treatments showed the largest effects. Figure 4 shows increasing chlorosis towards thebase of the canopy of “all-season” treated ‘Auxerrois’ grape plants from TV in 1991(Figure 4B) as compared to the commercial control-treated plants (Figure 4A). Inaddition, all lipid emulsion-treated grape berries exhibited a darker green, slightly42bronzed appearance, which can be seen in a comparison of the “all-season” treatedplants in Figure 4 to those whitish-green berries of the commercial control-treatedplants. The lipid emulsion-treated grape berries in all trials became bronzed particularlyin August, when ambient temperatures reached their highest in all three trials (Figure 5).The bronzing was observed at TV in 1991 at approximately the same time (aroundAugust 10) that the “all-season” treated plants had a higher stomatal resistance, whichwas followed by a slowed growth stage progression between August 10 and September7 (Table 12). Earlier in the growing season of 1992, there were no significant differencesobserved between treated plants for stomatal resistance (Table 13).43Figure 4. Comparison of the phytotoxic effects of (A) the commercial control and (B) the1.0% allseasonH jojoba wax sprays on ‘Auxerrois grape plants. Photographs takenAugust24, 1991 at TV,44Figure 5. Growing degree days for 1991, 1992 and previous 80-year mean, for theCentral Okanagan Region, in which are located both TV and ACRSTable 12. Percentage of experimental jojoba wax emulsion-treated field-grown‘Auxerrois’ grape plants observed in a growth stage, and stomatal resistancemeasurements of grape leavesGrowth stageTreatment August 10 - September 7 - Stomatal resistance on Augustcolour change berry ripening 10 (S mol-1 rn-2)Water control 39% A1 51% A 2.4 BCommercial control 36% A 49% A 2.1 B“All-season” 26% B 34% B 2.9 A“Bloom” 37% A 46% AB 2.4 B“Pre-veraison” 37% A 51% A 2.6 AB“Post-veraison” 38% A 38% AB 2.4 B1. n=24. Data with significant differences displayed only; remainder of data set isdisplayed in Appendices 2 and 3. Column means sharing the same letter are notsignificantly different according to Duncan’s multiple range test, p<0.05.0500450400350300250200150100500 IIILs—•-—-i 991• 1992—A-—--Means of last80yrsApr May Jun Jul Aug Sep OctMonth45Table 13. Stomatal resistance at mid-season of field-grown ‘Auxerrois’ grape leavestreated with experimental jojoba wax emulsionsStomatal resistance (S mol-1 m-2)July 4 July 11 July 25Treatment 0930 1200 0930 1200 0930 1200Water control 1.51 2.1 0.8 1.7 3.9 3.00.2% Triton X-100 control 2.3 2.3 1.3 2.1 3.6 4.01.0% jojoba wax “all-season” 2.2 2.2 1.1 1.7 3.1 3.41.0% jojoba wax “pre-veraison” 2.0 2.1 1.1 2.0 2.9 3.60.2% jojoba wax “all-season” 2.3 2.2 1.2 1.9 3.4 5.50.2% jojoba wax “pre-veraison” 2.1 2.2 1.1 1.9 2.4 2.01. n=1 6 for the Triton X-1 00 control, and both “Pre-veraison” treatments, n=24 for both“All-season” treatments, and n=64 for the water control.The raw growth data collected during the growing season from plants at TVin 1992 are displayed in Appendix 4. No data were collected after August 8 because theU. necator epiphytotic was observed to be a complicating factor with respect to theaccuracy of the measurements. The regression analysis followed by an ANOVA of theresiduals revealed that no significant effect on the number of leaves per shoot, numberof internodes per shoot, fifth and tenth internode length, tenth leaf area, berry volume,and rachis length could be attributed to any particular treatment. However, the 1.0%“pre-veraison”, 0.2% “all-season”, and 1.0% “all-season” treatments had significantlygreater effects on the amount of chlorophyll in the leaves as compared to othertreatment effects on July 10 (Figure 6). The effect of these three treatments onchlorophyll amount decreased over the three remaining sampling dates, with the 0.2%“all-season” treatment effect decreasing fastest, then the 1.0% “pre-veraison” treatmenteffect, and the 1.0% “all-season” treatment effect decreased slowest. The Triton X-1 00treatment had an intermediate effect on chlorophyll as compared to the othertreatments. The water control and 0.2% “pre-veraison” treatments had the least effect46Q.0I00.4-E4.-I-. 000E00C-I-.on chlorophyll throughout the sampling period; it was to their level of effect to which the0.2% “all-season”- and 1.0% “pre-veraison” treatments decreased.21.510.50-0.5—1-1.5—4---Water controlS 0.2% “Pre-veraison”—A-—i .0% “Pre-veraison”—E—0-2% “All-season”*: 1.0% “All-season”Triton X-i 00 control-2Jul-i 8 Jul-24Jul-i 0 Jul-31DateFigure 6. Residuals from stepwise multiple regression analysis showing jojoba waxemulsion treatment effect on ‘Auxerrois’ grape leaf chlorophyll at TV in 1992. Zero line isthe regression line representing leaf chlorophyll amount after the effects of the amountof powdery mildew, and number of leaves/number of internode ratio have beenremoved. Data points with the same letter are not significantly different according toDuncan’s multiple range test, p.<0.05. Where “. .. .“ is displayed, this indicatesoverlapping of Duncan’s letter categories; no significance between treatments indicatedcan be discerned.The detailed measurement of ‘Riesling’ grape plant modules made throughoutthe growing season at ACRS in 1992 showed little difference for growth between treatedplants for the per shoot number of leaves or number of internodes, fifth and tenthinternode length, tenth leaf area, rachis length, and berry volume measured (Appendix475). In addition, with respect to survival, the treated plants showed no statisticallysignificant differences in cane pruning weights in all three trials (Table 14).Table 14. Wet weight of experimental seed-storage lipid emulsion-treated ‘Auxerrois’and ‘Riesling’ grape plant cane prunings per plant before and after the growing seasonfor field trialsCane pruning weight (kg)1991 1992 1992‘Auxerrois’ ‘Auxerrois’ ‘R lesling’Treatments Before After Before After Before AfterWater control 0.8 2.2 0.7 1.6 - 1.0Commercial control 0.8 2.6 - - - 0.91.0% jojoba “all-season” 0.6 1.6 0.3 0.7 - -1.0% jojoba “bloom” 1.1 2.2 - - - -1.0% jojoba “pre-veraison” 0.9 2.0 0.7 1.9 - -1.0% jojoba “post-veraison” 0.6 2.1 - - - -0.2% Triton X-100 control - - 1.1 1.5 - -0.2% jojoba “all-season” - - 0.5 1.2 - -0.2% jojoba “pre-veraison” - - 0.6 2.0 - -1.0% jojoba (ACRS only) - - - - - 1.01.0% canola (ACRS only) - - - - - 0.90.5% canola (ACRS only) - - - - - 1.01. For 1991 samples, n=24. For 1992, the ‘Auxerrois’ grapes had n=16 for the Triton X100 control, and both “Pre-veraison” treatments, n=24 for both “All-season” treatments,and n=64 for the water control; the ‘Riesting’ grapes had n=1 2.3.2.3.2. YieldThere were no significant differences with respect to ‘Auxerrois’ grape berry orcluster weight, soluble solids concentration, titratable acidity or pH of whole berrysamples among treated plants at TV in 1991 (Table 15). There was a trend, however,towards slightly lower cluster weights for all lipid emulsion-treated plants compared tothose sprayed with the commercial control. ‘Auxerrois’ grape cluster weights for TV in481992 were complicated by the high degree of powdery mildew disease severity; thus,the data is not discussed here (Appendix 6).Table 15. Cluster weight and berry weight and composition measurements sampledfrom grapes harvested from experimental jojoba wax emulsions-treated field-grown‘Auxerrois’ plants in 1991Treatment Cluster Berry weight Soluble Titratable pHweight (g) (g) solids (°Brix) acidity (gIL)Water 120 B1 1.5 18.1 8.9 3.3Commercial 160 A 1.6 17.7 7.8 3.3MAll-season” 114 B 1.5 18.2 9.3 3.3“Bloom” 112 B 1.5 18.1 9.6 3.4“Pre-veraison” 141 AB 1.5 18.1 8.6 3.4“Post-veraison” 123 B 1.6 18.2 8.5 3.41. n=24. Column means sharing the same letter (or where not indicated) are notsignificantly different according to Duncan’s multiple range test, p.<0.05.At ACRS in 1992 (Table 16), there was no significant difference between treated‘Riesling’ grape plants with respect to grape berry or cluster weight, soluble solidsconcentration, titratable acidity or pH of whole berry samples (Table 16). However, therewas a trend to slightly lower berry weights for the lipid emulsion and commercial control-sprayed plants.Table 16. Yield, and berry cornposition of grapes harvested from experimental seed-storage lipid emulsions-treated field-grown ‘Riesling’ grape plantsTreatment Cluster Berry Soluble Titratable pHWeight Weight (g) solids Acidity (gIL) (-log[H])(g) (°Brix)1.0% Jojoba 231 1.1 B1 20.8 9.3 3.41.0%Canola 235 1.1 B 18.3 9.6 3.30.5% Canola 222 1.1 B 20.4 9.5 3.4Water control 299 1.3 A 19.5 10.3 3.3Commercial 258 1.3 AB 19.7 10.0 3.31. n=12. Column means sharing the same letter (or where not indicated) are notsignificantly different according to Duncan’s multiple range test, p.<0.05.494. DISCUSSIONThe overall objectives of this project were (1) to determine whether seed-storagelipid emulsions have potential as plant prophylactic substances and (2) to explore howlipid emulsions applied to plant epicuticles affect fungal plant pathogen infectionprocesses. The different emulsions had different effects on the various fungalpathogens studied; these differences indicated not only the degree but the nature of theinterference provided by the lipid emulsions against the fungal plant pathogens.4.1. Effects of Seed-Storage Lipid Emulsions as Phytoprotectants againstPowdery Mildews4.1.1. Efficacy of Lipid Emulsions against Powdery MildewsPlants sprayed with seed-storage lipid emulsions showed reduced powderymildew disease severity as compared to water control-treated plants. Cucumber plantssprayed with jojoba wax-containing emulsions showed a 77-92% reduction in powderymildew disease severity; plants sprayed with canola oil emulsions showed a 62-69%reduction (Table 4). Also, the 1.0% “all-season” jojoba wax emulsion-treated grapeplants showed a 92-100% reduction in powdery mildew disease severity on the leavesduring the growing season and reduced disease incidence on the fruit at harvest at TV(Table 6). Thus, emulsions of the two seed-storage lipids utilized are effectiveprophylactic mixtures against the powdery mildews studied. However, the lipidemulsions seemed to have prophylactic protective effects only, with no capacity toeradicate an existing infection. The 1.0% “pre-veraison” and “post-veraison” jojoba waxemulsion-treated grape plants at TV in 1991 were not significantly different from the501.0% “all-season-treated plants with respect to powdery mildew disease severity ofleaves or disease incidence of clusters, probably because the artificial inoculation tookplace after at least one spray had been applied to all of these plants (Table 6). However,the HbloomhItreated plants at TV in 1991 became diseased, probably because thesprays stopped before the majority of the plant canopy had grown; leaves which grewlater in the season were unprotected (Table 6). Also, the disease progression on thegrape clusters and leaves which existed previous to sprays of the jojoba wax emulsionsat TV in 1992 was unimpeded by subsequent sprays; only on plant material not alreadydiseased were the subsequent sprays able to provide any protection against the fungalpathogen. This solely prophylactic protection has also been observed fortriacylglyceridic lipid emulsion sprays on grape plants by Northover et al. [1993], wherethe emulsions also provided no eradicant type of control for Uncinula necator.Slight physiological damage consistent with increases In leaf and fruittemperature as a result of decreased transpiration [Quarles 1991] were concurrent withthe beneficial prophylactic effects of the lipid emulsion sprays. Damage to grape leavesoccurred at controlled greenhouse temperatures when emulsions containing >1.0%jojoba wax were applied (Table 10). These concentrations (higher than theconcentrations used for testing the prophylactic effects of the lipids against fungalpathogens) probably inhibited transpiration which raised leaf temperature sufficiently tocause observable damage, as opposed to the transpiration inhibition caused by lowerLipid concentrations. However, even the lower concentrations of lipids (1 .0%) seemedto cause some damage where ambIent temperatures were already higher, as higherstomatal resistances and berry bronzing were observed on lipid emulsion-sprayed grapeplants (Table 12, Figure 4) with the onset of hotter late-summer temperatures in fieldtrials (Figure 5). This did not occur earlier in the season, when it was cooler (Table 13),nor did it occur on water control-treated plants (Table 12). Berry bronzing and51subsequent desiccation might also account for the slightly lower cluster and berryweights of lipid emulsion-treated plants (Table 15 and 16).Treatment effects on vegetative growth related to the slight physiologicaldamage observed were subtle (Table 12, Figure 6). Growth stage progression wasslightly diminished for the 1.0% “all-season” treated-plants at TV in 1991 (Table 12). AtTV in 1992, the effect of treatments with higher concentrations of jojoba wax or moresprays of the jojoba wax emulsion on the amount of leaf chlorophyll was greater thanother treatments with lower concentrations or fewer sprays (Figure 7). These subtleeffects may have been stimulated by hormone production in damaged leaves [Quarles1991, Kastori et a!. 1991]. Increased leaf temperature due to decreased transpirationwould have resulted in damage to the photosynthetic apparatus [Nover 1989]. However,there was no long term effect on survival, as measured using field-grown grape plants(Table 14).Both grape (Figure 3) and cucumber leaves were observed to have a slightchlorosis which was not correlated with lipid emulsion treatment, but rather theapplication of Triton X-series surfactants. Surfactants of the Triton X-series type areknown to induce ethylene formation, producing symptoms of senescence [Lownds andBukovac 1989]. A surfactant effect may account for the small amount of chlorosisobserved.Slight damage was observed on the plants treated with the seed-storage lipidemulsions; there were no large, general effects on growth or yield observed. Therefore,the advantages of the prophylactic usage of seed-storage lipid emulsions outweigh thesmall damage caused by the sprays.524.1.2. Mechanism of Lipid Emulsions against Powdery MildewsThe two seed-storage lipids may have theoretically affected any of the fourinfection processes of the powdery mildew fungi. For both seed-storage lipids,germination interterence can be ruled out as a mechanism, as SEM analyses showedthat germination of E. cichoracearum spores on leaves treated with these emulsionswas as frequent as those on leaves treated with water alone (Figure 2A, D, Table 5). Incontrast, spores observed on WiltPruf-treated leaves did not germinate, a phenomenonwhich has been observed previously [Ziv and Fredericksen 1983, Elad et al. 1989,1990]. However, while 60-100% of the samples of the water and surfactant control-treated leaves showed appressorium formation at 48 hours, samples of cucumberleaves treated with the jojoba wax- or canola oil-containing emulsions showedgerminated spores with no appressorium formation. This observation indicates that thesurfactants had no effect on the incidence of appressorium formation, a fact whichimplicates the action of the seed-storage lipids alone on incidence of appressoriumformation at 48 hours. However, the lack of appressoria on leaves treated with the lipidemulsions could be explained by inhibition of germ tube growth, differentiation, or therecognition process.If the seed-storage lipids interfered with the growth process, meaning thatappressorium development had simply not yet occurred at 48 hours because of a directaction of the lipids on the germ tube, then one would expect the difference in germ tubelength to vary with the presence or absence of the lipids. However, the Ecichoracearum germ tubes of spores on Triton X-100 control- and seed-storagelipid/Triton X-1 00-treated leaves showed diminished growth as compared to water-sprayed leaves (Figure 2C), whereas germ tubes on leaves treated with ISOL-RBcontrol- and jojoba wax/1SOL-RB-treated leaves showed no diminished growth (Figure532A). This seems to indicate that Triton X-100, both alone and in the lipid emulsions, hadthe effect on germ tube growth. A retardation of growth can be mitigated by time, andeventually disease signs were observed, as noted on the greenhouse cucumber plantstreated with Triton X-1 00-containing treatments 17 days after inoculation (Table 4).Moreover, the fact that the germ tubes of spores on the jojoba waxIlSOL-RB-treatedleaves showed no appressorium formation at 48 hours in spite of vigourous germination(Table 5) and germ tube growth (data not shown) indicates that the absence ofappressoria on jojoba wax- (and perhaps canola oil-) treated leaves was due tointerference with differentiation or the recognition interference, not due to growthinterference. The interference of the seed-storage lipids was not diminished in time, asthe plants treated with the seed-storage lipid emulsions, and especially those containingjojoba wax, had few disease signs after 17 days (Table 4).The Triton X-100 surfactant effect of reducing powdery mildew mycelialgrowth (Table 5) has been observed previously in rice blast fungi (Pyricularia oryzaeCay.) in vivo [Kim et al. 1989] and Mucor mucedo (L. ex Fr.) in vitro [Reyes 1992].Cucumber and grape plants treated with the Triton X-1 00 controls had fewer powderymildew colonies than water treated plants (Table 4 and 6), and SEM studies showedthat E. cichoracearum spores on Triton X-1 00-treated leaves had limited germ tubegrowth, and that appressorium development was exaggerated (Figure 2C). In addition,the lower powdery mildew disease incidence observed on the plants treated with the1.0% and 0.2% ‘pre-veraison’1and the 0.2% “all-season” treatments in the ‘Auxerroisgrape field trials at TV may have been due to Triton X-100 alone, as the reductionobserved for the Triton X-1 00-treated plants was not significantly different from that ofthese jojoba wax emulsion-treated plants (Table 6). Inhibition may have been due to asolubilizing effect of Triton X-100 micelles on the plasma membrane of the growinggerm tube. The exaggerated appressorium growth observed seems to corroborate this54idea. Since appressoria are largely anchored to the epicuticle by water tension [Howardet aL 1991], the presence of a larger appressorium may have countered the effect ofthe surfactant in reducing water tension. These actions of the micellar contents on thegerm tube plasma membrane would result in the inhibition of germ tube extension, theuseless expenditure of cell energy, and the consequent reduction of growth.The seed-storage lipids must have had an effect either by inhibiting differentiationof the germ tube into appressoria, or by interfering with the recognition processes whichtriggers differentiation. One possibility for differentation inhibition is that seed-storagelipids interefered with differentiation via a plant-mediated response. Recent researchhas discussed jasmonic acid as the plant growth regulator [Meyer et al. 1984] whichmediates plant wound responses [Farmer and Ryan 1992]. Occurring in plants as aresponse to wounding [Creelman et a!. 1992], jasmonic acid induces the formation ofextracellular proteins which have no antifungal activity on their own [Schweizer et a!.1993, Reinbothe et a!. 1992]. However, jasmonic acid itself may interfere with theappressorial differentiation of the powdery mildew fungal germ tube, as suggested for E.graminis [Schweizer et a!. 1993]. It is a possibility that the seed-storage lipids couldhave interfered with germ tube differentiation by eliciting a wound response, or byproviding a sufficient quantity of the octadecanoid precursors required for the jasmonicacid pathway [Farmer and Ryan 1992]. However, Triton X-1 00 has been shown to elicitwound responses in plants [Lownds and Bukovac 1989], and canola oil contains largeproportions of the 18:2 linoleic and 18:3 linolenic fatty acid precursors used by thejasmonic acid pathway [Ackman 1990, Farmer and Ryan 1992]. If the seed storagelipids were eliciting a wound response, then the leaves treated with Triton X-1 00- orcanola oil-containing emulsions might have shown less powdery mildew diseaseincidence. This was not the case; leaves treated with the Triton X-100- or canola oilcontaining treatments showed significantly more powdery mildew disease than the55jojoba wax emulsion-treated plants (Table 4). If it can be assumed that jasmonic acidwould have the same effect on the differentiation processes of E. cichoracearum andother powdery mildews, then it seems unlikely that the seed-storage lipids have asignificant plant-mediated effect on the differentiation process which produces anappressorium.While seed-storage lipids could interfere with differentiation of the appressoriumby acting on the germ tube directly, this action is morphologically indistinguishable fromrecognition interference, which also results in no appressorium formation. Thus, theseed-storage lipid emulsions may contain compounds which directly interfere with theproduction of the appressorium, or they may interfere with the environmental conditions(>99% relative humidity without free moisture [Blaich et at 1989, Heintz 1986, Carver eta!. 19901) required for appressorium formation, perhaps by modulating the transpirationgradient as suggested by Waard [1971]. Both types of interference preclude anypenetration interference by these lipids (penetration happens after recognition).Therefore, at least for E. cichoracearum, and perhaps other powdery mildews, the onlything that can be said for the seed-storage lipids is that they interfere with thedevelopment of fungal appressoria.The efficacy of the lipids in inhibiting appressorium formation may have been afunction of the amount of lipid which was released from the spray solution to thesurface. Both the canola oil emulsion- and Folicote-treated cucumber leaves showed aslittle E. cichoracearum appressorium formation in the SEM studies as leaves treatedwith the jojoba wax emulsions (Table 5). However, the canola oil emulsion, Folicote, andeven jojoba wax with ISOL-RB did not inhibit E. cichoracearum colony formation to thesame degree as jojoba wax emulsified with Triton X-1 00 (Table 4). The varying efficacyof the lipid emulsions may have depended on the way in which each lipid emulsified.56Emulsions are suspensions of particles of one liquid in another liquid in which the formeris immiscible; this causes a concomitant increase in the interface area between the two,a state which is inherently thermodynamically unstable [Rosen 1978]. The surfactantforms a micellar film between the two liquids as a consequence of its amphipathicstructure, grading from one liquid to the other. This stabilizes the dispersion, as long ashydrogen bonds or Van der WaaI forces hold the surfactant micelle together. The factthat the canola oil and Folicote suspensions were stable and semitransparentcontributes to the idea that these lipids were not decreasing the stability of the micelles,whereas jojoba wax formed an opaque, unstable suspension with Triton X-1 00 andISOL-RB. When the canola oil or Folicote emulsions were dispersed onto the leafsurface, the canola oil and the paraffin wax in the Folicote would be more likely to stayin the micelles of pure surfactant on the epicuticle, and thus be less of an impedance tofungal infection processes. Folicote has previously been observed to cover epicuticles towhich it is applied in a discontinuous fashion [Zekaria-Oren et a!. 1991]; thediscontinuities may be due to the lack of separation of Folicote from micelles. The jojobawax would come out of suspension soon after dispersion, and, when the spray waterhad evaporated, the jojoba wax would be more likely to coat the epicuticular surface,with micelles of either the Triton X-100 or the ISOL-RB surfactant sitting on top of thecoating. (The ISOL-RB-containing suspension was observed to be slightly more stablethan the Triton X-100 suspension; thus it likely differed in the actual micellarconstruction, which may have resulted in its lower efficacy). If this idea is true, thenchanging the type and length of the chains of the lipid and the surfactant (as long asthey are compatible with the plant) may result in increased phytoprotection againstpowdery mildews and related pathogens.574.2. Effects of Seed-Storage Lipid Emulsions as Phvtoprotectants against Rot-Causing FungiIn vitro, the seed-storage lipids seemed to have no efficacy in reducing themycelial growth of either B. cinerea and D. bryoniae (Table 7). Triton X-1 00 was thecommon factor where the greatest reduction (75-92%) of mycelial growth was observed(Table 7). This mycelial growth inhibition by Triton X-1 00 is consistent with the inhibitionobserved for powdery mildew germ tubes, as discussed above in section 4.1.2. The factthat the addition of lipids with the Triton X-100 to agar petri dishes seemed to slightlylessen the inhibition of growth observed for the Triton X-100 control-treated petri dishes(Table 7) suggests that the lipids in the surfactant micelles were interfering with theeffects of Triton X-1 00 against the fungal germ tubes, perhaps by interfering with thestability of the micelles, or simply getting in the way.However, the in vivo efficacy of Triton X-1 00 was not consistent between the B.cinerea-grape and the D. bryoniae-cucumber systems. One factor which may havecontributed to the efficacy of the Triton X-1 00-containing treatments was the moisture inthe system. The cucumber plants treated with the lipid emulsions showed no reductionin black rot disease incidence (Table 8), whereas grape clusters treated with lipidsemulsified with Triton X-1 00 at ACRS showed a —80% reduction in bunch rot diseaseincidence, as compared to water and commercial control-treated berry clusters (Table9). Indeed, plants treated with any of the three lipid-containing treatments at ACRSshowed equivalent reductions in the amount of bunch rot disease incidence in spite ofthe varying concentrations of lipid (Table 9), a fact which suggests that the emulsifyingsurfactant, Triton X-100, was responsible for the reduction. The contrast between thetwo in viva systems is in spite of the fact that the amount of either D. bryoniae or B.cinerea- growth for the lipid emulsions-treated petri dishes was equivalently reduced in58the in vitro trials (Table 7). However, the D. bryoniae-cucumber petiole system isinherently wet; the petioles were observed to bleed sap for up to 24 hours after theinjuries were caused, meaning the added lipid emulsions were probably washed awayby the sap. This is opposed to the dry nature of the B. cinerea-grape berry system.Future trials could take place on systems of the latter character, not the former, in orderto check the effect of moisture on the efficacy of Triton X-100 in reducing mycelialgrowth.4.3. Overall DiscussionThe seed-storage lipid emulsions tested did provide protection against diseasecaused by fungal pathogens, protecting the grape and cucumber plants sprayed by atleast 60% as compared to water control-treated plants, and often protecting the plantsat a degree greater than 90%. However, this control did depend on the plants being freeof the fungal pathogen in question previous to spraying, as only plants completelycovered with the lipid emulsions remained free from disease. Also, control was wroughtby different parts of the emulsion for different pathogens. For the cucumber powderymildew at least, the lipid component of the emulsion provided the majority of theprotection, whereas the surfactant component of the emulsion provided the protectionagainst the rot-causing fungi. This was likely due to differences in the nature of the twoplant fungal systems, and the pre-penetrative mechanism by the two different classes offungi.For the powdery mildew systems, the evaporation of the spray water from thelipid emulsions probably left a layer of lipid with isolated surfactant micelles sitting on theepicuticular surface. The differentiation of the germinated powdery mildew spores, whileencountering occasional surfactant micelles, seemed mostly affected by the lipid layer,59which may have directly interfered with appressorium differentiation, or alteredenvironmental conditions required for appressorium formation. Alternatively, the rot-causing fungal system required free moisture for germination; this moisture (as long asit did not wash the lipid emulsion away) would preclude the formation of a lipid layer,and the surfactant micelles would be able to move. The differentiation of the germinatedrot-causing fungal spores seemed mostly affected by the surfactants, which may havesolubilized the plasma membrane of the growing germ tubes. In both systems, the lipidemulsions effected control, but different components of the emulsions were the activecomponent in each case. Because of the ability of the lipid emulsion (and particularlythe jojoba wax emulsion) to affect both classes of fungi studies, these emulsions may beused agricultural situations on plants which are attacked by fungi of both types. In fact,the jojoba wax/Triton X-1 00 emulsion used in this study has been patented under thetrademark JojobaShield.These emulsions may be used to suggest the role of various epicuticularcuticular lipids in plant-fungal interactions. For instance, jojoba wax is composed of anester similar in chain length to esters found in the epicuticle, whereas Folicote andcanola oil are similar to compounds found in the cuticle (paraffin wax, a long chainhydrocarbon, and oleic acid triacyglycerides, respectively). Perhaps the particularefficacy of the jojoba wax emulsion against powdery mildews can be attributed to asimilarity between the jojoba wax and the ester components of plant epicuticles. Afurther extension would be to suggest that esters in the epicuticle contribute particularlyto the natural passive defense of plants against powdery mildews. This idea could betested by spraying plant epicuticles with synthetic versions of components of theepicuticle, and determining which are most protective against powdery mildews.60The lipid emulsions might also be used as tools to discover what triggers areused to initiate germ tube morphogenesis for different fungal pathogens. For instance,surfactants like Triton X-100 might be used to determine the amount of surface tensionrequired between an appressorium and an epicuticular surface before penetration isattempted. Also, fungi similar to the rot-causing fungi studied here might be placed onsurfactant-treated host plants to determine the amount of the susceptibility to theproposed solubilization of the germ tube plasma membrane. These experiments (andcertainly others) would provide additional knowledge as to how fungi interact with theirplant hosts, and which possible interferences of those interactions might be mosteffective.However, with respect to the effect of the seed-storage lipids on the powderymildew fungi, this research indicated the need to discover the cause of theappressorium formation inhibition. An in vitro experiment on lipid-emulsion-coveredmedia done under optimum environmental conditions for appressorium formation shouldclear up the question. If appressorium formation occurs, then a direct inhibition of theseed-storage lipid emulsions on appressorium differentiation can be ruled out. Also, ifappressorium formation does not occur, the interference of the microenvironmentalrecognition factors by the lipid emulsions can be ruled out. This experiment would makemore certain the effects of the seed-storage lipid emulsions, and its results would directthe application of the seed-storage lipid emulsions to further research concerning thepowdery mildew fungi.615. LITERATURE CiTEDACKMAN, R.G. 1990. Canola fatty acids - an ideal mixture for health, nutrition and fooduse. In Canola and Rapeseed-Production, Chemistry, and Nutrition. Editedby F. Shahidi. Van Nostrand Reinhold, NY. pp8l -98.AIST, J.R. and W.R. BUSHNELL. 1991. Invasion of plants by powdery mildew fungi, andcellular mechanisms of resistance. In in Plants and Animals. EditedbyG.T.Cole and H. Hoch. Plenum Press, NY. pp321 -339.AGRIOS, G.N. 1988. Plant Pathology. 3 ed. Academic Press, NY. pp63-84, 97-107BERGSTROM, G, KNAvEL, D.E. and KUC, J. 1982. Role of insect injury and powderymildew in the epidemiology of gummy stem blight disease of cucurbits. P1.Dis.. 66: 683-686.BLAICH, R., HEINTZ, C. and WIND, R. 1989. Studies on conidial germination and initialgrowth of the grapevine powdery mildew Uncinula necator on artificialsubstrates. Appl. Microbiol. Biotech. 30: 415-421.BLAICH, R., STEIN, U. and WIND, R. 1984. Perforationen in der cuticula con weinbeerenals morphologischer faktor der botrytisresistenz. Vitis. 23: 242-256.BROWN, W. and HARVEY, C. 1927. Studies in the physiology of parasitism. X. On theentrance of parasitic fungi into the host plant. Ann. Bot. (Konig & Sims). 41:643-662.BULIT, J. and DuBols, B. 1988. Fruit and foliar diseases caused by fungi - BotrytisBunch Rot and Blight. In Compendium of Grape Diseases. Edited by R.Pearson, and A. Goheen. APS Press, St. Paul, Minn. pp13-15.BULIT, J. and LAFON, R. 1978. Powdery mildew of the vine. In The Powdery Mildews.Edited by D.M. Spencer. Academic Press, London. pp525-547.CARVER, T.L.W. and INGERSON, S.M. 1987. Response of Erysiphe graminis germlings tocontact with artificial and host surfaces. Physiol. Molec. P1. Pathol. 30: 359-372.CARVER, T.L.W. and THOMAS, B.J. 1990. Normal germling development by Etysiphegraminis on cereal leaves freed of epicuticular wax. P1. Pathol. 39:367-375.CARVER, T.L.W., THOMAS, B.J., INGERSON-MORRIS, S.M. and RODERICK, H.W. 1990.The role of abaxial leaf surface waxes of Lolium spp. in resistance toErysiphe graminis. Pt. Pathol. 39: 573-583.CHIU, W.F. and WALKER, J. 1949. Physiology and development of the cucurbit black-rotfungus. J. Agric. Res. 78: 589-615.62COOMBE, B.G. 1992. Research on Development and ripening of the grape berry. Amer.J. Enol. Vitic. 43:105-110.CREELMAN, R.A., TIERNEY, M.L., and MULLET, J.E. 1992. Jasmonic acidimethyljasmonate accumulate in wounded soybean hypocotyls and modulate woundgene expression. Proc. Nati. Acad. Scie. U.S.A. 89:4938-4941.DAS, V.S.R. and RAGHAVENDRA, A.S. 1979. Antitranspirants for improvement of wateruse efficiency of crops: Field, vegetable and fruit crops. Outlook Agric. 10:92-98.DEISING, H., NICHOLSON, R.L., HAUG, M., HOWARD, R.J. and MENDGEN, K. 1992.Adhesion pad formation and the involvement of cutinase and esterases in theattachment of uredospores to the host cuticle. P1. Cell. 4:1101-1111.DE NEERGAARD, E. 1989. Histological investigation of flower parts of cucumber infectedby Didymella byroniae. Can. J. P1. Pathol. 11:28-38.DESERT KING JOJOBA CORP. 1990. Product report: EXTRAN(TM)-series jojoba waxes.In House. Chula Vista, CA.DUGAN, F. and BLAKE, G. 1989. Penetration and infection of western larch seedlings byBotrytis cinerea. Can. J. Bot. 67: 2596-2599.ELAD, Y., AYIsH, N., ZIV, 0. and KATAN, J. 1990. Control of grey mould (Botrytis cinerea)with film-forming polymers. P1. Pathol. 39: 249-254.ELAD, Y., Ziv, 0., AYISH, N. and KATAN, J. 1989. The effect of film-forming polymers onpowdery mildew of cucumber. Phytopathologica. 17: 279-288.EMME1T, R.W. and PARBERY, D.G. 1975. Appressoria. Annual Rev. Phytopathol. 13:147-167.EPSTEIN, L., LACCETTI, L., STAPLES, R. and HOCH, H.C. 1987. Cell-substratum adhesiveprotein involved in surface contact responses of the bean rust fungus.Physiol. Molec. P1. Pathol. 30: 373-388.FARMER, E. E. and RYAN, C.A. 1992. Octadecanoid precursors of jasmonic acid activatethe synthesis of wound-inducible proteinase inhibitors. Pt. CeLl 4: 129-134.GALE, J. and HAGAN, R.M. 1966. Plant antitranspirants. Annual Rev. P1. Physiol. 17:269-279., J. and PEARCE, R. 1984. The structure of plant surfaces. In Infection Processes ofFungi. Edited by D.W. Roberts and J.R. Aist. Rockefeller Foundation, NY.ppl6-30.63GOODAY, G.W. and TRINCI, A.P.J. 1980. Wall structure and biosynthesis in fungi. In TheEukaryotic Microbial Cell. Edited by G.W. Gooday, D. Lloyd, and A.P.J.Trinci. Cambridge University Press, Cambridge. pp207-252.GUTIERREZ-GRANDA, M.J. and MORRISON, J. 1992. Solute distribution and malic enzymeactivity in developing grape berries. Amer. J. Enol. Vitic. 43: 323-328.HEINTZ, C. 1986. Infection mechanisms of grapevine powdery mildew (Oldium tuckeri):Comparative studies of the penetration processes on artificial membranesand leaf epidermis. Vitis. 25: 215-225.HEINTZ, C. and BLAICH, R. 1989. Structural characters of epidermal cell walls andresistance to powdery mildew of different grapevine cultivars. Vitis. 28: 153-160.HOCH, H.C. and STAPLES, R. 1991. Signaling for infection structure formation in fungi. InThe Fungal Spore and Disease Initiation in Plants and Animals. Edited byG.T. Cole and H.C. Hoch. Plenum Press, NY. pp25-42.HOWARD, R.J., FERRARI, M.A., ROACH, D.H. and MONEY, N.P. 1991. Penetration of hardsubstrates by a fungus employing enormous turgor pressures. Proc. Nati.Acad. Sd. U.S.A. 88:11281-11284.INSKEEP, W.P. and BLOOM, P.R. 1985. Extinction coefficients of chlorophyll a and b inN,N-dimethylformamIde and 80% acetone. P1. Physiol. 77: 483-485.JARVIS, W.R. 1977. Botryotina and Botrytis species: Taxonomy, physiology andpathogenicity. Monograph No. 15. Research Branch, Canada Department ofAgriculture, Ottawa.JARvIs, W.R. and Nuttall, V.W. 1979. Cucumber diseases. Publication 1684. InformationServices, Canada Department of Agriculture, Ottawa.JOHNSON, J.D. and HINMAN, C.W. 1980. Oils and rubber from arid land plants. Science.208: 460-462.KAMP, M. 1985. Control of Erysiphe cichoracearum on Zinnia elegans with a polymer-based antitranspirant. Hortscience 20: 879-881.KASTORI, R, PETROVIC, N. and STANKOVIC, Z. 1991. Possibilities of use antitranspirantsin plant protection. Biol. Vestn.. 39: 109-114. (Abstr.)KENNEDY, M.J. 1990. Models for study the role of fungal attachment in colonization ofpathogens. Mycopathologica. 109:177-182.KIM, B.S., C HUNG, Y.R. and CHQ, K.Y. 1989. Influence of several surfactants, solvents,and fungicides on the activity of Pyricularia oryzae cavara and the rice blastseverity. Kor. J. P1. Pathol. 5:168- 173.64KOLLATUKUDY, P.E. 1984. How do plant pathogenic fungi break the plant cuticularbarrier? In Infection Processes of Fungi. Edited by D.W. Roberts and J.R.Aist. Rockefeller Foundation, NY. pp3l -37.KOLLATUKUDY, P.E. 1985. Enzymatic penetration of the plant cuticle by fungalpathogens. Annual Rev. Phytopathol. 23: 223-250.KOLLATUKUDY, P.E., E111NGER, W.F. and SEBASTIAN, J. 1987. Cuticular lipids in plant-microbe interaction. In The Metabolism, Structure and Function of PlantLipids. Edited by P.Strumf, J. Mudd and W. Nes. Plenum Press, NY. pp 473-480.KOLLER, W. 1991. The plant cuticle: A barrier to be overcome by fungal plantpathogens. In The Fungal Spore and Disease Initiation in Plants andAnimals. Edited by GT. Cole and H.C. F-loch. Plenum Press, NY. pp2l 9-240.KOLLER, W., ALLAN C.R. and KOLLATUKUDY, P.E. 1982. Inhibition of cutinase andprevention of fungal penetration into plants by benomyl - A possibleprotective mode of action. Pestic. Biochem. Physiol. 18:15-25.KUNOH, H. 1984. Cytological aspects of penetration of plant epidermis by fungi. InInfection Processes of Fungi. Edited by D.W. Roberts and J.R. Aist.Rockefeller Foundation, NY. ppl 37-146.KUNOH, H., NICHOLSON, R.L. and KOBAYASHI, I. 1991. Extracellular materials of fungalstructures: Their significance at prepenetration stages of infection. InElectron Microscopy of Plant Pathogens. Edited by K. Mendgen and D.E.Lesemann. Springer-Verlag, Berlin. pp223-230.LESKI, B. 1984. Black fruit- and stem- rot caused by Didymeila byroniae - an importantdisease of glasshouse cucumbers - New to Poland. Acta Hort. 156: 245-247.Li, C.C. 1975. Path Analysis - a Primer. Boxwood Press, Pacific Grove, CA. ppl 24.LOWNDS, N.K. and BUKOVAC, M.J. 1989. Surfactant-induced ethylene production by leaftissue. J. Amer. Soc. Hort. Sci. 114: 449-454.MACKO, V. 1981. Inhibitors and stimulants of spore germination and infection structureformation in fungi. In The Fungal Spore: Morphogenetic Controls. Edited byG. Turian and H.R. HohI. Academic Press, London. pp565-584MAROIS, J.J., NELSON, J.K., MORRISON, J., LiLE, L.S. and BLEDSOE, A.M. 1986. Theinfluence of berry contact within grape clusters on the development ofBotrytis cinerea and epicuticular wax. Amer. J. Enol. Vitic. 37: 293-296.MATTICK, L.R. 1983. A method for the extraction of grape berries used in total acid,potassium, and individual acid analysis. Amer. J. Enol. Vitic. 34: 49.65MCKEEN, W. 1974. Mode of penetration of epidermal cell walls of Vicia faba by Botiytiscmnerea. Phytopathol. 64: 455.MEYER, A., MIERSCH, 0., BUTTNER, C., DATHE, W., SEMBDNER, G. 1984. Occurrence ofthe plant growth regulator jasmonic acid in plants. J. Pt. Growth. Regul. 3: 1-8.MIMS, C.W., TAYLOR, J. and RICHARDSON, E.A. 1989. Ultrastructure of early stages ofinfection of peanut leaves by the rust fungus Puccinia arachidis. Can. J. Bot.67:3570-3579.NATIONAL RESEARCH COUNCIL (US). 1985. Jojoba: New Crop for Arid Lands, NewMaterial for Industry. Academy Press, Washington, D.C. pp37-46.NICHOLSON, R.L. 1984. Adhesion of fungi to the plant cuticle. In Infection Processes ofFungi. Edited by D.W. Robert and J.R. Aist. Rockefeller Foundation, NY.pp74-89.NICHOLSON, R.L. 1990. Functional significance of adhesion to the preparation of theinfection court by plant pathogenic fungi. ACS Sym. Ser. Am. Chem. Soc.439: 21 8-238.NICHOLSON, R.L. and EPSTEIN, L. 1991. Adhesion of fungi to the plant surface:Prerequisite for pathogenesis. In The Fungal Spore in Disease Initiation inPlants and Animals. Edited byG.T. Cole and ftC. Hoch. Plenum Press NY.pp3-19.NORTHOVER, J., SCHNEIDER, K. and STUBBS, L. 1993. Control of grapvine diseases withoils. 6th International Congress of Plant Pathology. July 28-August 6, 1993.Montreal, Quebec. Abstract 3.4.15.NOVER, L. 1989. Other plant stress response systems. In Heat Shock and Other StressReponse Systems of Plants. Edited by L. Nover, D. Neumann, and K. Scharf.Springer-Verlag, Berlin. pp82, 85, 92-93.OCONNELL, R.J., BAILEY, J.A.and RICHARDSON, D.V. 1985. Cytology and physiology ofinfection of Phaseolus vulgaris by Colletotrichum Iindemutliianum. Physiol.P1. Pathol. 27:75-98.PADGETT, M. and MoRRIsoN, J. 1990. Changes in grape berry exudates during fruitdevelopment wid their effect mycelial growth of Botrytis cinerea. J. Amer.Soc. Hort. Sci. 115: 269- 273.PASCHOLATI, S.F., YOSHIOKA, H., Kunoh, H. and NICHOLSON, R.L. 1992. Preparation ofthe infection court by Erysiphe graminis f. sp. hordei.:: cutinase is acomponent of the conidiaf exudate. Physiol. Molec. Pt. Pathol. 41:53-59.66PEARSON, R. 1988. Fruit and foliar diseases caused by fungi - Powdery mildew. InCompendium of Grape Diseases Edited by R.C. Pearson, and A. Goheen.APS Press, St. Paul, Minn. pp9-1 1.PEARsoN, R. and GADOURY, D.M. 1987. Cleistothecia, the source of primary inoculumfor grape powdery mildew in New York. Phytopathol. 77:1509-1514.PEARSON, R. and GOHEEN, A. 1988. Introduction In Compendium of Grape DiseasesEdited byR.C. Pearson, and A. Goheen. APS Press, St. Paul, Minn. ppl-8.PODILA, G., ROGERS, L. and KOLLATUKUDY, P.E. 1993. Chemical signals from avocadosurface wax triggers germination and appressorium formation inColletotrichum g!oeosporioides. P1. Physiol. 103: 267-272.QUARLES, W. 1991. Antitranspirants show promise as nontoxic fungicides. 1PMPractitioner. 8:1-10.REYES, A.A. 1992. Comparative effects of an antitranspirant, surfactants and fungicideson Mucor rot of tomatoes in storage. Microbios. 71: 235-241.RIEDERER, M. and ScH0NHERR, J. 1990. Effects of surfactants on water permeability ofisolated plant cuticles and on the composition of their cuticular waxes. Pestic.Sd. 29:85-94.REINBOTHE, S., REINBOTHE, C., LEHMANN, J., PARTHIER, B. 1992. Differentialaccumulation of methyl jasmonate-induced mRNAs in response to abscisicacid and desiccation in barley (Hordeum vulgare). P1. Physiol. 86: 49-56.ROSEN, M.J. 1978. Surfactants and Interfacial Phenomenon. John Wiley and Sons, NY.ppl-3, 83-88, 224-226.SALINAS, J., WARNAAR, F. and VERHOEFF, K. 1986. Production of cutin hydrolyzingenzymes by Botiytis cInerea in vitro. J. Phytopathol. 116: 299-307.SAMUELS, A.L., GLASS, A.D.M., EHERT, D.L. and MENZIES, J.G. 1991. Distribution ofsilicon in cucumber leaves during infection by powdery mildew fungus(Sphaerotheca fuliginea). Can. J. Bot. 69:140-146.SAS INSTITUTE, INC. 1985. SAS UsertsGuide: Statitistics Version 5 Edition. Cary, NC.SHISHIYAMA, J., ARAKI, F. and AKAI, S. 1970. Studies on cutin-esterase. II.Characteristics of cutin-esterase from Botrytis cinerea and its activity ontomato cutin. P1. Cell Physiol. 11: 937-945.SITTERLY, W.R. 1978. Powdery mildews of cucurbits. In The Powdery Mildews Edited byD.M. Spencer. Academic Press, London. pp359-377.STAPLES, R. and HOCH, H. 1987. Topical review: Infection structure - form and function.Exp. Mycol. 11:163-169.67STEINBURG, S.L., MCFARLAND, M.J. and WORTHINGTON, J.W. 1990. Antitranspirantreduces water use by peach trees following harvest. J. Amer. Soc. Hort. Sd.115:29-24.SUSSMAN, A.S. 1966. Dormancy and spore germination. In The Fungi. Edited by G.Ainsworth and A.S. Sussman. Academic Press, London. pp733-764.SUSSMAN, A.S. and DOUTHIT, H.A 1973. Dormancy in microbial spores. Annual Rev. P1.Physiol. 24: 311-352.SVEDELUS, G. 1990. Effects of environmental factors and leaf age on growth andinfectivity of Didymelia byroniae. Mycol. Res. 94: 885-889.SVEDELIUS, G. and UNESTAM, T. 1978. Experimental factos favouring infection ofattached cumuber leaves by Didymella byroniae. Trans. Brit. Mycol. Soc. 71:89-97.SCHWEIZER, P., GEES, R., and MOSINGERM E. 1993. Effect of jasmonic acid on theinteraction of barley (Hordeum vulgare L.) with the powdery mildew Erysiphegraminisf.sp. hordel. P1. Physiol. 102:503-511.TULLOCK, A.P. 1976. Chemistry of plant waxes. In Chemistry and Biochemistry ofNatural Waxes. Edited by P. E. Kollatukudy. ELsevier Pubs, NY. pp235-289.TUNLID, A., NIVENS, D.E., JANSSON, H.B. and WHITE, D. 1991. Infrared monitoring of theadhesion of Catenaria anguillu!ae zoospores to solid surfaces. Exp. Mycol.15: 206-214.VAN ETTEN, J.L., DAHLBERG, K.R. and RUSSO, G.H. 1983. Fungal spore germination. InFungal Differentiation. Edited byJ. E. Smith. Marcel Dekker, Inc., NY.VAN STEEKELENBURG, N.A.M. 1985. Influence of humidity on incidence of Didymellabyroniae on cucumber leaves and growing tips under controlledenvironmental conditions. Netherlands J. P1. Pathol. 91: 277-283.VIELVOYE, J. 1984. Production Guide - Grape: For Commercial Growers. BritishColumbia Ministry of Agriculture, Fisheries and Foods, Queen’s Printer,Victoria, B.C.WAARD, M.A. de. 1971. Germination of powdery mildew conidia in vitro on cellulosemembranes. Netherlands J. P1. Pathol. 77:6-13. (Abstr.)WINKLER, A.J., COOK, J.A., KLIEWER, W.M. and LIDER, L.A. 1974. General Viticulture.2nd edition. UC Press, Berkley, CA. pp138-187.ZEKARIA, O.J., EYAL, Z. and Ziv, 0. 1991. Effect of film-forming compounds on thedevelopment of leaf rust on wheat seedLings. Pt. Dis. 75: 231-234.68Ziv, 0. and FREDERIKSEN, R.A. 1983. Control of foliar diseases with epidermal coatingmaterials. P1. Dis. 67: 21 2-214.Ziv, 0. and FREDERIKSEN, R.A. 1987. The effect of film-forming antitranspirants on leafrust and powdery mildew incidence on wheat. P1. Pathol. 36: 242-245.696.APPENDICESAppendix 1. Number of E. cichoracearum colonies per six-week old “Chicago Pickling”cucumber leaf (mean for three samples per plant) both two weeks after application ofexogenous lipid emulsions1992 TrialsTreatment Trial 1 Trial 2Water control 46.9 A1 39.4 A0.5% ISOL-RB control 39.7 ABC 31.4 ABC0.5% Triton X-100 control 42.3 AB 27.1 BCD4.O%Folicote - 25.4 CD1.0% CanolaiO.5% Triton 31.3 BC 19.9 DE10% WiltPruf - 3.8 FG1.0% Jojobalo.5% ISOL-RB 39.3 ABC 11.9 EF1.0% JojobaiO.5% Triton 26.9 C 9.4 FGCommercial control 3.1 D 0.0 G1. For trial 1, n=6; for trIal 2, n=8: Column meañishàring the same letter are notsignificantly different according to Duncan’s multiple range test, p<0.05.70Appendix 2. Percent of experimental jojoba wax emulsion-treated field-grown ‘Auxerrois’grape plants in a particular growth stage as recorded throughout the growing season in1991Treatments appliedDate Water Commercial “All- “Bloom” “Pre- “Post-control control season” veraison” veraison”Mäy9 56%1 88% 58% 55% 28% 48%3-8 cm Bud burst 3-8 cm 3-8 cm 3-8 cm 3-8 cmshoots shoots shoots shoots shootsM15 29% 97% 17% 27% 10% 15%tight duster 3-8 cm tight duster tight duster tight duster tight dustershootsMay Z3 29% 26% 23% 46% 10% 60%tight duster tight duster tight duster tight duster tight duster tight dusterJtn5 20% 10% 8% 8% 17% 10%bloom bloom bloom bloom bloom bloomJii7 29% 29% 25% 28% 31% 26%berry set berry set berry set berry set berry set berry setJii27 26% 24% 22% 26% 31% 26%colour colour colour colour colour colourchange change change change change changek 10 39% 36% 26% 37% 37% 38%colour colour colour colour colour colourchange change change change change changeSep7 51% 49% 34% 46% 51% 52%berry berry berry berry berry berryripening ripening ripening ripening ripening ripeningSep14 58% 58% 52% 50% 53% 55%berry berry berry berry berry berryripening ripening ripening ripening ripening ripeningSep23 69% 71% 63% 65% 68% 65%berry berry berry berry berry berryripening ripening ripening ripening ripening ripening1 n=24. Data with significant differences between treatments are Listed in Table 13.Growth stage was measured by determining for the most advancedstage the number ofvines per treatment that were in that stage.71Appendix 3. Stomatal resistance (S mo11 rn-2) measurements from the centre ofexperimental jojoba wax emulsion-treated field-grown ‘Auxerrois’ grape leaves on thesouth and outer side of grape plant canopies as recorded throughout the growingseason in 1991Treatments appliedDate Water Commercial “All- “Bloom” “Pre- “Post-control control season” veraison” veraison”Jun 5 2.821 2.83 2.85 2.85 2.71 2.70Jun 15 4.11 3.95 4.29 4.42 4.28 4.21Jun29 2.08 2.11 2.07 2.14 2.25 1.94Jul 7 3.98 3.88 4.38 4.13 4.45 4.13Jul 20 5.77 5.09 4.92 5.52 5.10 5.21Jul 27 3.25 2.89 3.31 3.71 3.49 3.63Aug 10 240 2.13 2.91 2.40 2.56 2.431. n=24. Data showing significant differences between treatments are listed in Table 13.Appendix 4.1. Number of leaves per shoot of experimental jojoba wax emulsion-treatedfield-grown ‘Auxerrois’ grape plants throughout the growing season in 1992Treatments appliedDate Water 0.2% “pre- 1.0% “all- 0.2% “all- 1.0% “pre- Triton Xcontrol veraison season” season” veraison” 100 controlJun 12 15.41 14.3 15.5 15.0 13.9 16.1Jun 18 19.8 20.9 20.2 20.5 18.9 21.6Jun 26 33.8 33.2 33.0 32.9 31.3 36.1Jul3 46.6 42.5 41.9 40.2 40.0 46.7Jul 10 49.4 46.9 47.9 46.3 43.7 50.4Jul 18 38.6 40.8 43.0 44.8 42.9 48.4Jul 24 53.7 54.6 56.4 54.5 52.7 60.5Jul 31 45.2 48.4 54.1 50.4 50.8 55.91. n=1 6 for the Triton X-1 00 control, and both “Pre-veraison” treatments, n=24 for both“All-season” treatments, and n=64 for the water control. Means are averages of twoshoots per plant.72Appendix 4.2. Number of internodes per shoot of experimental jojoba wax emulsion-treated field-grown ‘Auxerrois’ grape plants throughout the growing season in 1992 -Treatments appliedDate Water 0.2% “pre- 1.0% “all- 0.2% “all- 1.0% “pre- Triton Xcontrol veraison” season” season” veraison” 100 controlJun12 12.3 12.5 11.7 12.0 11.5 11.9Jun 18 13.5 14.0 13.7 13.7 13.8 14.2Jun 26 18.4 18.0 17.3 17.5 17.3 18.0Jul 3 22.0 20.8 20.4 20.6 20.4 21.8Jul 10 23.4 21.9 22.0 21.8 21.8 24.0Jul 18 21.0 19.7 21.0 21.3 21.7 23.7Jul 24 22.8 22.5 22.2 22.0 22.7 23.9Jul31 22.3 22.0 23.5 21.1 23.1 24.41. n=1 6 for the Triton X-1 00 control, and both “Pre-veraison” treatments, n=24 for both“All-season” treatments, and n=64 for the water control. Means are averages of twoshoots per plant.Appendix 4.3. Length of the fifth internode (cm) on shoots of experimental jojoba waxemulsion-treated field-grown ‘Auxerrois’ grape plants throughout the growing season in1992Treatments appliedDate Water 0.2% “pre- 1.0% “all- 0.2% “all- 1.0% “pre- Triton X_____control veraison” season” season” veraison” 100 controlJun 12 7.61 7.4 5.5 8.7 7.8 9.3Jun 18 7.7 7.1 6.1 8.4 8.2 9.3Jun 26 8.2 7.6 6.1 8.7 8.3 9.6Jul 3 8.5 7.7 6.2 8.1 8.7 9.6Jul 10 8.4 7.7 6.6 8.9 8.4 9.8Jul 18 7.8 7.0 5.9 8.5 8.0 8.5Jul 24 8.1 7.2 6.1 8.6 8.3 9.7Jul 31 7.5 6.9 6.0 8.7 8.0 8.41. n=1 6 for the Triton X-1 00 control, and both “Pre-veraison” treatments, n=24 for both“All-season” treatments, and n=64 for the water control. Means are averages of twoshoots per plant.73Appendix 4.4. Length of the 10th internode (cm) on shoots of experimental jojoba waxemulsion-treated field-grown ‘Auxerrois’ grape plants throughout the growing season in1992Treatments appliedDate Water 0.2% “pre- 1.0% “all- 0.2% “all- 1.0% “pre- Triton Xcontrol veraison” season” season” veraison” 100 controlJun 12 6.81 5.6 6.0 6.1 6.2 6.9Jun 18 8.0 6.6 6.7 7.2 6.6 8.2Jun 26 7.9 6.8 6.5 7.4 6.3 7.7Jul 3 7.8 6.9 6.6 7.6 6.8 7.9Jul 10 8.5 7.0 6.5 7.6 6.5 8.2Jul 18 8.2 6.3 6.2 7.9 7.2 8.1Jul 24 7.8 7.1 6.1 7.4 6.5 7.9Jul 31 8.3 6.0 6.1 7.7 7.1 8.21. n=1 6 for the Triton X-1 00 control, and both “Pre-veraison” treatments, n=24 for both“All-season” treatments, and n=64 for the water control. Means are averages of twoshoots per plant.Appendix 4.5. Mean area of the 10th leaf (cm2) on shoots of experimental jojoba waxemulsion-treated field-grown ‘Auxerrois’ grape plants throughout the growing season in1992Treatments appliedDate Water 0.2% “pre- 1.0% “all- 0.2% “all- 1.0% “pre- Triton Xcontrol veraison” season” season” veraison” 100 controlJun 12 114.5 108.2 81.7 107.7 105.5 130.4Jun 18 143.5 140.4 106.3 143.3 143.5 168.0Jun26 166.7 168.0 128.8 175.6 177.7 196.6Ju13 173.7 176.1 133.2 180.1 185.8 198.8Jul10 179.8 165.9 135.0 176.6 175.0 194.9Jul18 159.8 169.0 141.9 177.7 176.6 191.3Jul24 178.5 182.3 131.6 177.7 180.1 192.7Jul31 164.4 169.0 143.3 163.3 169.0 191.31. n=1 6 for the Triton X-1 00 control, and both “Pre-veraison” treatments, n=24 for both“All-season” treatments, and n=64 for the water control. Means are averages of twoshoots per plant.74Appendix 4.6. First cluster rachis length (cm) on shoots of experimental jojoba waxemulsion-treated field-grown ‘Auxerrois’ grape plants throughout the growing season in1992Treatments appliedDate Water 0.2% “pre- 1.0% “all- 0.2% “all- 1.0% “pre- Triton Xcontrol veraison” season” season” veraison” 100 controlJun 12 9•41 7.9 6.2 9.2 7.9 10.2Jun 18 10.3 8.6 6.5 9.8 8.6 10.6Jun 26 10.9 9.6 7.5 11.1 9.2 11.7Jul 3 12.1 10.3 8.9 11.8 10.8 13.3Jul 10 12.7 10.2 8.9 12.9 10.9 13.5Jul18 12.6 10.4 8.8 12.7 11.9 12.5Jul24 11.8 10.6 9.1 13.0 11.5 13.8Jul 31 13.0 10.8 9.9 12.7 12.6 13.61. n=1 6 for the Triton X-1 00 control, and both “Pre-veraison” treatments, n=24 for both“All season” treatments, and n=64 for the water control. Means are averages of twoshoots per plant.Appendix 4.7. Berry volume in mm3 from berries sampled at the bottom of first clusterson shoots of experimental jojoba wax emulsion-treated field-grown ‘Auxerrois’ grapeplants throughout the growing season in 1992Treatments appliedDate Water 0.2% “pre- 1.0% “all- 0.2% “all- 1.0% “pre- Triton Xcontrol veraison” season” season” - veraison” 100 controlJun 12 0.0 0.0 0.0 0.0 0.0 0.0Jun 18 0.0 0.0 0.0 0.0 0.0 0.0Jun 26 0.0 0.0 0.0 0.0 0.0 0.0Jul3 110.6 140.6 68.9 157.5 91.1 157.5Jul10 262.1 373.2 185.2 421.9 300.8 389.0Jul18 250.0 456.5 262.1 493.0 571.8 493.0Jul 24 274.6 571.8 - 830.6 493.0 857.4Jul31 456.5 551.4 493.0 884.7 804.4 941.21. n=1 6 for the Triton X-1 00 control, and both “Pre-veraison” treatments, n=24 for both“All-season” treatments, and n=64 for the water control. Means are averages of twoshoots per plant.75Appendix 4.8. Amount of chlorophyll in mg/cm2 for experimental jojobatreated field-grown ‘Auxerrois’ grape leaf samples taken from the southgrape canopies at the 1.5 m level throughout growing season in 1992Treatments appliedDate Water 0.2% “pre- 1.0% “all- 0.2% “all- 1.0% “pre- Triton Xcontrol veraison” season” season” veraison” 100 controlJun 12 0.39 0.45 0.48 0.50 0.45 0.49Jun 18 0.53 0.52 0.51 0.54 0.52 0.51Jun 26 0.52 0.51 0.53 0.53 0.51 0.54Jul 3 0.53 0.59 0.55 0.60 0.55 0.59Jul 10 0.50 0.54 0.54 0.56 0.53 0.49Jul 18 0.47 0.51 0.55 0.57 0.52 0.53Jul 24 0.42 0.49 0.49 0.49 0.55 0.47Jul 31 0.49 0.44 0.39 0.44 0.45 0.45• n=1 6 for the Triton X-1 00 control, and both “Pre-veraison” treatments, n=24 for both“All-season” treatments, and n=64 for the water control. Means are averages of twoshoots per plant.Appendix 5.1. Number of leaves per shoot of experimental seed-storage lipid emulsion-treated field-grown ‘Riesling’ grape plants throughout the growing season in 1992Treatments appliedDate 1.0% jojoba 1.0% canola Commercial Water control 0.5% canolawax oil control oilJun 12 13.31 13.3 14.0 14.3 13.3Jun 18 21.5 21.4 21.6 23.7 30.9Jun 26 32.0 34.1 33.4 37.1 33.9Jul 3 35.6 36.1 36.3 40.5 37.1Jul 10 38.3 40.5 41.9 46.3 42.8Jul 18 55.3 52.4 50.6 56.9 56.3Jul 24 47.2 49.6 50.8 54.8 54.4Jul 31 49.1 53.0 51.3 55.7 59.7Aug 12 51.9 58.3 53.7 58.7 65.5Aug 21 59.2 63.9 55.1 58.6 64.3Sep 7 68.7 68.7 57.8 64.3 79.01. n=12wax emulsionand outside of76Appendix 5.2. Number of internodes per shoot of experimental seed-storage lipidemulsion-treated field-grown ‘Riesllng’ grape plants throughout the growing season in1992Treatments appliedDate 1.0% jojoba 1.0% canola Commercial Water control 0.5% canolawax oil control oilJun 12 12.21 12.6 13.1 13.3 12.3Jun 18 14.7 14.8 15.7 15.9 14.9Jun 26 19.0 20.3 20.5 20.7 19.7Jul 3 22.3 23.1 22.8 22.9 22.4Jul10 21.5 23.6 23.0 23.7 22.4Jul 18 24.8 24.2 25.2 25.5 23.9Jul 24 22.2 24.6 24.6 24.2 23.7Jul 31 22.5 24.8 25.0 24.2 24.9Aug 12 21.7 25.1 25.2 23.8 25.0Aug 21 23.9 27.8 25.7 24.2 27.3Sep 7 26.6 26.5 26.0 25.0 25.31. n=12Appendix 5.3. Length of the fifth internode (cm) on shoots of experimental seed-storagelipid emulsion-treated field-grown ‘Riesling’ grape plants throughout the growing seasonin 1992Treatments appliedDate 1.0% jojoba 1.0% canola Commercial Water control 0.5% canolawax oil control oilJun 12 5.1 1 5.9 5.6 6.6 5.4Jun 18 - 6.3 6.4 6.4 5.6Jun 26 5.4 6.4 6.3 6.3 5.5Jul 3 5.6 6.3 6.5 6.4 5.7Jul 10 5.6 6.3 6.3 6.3 5.6Jul 18 6.4 6.6 6.7 7.0 5.9Jul 24 5.5 6.4 6.5 6.4 5.6Jul31 5.5 6.1 6.3 6.3 5.4Aug 12 5.1 6.1 6.3 5.9 5.4Aug 21 5.4 6.4 6.4 6.3 5.2Sep 7 6.2 6.5 6.6 6.6 5.41. n=1277Appendix 5.4. Length of the 10th internode (cm) on shoots of experimental seed-storagelipid emulsion-treated field-grown ‘Riesling’ grape plants throughout the growing seasonin 1992Treatments appliedDate 1.0% jojoba 1.0% canola Commercial Water control 0.5% canolawax oil control oilJun 12 371 4.2 4.8 5.6 4.5Jun 18 4.4 4.4 5.1 6.1 5.0Jun 26 4.5 4.8 4.9 6.1 5.3Jul 3 4.5 4.8 5.3 3.0 5.3Jul 10 4.4 5.1 5.2 6.1 5.3Jul 18 5.1 5.0 5.5 6.8 5.6Jul 24 4.5 5.0 5.2 6.1 5.3Jul 31 4.5 4.9 5.0 6.0 5.1Aug 12 4.8 - 5.1 6.4 5.4Aug 21 4.3 4.8 5.0 5.9 5.1Sep 7 5.0 4.7 5.2 6.5 5.51. n=12Appendix 5.5. Area of the 10th leaf (cm2)on shoots of experimental seed-storage lipidemulsion-treated field-grown ‘Riesling’ grape plants throughout the growing season in1992Treatments appliedDate 1.0% jojoba 1.0% canola Commercial Water control 0.5% canolawax oil control oilJun 12 59.01 63.1 769 77.3 76.3Jun 18 86.0 86.9 94.4 106.1 98.9Jun26 93.4 94.4 110.6 119.3 116.6Jul3 96.0 97.6 106.1 116.2 114.1Jul 10 95.5 98.5 109.3 120.6 115.7Jul18 111.3 105.3 119.7 133.3 126.6Jul24 95.8 104.9 112.1 120.3 117.2Jul31 96.8 103.6 111.1 117.8 115.6Aug 12 92.5 99.0 112.7 120.7 126.0Aug21 101.9 99.9 110.5 117.6 112.8Sep7 108.4 94.1 120.7 128.7 128.51. n=1278Appendix 5.6. First cluster rachis length (cm) on shoots of experimental seed-storagelipid emulsion-treated field-grown Rieslingtgrape plants throughout the growing seasonin 1992Treatments appliedDate 1.0% jojoba 1.0% canola Commercial Water control 0.5% canolawax oil control oilJun 12 6.71 7.2 6.5 7.1 7.0Jun 18 7.4 8.0 7.4 8.1 7.8Jun 26 8.0 8.7 8.0 8.9 9.0Jul 3 8.3 8.9 8.7 9.1 9.1Jul 10 8.9 9.3 8.6 9.4 9.8Jul 18 9.2 9.7 7.3 8.8 9.8Jul 24 9.0 - 9.1 9.3 10.1Jul 31 8.9 9.4 8.5 9.5 9.7Aug 12 8.9 9.5 9.0 10.2 9.8Aug 21 9.0 9.6 8.8 9.7 10.0Sep 7 9.7 10.1 8.6 9.3 10.7• n=12Appendix 5.7. Berry volume (mm3) from berries sampled at the bottom of first clusterson shoots of experimental seed-storage lipid emulsion-treated field-grown ‘Rieslinggrape plants throughout the growing season in 1992Treatments appliedDate 1.0% jojoba 1.0% canola Commercial Water control 0.5% canolawax oil control oilJun 12 0.01 0.0 0.0 0.0 0.0Jun 18 0.0 0.0 0.0 0.0 0.0Jun 26 39.4 45.5 45.1 46.5 60.6Jul3 184.6 183.5 168.3 191.1 178.3Jul 10 283.9 264.3 274.8 272.1 292.2Jul 18 313.6 369.7 300.7 351.4 350.4Jul24 428.5 395.0 416.6 441.2 490.0Jul 31 400.4 355.6 396.3 414.0 365.2Aug 12 - 564.5 466.7 545.0 -Aug 21 532.5 514.1 544.2 619.8 549.9Sep7 664.2 676.7 607.6 674.3 683.91. n=1279Appendix 5.8. Amount of chlorophyll in mg/cm2 for experimental seed-storage lipidemulsion-treated field-grown ‘Riesling’ grape leaf samples taken from the south andoutside of grape canopies at the 1.5 m level throughout growing season in 1992Treatments appliedDate 1.0% jojoba 1.0% canola Commercial Water control 0.5% canolawax oil control oilJun 12 0.49 0.50 0.48 0.50 0.46Jun 18 0.51 0.48 0.49 0.44 0.47Jun 26 0.50 0.53 0.53 0.54 0.55Jul 3 0.56 0.53 0.54 0.60 0.55Jul 10 0.48 0.51 0.51 0.54 0.51Jul 18 0.54 0.56 0.53 0.53 0.51Jul 24 0.58 0.57 0.56 0.58 0.58Jul 31 0.53 0.55 0.53 0.54 0.54Aug 12 0.57 0.48 0.58 0.54 0.58Aug 21 0.47 0.50 0.49 0.49 0.48Sep 7 0.47 0.47 0.46 0.49 0.491. n=12Appendix 6. Weight of harvested grape clusters sampled from experimental seed-storage hpid emulskn-treated field-grown ‘Auxerrois’ grape plants in 1992Treatment Cluster weight (g)0.2% “All-season” 77 A11.0% “All-season” 60 AB0.2% Triton X-100 control 60 AB1.0% “Pre-veraison” 69 AB0.2% “Pre-veraison” 54 BWater control 19 C1. n=1 6 for the Triton X-1 00 control, and both “Pre-veraison” treatments, n=24 for both“All-season” treatments, and n=64 for the water control. Means are averages of twoshoots per plant. Column means sharing the same letter are not significantly differentaccording to Duncan’s multiple range test, p<0.05.80

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0087345/manifest

Comment

Related Items