In order to aid the booming wine industry in the state of Virginia, U.S.A., we developed a series of studies to provide a deeper understanding of the viruses and vectors for management of virus diseases and development of better tools for grapevine virus diagnostics. A statewide survey for 14 different grapevine viruses between 2009 and 2014 was conducted: 721 samples were collected from 116 vineyards in the period. Among the 12 viruses identified, Grapevine leafroll associated virus-3 (GLRaV-3), Grapevine rupestris stem-pitting associated virus (GRSPaV), and Grapevine red blotch-associated virus (GRBaV) were most commonly present. A new real-time PCR method for the detection of the V2 gene of GRBaV was developed. The resulting method takes less time for more accurate diagnostics than conventional PCR. Evaluation of insecticide effectiveness on GLRaV-3 vectors (mealybugs) and the spread of GLRaV-3 were examined: Four trials conducted from 2012 to 2014 revealed that despite successful control of mealybugs, GLRaV-3 is spread at a very rapid rate. A new sampling technique for efficient nucleic acid storage and testing was developed: the nitrocellulose membrane-based method allows simpler extraction of nucleic acid and provides a storage medium that can hold viable RNA/DNA at room temperature for up to 18 months. An investigation of multiple virus-infected vines and the impact of these co-infections on grapevine fruit chemistry was conducted. GLRaV-3, GRBaV, GRSPaV, and co-infections of the 3 all negatively impacted Brix, pH, titratable acidity, and anthocyanin levels. / Ph. D.

Grapevine leafroll-associated virus-2 (GLRaV-2), GLRaV-3, and grapevine fleck virus (GFkV) are widespread in grapes around the world. These viruses can cause significant crop loss and affect wine quality by reducing sugar accumulation and compromising skin color. Mealybugs are vectors of grapevine leafroll-associated viruses (GLRaVs). A statewide survey of commercial and wild grapevines in Virginia was conducted during 2009 through 2011. Also, vector management options were tested in two field studies. GLRaV-2, GLRaV-3, and GFkV were detected in 8%, 25%, and 1%, respectively, of over 1,200 vine samples (41 wine grape varieties) from 77 locations, and 64% of vineyards were positive for at least one of the tested viruses. All 100 wild grapevines tested were free of these three viruses, indicating that they are not alternative hosts. The majority of infected vines from commercial vineyards were planted prior to the 1990\'s; however, some new plantings were also found to be positive, indicating movement of the viruses among vineyards and also potential infection prior to planting. The high frequency of virus-infected vines emphasizes the importance of clean plant materials, as well as management of vector insects. The insecticide trials resulted in promising vector control with dinotefuran and spirotetramat; however, acetamiprid and pryrethroid resulted in an increase in mealybug population. This study is the first to examine multiple grape viruses in VA. It will aid in developing better strategies aimed at controlling mealybugs to restrict the movement of viral diseases. / Master of Science in Life Sciences

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Grapevine trunk diseases are a cause of decline and loss of productivity in grapevines at all stages of growth. These diseases are caused by a complex of wood-inhabiting fungi that infect mainly through pruning wounds. The management of these diseases relies on wound protection to prevent infection since there are no eradicative control measures to cure infected vines. There are few or no fungicides registered for grapevine pruning wound protection in most countries, while Trichoderma biocontrol agents are often available. This study aimed at improving grapevine wound protection by Trichoderma (T.) spp. and to gain a better understanding of the factors and mechanisms involved in biocontrol. The effect of pruning time (early or late) and five timings of application of the biocontrol agent after pruning on pruning wound colonisation by T. atroviride and T. harzianum were determined. Chenin blanc and Cabernet Sauvignon vineyards were pruned in July (early) and August (late) of 2011 and 2012, and pruning wounds were treated with suspensions of the Trichoderma spp. at various times (0, 6, 24, 48 and 96 hours) after pruning. Wound colonisation was depended on the physiological state of the vine at pruning for both cultivars. However, for the 2012 season in Chenin blanc, wound colonisation was similarly high for both pruning times, which was attributed to high rainfall and humidity. Application of the biocontrol agents 6 hours after pruning consistently resulted in high wound colonisation by the Trichoderma spp. in both cultivars and pruning times. In both cultivars, pruning wound infection due to natural inoculum was higher in wounds made in late winter than those made earlier. The effect of conidial formulation in nutritional (glucose, yeast extract and urea) and bio-enhancing (chitin and cell free culture filtrates) additives, on pruning wound colonisation by T. atroviride was also investigated. Nutritional additives increased the extent of pruning wound colonisation by T. atroviride compared to the un-amended conidial suspensions in a glass house study. The additives as well as Garrison, a fungicide containing pruning wound paint, and Eco77®, a registered T. harzianum biocontrol product, were tested in field trials for wound protection from infection by Phaeomoniella (Pa.) chlamydospora. In 2011, the pathogen was inoculated a day after pruning and all the Trichoderma spp. treatments similarly reduced Pa. chlamydospora infection by 75% to 90% in Thompson Seedless, while control was less in Chenin blanc and ranged from 40% to 74%. In 2012, the trial was carried out on Chenin blanc only and the pathogen was inoculated at intervals of 1, 3 and 7 days after pruning. Wound protection by the Trichoderma treatments was highest when wounds were inoculated with Pa. chlamydospora seven days after pruning. Two conidial formulations, a culture filtrate made from a chitin based medium and a combination of yeast extract, urea and glucose, consistently enhanced biocontrol efficacy. These formulations reduced Pa. chlamydospora infection to levels similar to those of Garrison. The integration of chemical and biological wound protection could provide both immediate and long term wound protection, but is limited by the sensitivity of the biocontrol agent to fungicides. Benzimidazole resistant Trichoderma strains were generated by gamma irradiation from the wild type isolates of T. atroviride (UST1 and UST2) and T. harzianum (T77). Mutants from UST1 and UST2 were of similar biological fitness as the wild type isolates and retained their in vitro antagonistic activity against grapevine trunk pathogens, while the mutant from T77 had reduced fitness and was not antagonistic to the pathogens. The wild type, UST1, and its mutant were tested alone and in combination with thiophanate methyl and carbendazim, respectively, for their ability to prevent pruning wound infection by Pa. chlamydospora. The combination of the UST1 mutant and carbendazim was the most effective treatment and gave the highest reduction in Pa. chlamydospora infection (70% to 93% control). Grapevine cell cultures were used to compare the response of grapevines to T. atroviride and Eutypa (E.) lata as a first step to determining the importance of Trichoderma-grapevine interactions in pruning wound bio-protection. The expression of genes coding for enzymes of the phenylpropanoid pathway and pathogenesis related (PR) proteins was profiled over a 48-hour period using quantitative reverse transcriptase PCR. The cell cultures responded to fungal elicitors in a hypersensitive-like response that lead to a decrease in cell viability. Fungal elicitors from both fungi triggered the same genes and caused up-regulation of phenylalanine ammonia-lyase (PAL), 4 coumaroyl Co-A ligase (CCo-A), stilbene synthase (STS), chitinase class IV (CHIT IV), PR 3 and PR 4, and a down regulation of chalcone synthase (CHS) genes. Higher expression of PAL and CHIT IV in cell cultures treated with the T. atroviride elicitor led to a significantly higher (P < 0.05) total phenolic content and chitinolytic enzyme activity of the cell cultures compared to cell cultures treated with the E. lata elicitor. The response of the cell cultures to the T. atroviride elicitor signifies that the induction of grapevine resistance may be involved in wound bio-protection. The role of secondary metabolites produced by Trichoderma spp. used in pruning wound protection was also investigated. A volatile antimicrobial compound, 6-pentyl α-pyrone (6PP), was isolated and found to be the major secondary metabolite from the T. atroviride (UST1 and UST2) and T. harzianum (T77) isolates. This metabolite was found to inhibit mycelial growth, spore and conidia germination of E. lata, Neofussicocum (N.) australe, N. parvum and Pa. chlamydospora. The production of 6PP was induced when the T. atroviride isolates were grown in a grapevine wood extract medium while for UST1, the 6PP concentration was further doubled when it was co-cultured with N. parvum. Results therefore, indicate that 6PP is involved in the Trichoderma-pathogen interactions on pruning wounds. The results of this study have provided new information in regards to the application of Trichoderma-based pruning wound products. The best time of application proved to be 6 hours post pruning. The formulation of conidial suspensions of Trichoderma spp. with nutritional additives and in protein extracts of the biocontrol agent showed potential in reducing variability of wound bio-protection. However, further research would be necessary to develop commercial products. The application of a fungicide together with Trichoderma spp. in the field holds promise to improve control, but would require further trials for possible commercialisation. This study is the first to report on grapevine host defence genes that are activated by the Trichoderma spp. used in pruning wound protection. Together with the characterisation of the major secondary metabolite produced by these Trichoderma spp., this information aids in understanding the mechanisms involved in the complex interaction between the biocontrol agent, the host and the pathogen. / AFRIKAANSE OPSOMMING: Wingerdstamsiektes veroorsaak terugsterwing en verlies aan produktiwiteit in wingerdstokke gedurende alle groeifases. Hierdie siektes word veroorsaak deur „n verskeidenheid van hout-koloniserende swamme wat die wingerdstok meestal deur snoeiwonde infekteer. Die bestuur van hierdie siektes is afhanklik van wondbeskerming om infeksie te verhoed, omdat daar geen uitwissende beheermetodes na infeksie bestaan nie. In meeste lande is daar min of geen swamdoders geregistreer vir snoeiwond beskerming, terwyl Trichoderma biobeheer agente gereëld beskikbaar is. Hierdie studie poog om wingerd wondbeskerming deur Trichoderma (T.) spp. te verbeter en „n meer volledige begrip van die faktore en meganismes betrokke by biologiese beheer te ontwikkel. Die effek van die tydsberekening van snoei (vroeg of laat) en vyf behandelingstye van die biobeheer agent na snoei op die kolonisering van snoeiwonde deur T. atroviride en T. harzianum is bepaal. Chenin blanc en Cabernet Sauvignon wingerde is gesnoei gedurende Julie (vroeg) en Augustus (laat) in 2011 en 2012, en snoeiwonde is behandel met Trichoderma spp. suspensies op verskillende tydspunte (0, 6, 24, 48 en 96 ure) na snoei. Wond-kolonisering was afhanklik van die fisiologiese toestand van die wingerdstok gedurende snoei vir albei kultivars. Gedurende die 2012 seisoen was wond-kolonisering ewe hoog vir albei snoeitye op Chenin blanc. Dit is verklaar deur hoë reënval en humiditeit gedurende daardie seisoen. Die aanwending van biobeheer agente 6 ure na snoei het konsekwent hoë kolonisering deur Trichoderma spp. tot gevolg gehad op albei kultivars en albei snoeitye. In albei kultivars is wondinfeksie as gevolg van natuurlike inokulum hoër gewees in wonde gemaak gedurende laat winter as in wonde wat vroeër in die seisoen gemaak is. Die effek van konidia formulasie in voeding (glukose, gisekstrak en urea) en bioverbetering (chitien en sel-vrye kultuurfiltraat) toevoegings op snoeiwond-kolonisering deur T. atroviride is ook ondersoek. Voeding toevoegings het die omvangs van snoeiwond-kolonisering deur T. atroviride vergroot in vergelyking met ongewysigde konidia suspensies gedurende „n glashuis studie. Die toevoegings, sowel as Garrison, „n snoeiwond verf wat „n swamdoder bevat, en Eco77®, „n geregistreerde T. harzianum biobeheer produk, is getoets in veldproewe vir wondbeskerming teen infeksie deur Phaeomoniella (Pa.) chlamydospora. In 2011 is die patogeen geïnokuleer „n dag na snoei en al die Trichoderma spp. behandelings het infeksie verminder met 75% tot 90% op Thompson Seedless. Beheer was minder suksesvol op Chenin blanc, waar slegs 40% tot 74% beheer behaal is. In 2012 is die proef uitgevoer slegs op Chenin blanc en die patogeen is geïnokuleer teen intervalle van 1, 3 en 7 dae na snoei. Wondbeskerming by die Trichoderma behandelinge was die hoogste wanneer wonde sewe dae na snoei geïnokuleer is met Pa. chlamydospora. Twee konidia formulasies, „n kultuurfiltraat wat bestaan het uit „n chitien-gebaseerde medium en „n kombinasie van gisekstrak, urea en glukose het deurlopend die effektiwiteit van biobeheer verbeter. Hierdie formulasies het Pa. chlamydospora infeksie verminder tot soortgelyke vlakke behaal deur Garrison. Die integrasie van chemiese- en biobeheer in wondbeskerming kan onmiddelike en langtermyn wondbeskerming bied, maar is beperk deur die sensitiwiteit van die biobeheer agent teen swamdoders. Benzimidazole-weerstandbiedende Trichoderma isolate is ontwikkel deur gamma-bestraling van die wilde-tipe isolate van T. atroviride (UST1 en UST2) en T. harzianum (T77). Mutante van UST1 en UST2 het soortgelyke biologiese fiksheid getoon as die wilde-tipe en het hul in vitro antagonistiese aktiwiteit teen wingerd stampatogene behou, terwyl die mutant van T77 verminderde fiksheid getoon het en nie meer antagonisties teen patogene was nie. Die wilde-tipe, UST1, en sy mutant is apart en in kombinasie met thiofanaatmetiel en carbendazim, respektiewelik, getoets vir die vermoë om snoeiwonde te beskerm teen Pa. chlamydospora. Die kombinasie van die UST1 mutant met carbendazim was die mees effektiewe behandeling en het die hoogste vermindering in Pa. chlamydospora infeksie gelewer (70 tot 93% beheer). As „n beginpunt om die belang van Trichoderma-wingerd interaksies in snoiewondbeheer te bepaal, is die invloed van T. atroviride en Eutypa (E.) lata op somatiese selkulture van wingerd vergelyk. Die effek van dié behandelings op ensieme in die fenielpropanoïedweg en patogenese-verwante (PR) proteïene is bepaal deur intydse PKR (real time PCR) van die korresponderende gene oor „n 48 uur tydperk. Die swam-afkomstige ontlokkers het „n hipersensitiewe-tipe reaksie in die selkulture ontlok, wat tot „n afname in sellewensvatbaarheid gelei het. Ontlokkers afkomstig van beide swamme het dieselfde gene aangeskakel en het induksie van fenielalanien ammoniak-liase (PAL), 4 kumaroïel Ko-A ligase (CCo-A), stilbeen sintase (STS), chitienase klas IV (CHIT IV), PR 3 en PR 4 veroorsaak en „n onderdrukking in chalkoon sintase (CHS) gene tot gevolg gehad. Hoër uitdrukking van PAL en CHIT IV in selkulture behandel met die T. atroviride ontlokker het gelei tot „n beduidende hoër (P < 0.05) totale fenoolinhoud en chitienolitiese aktiwiteit in selkulture in vergelyking met selkulture wat behandel is met die E. lata ontlokker. Die reaksie van die selkulture op die T. atroviride ontlokker dui daarop dat die induksie van wingerd weerstandbiedenheid betrokke mag wees in wond biobeheer. Die rol van sekondêre metaboliete geproduseer deur Trichoderma spp. wat gebruik word in snoeiwond beheer is ook ondersoek. „n Vlugtige antimikrobiese verbinding, 6-pentiel α-pyroon (6PP) is geïsoleer en bepaal om die hoof sekondêre metaboliet afkomstig vanuit die T. atroviride (UST1 en UST2) en T. harzianum (T77) isolate te wees. Hierdie metaboliet is betrokke by inhibisie van miselium groei, spoor en konidium ontkieming van E. lata, Neofusicoccum (N.) australe, N. parvum en Pa. chlamydospora. Die produksie van 6PP is geïnduseer deur die T. atroviride in wingerd hout ekstrak te kweek. In die geval van UST1, is die 6PP konsenstrasie verdubbel deur die isolaat met saam met N. parvum te kweek. Hierdie resultaat is „n aanduiding dat 6PP betrokke is in die Trichoderma-patogeen interaksie op snoeiwonde. Die resultate van hierdie studie het nuwe inligting met betrekking tot die aanwending van Trichoderma-gebaseerde snoeiwond produkte verskaf. Die beste tyd vir aanwending van sulke produkte was 6 ure na snoei. Die formulasie van konidia suspensies van Trichoderma spp. met voeding toevoegings en in proteïen ekstrakte van die biobeheer agent het potensiaal getoon in die vermindering van variasie in wondbeskerming deur biobeheer agente. Verdere navorsing sal nodig wees om kommersiële produkte te ontwikkel. Die aanwending van „n swamdoder saam met Trichoderma spp. in die wingerd is belowend om beheer te verbeter, maar het meer proewe nodig voor kommersialisering. Hierdie studie is die eerste om wingerd beskerming gene wat deur Trichoderma spp. geaktiveer word aan te meld. Laasgenoemde, saam met die beskrywing van die hoof sekondêre metaboliete wat deur hierdie Trichoderma spp. geproduseer word, dra by tot „n meer volledige begrip van die meganismes betrokke by die komplekse interaksie tussen die biobeheer agent, die gasheer en die patogeen.

Caractérisation d'inhibiteurs de protéases lors de l'interaction entre la vigne et Botrytis cinerea. Il a été montré que lors de l'infection de la baie de raisin par B. cinerea, des protéases fongiques pourraient être à l'origine de la dégradation d'une des protéines PR majoritaires de la baie mûre, la chitinase VvChi4D (Thèse S. Colas, 2012 ; van Sluyter et al., 2013). L'hypothèse émise lors de notre étude est que des inhibiteurs de protéases de la vigne pourraient empêcher la dégradation de cette protéine de défense par les protéases de B. cinerea.L'expression de deux inhibiteurs de protéases, un Potato Inhibitor I (VvPin) et un Kunitz (VvKun), ainsi que celle de trois protéases fongiques, une protéase aspartique (BcAp8), une protéase glutamique (BcAcp) et une protéase à sérine (BcSer), ont été suivies lors de l'infection de la baie de Pinot noir et de la feuille de vitroplants. Les résultats obtenus montrent que l'expression des deux IP est induite en même temps que celle de la protéase à sérine mais après celle des deux protéases acides du champignon. La production en système hétérologue des deux IP ainsi que l'obtention de protéases acides et de protéases à sérine de B. cinerea a permis de montrer que la protéine VvKun est capable d'inhiber les protéases à sérine du champignon. En revanche, aucun des deux IP n'est capable d'inhiber les protéases acides du champignon, protéases responsables de la dégradation de la chitinase VvChi4D. / Characterization of protease inhibitors in the interaction between Vitis vinifera and Botrytis cinerea.It has been shown that upon infection of the grape berry by B. cinerea, fungal proteases may be responsible for the degradation of a PR protein of the mature berry VvChi4D chitinase (Thesis S. Colas, 2012; van Sluyter et al, 2013). The hypothesis of our study is that protease inhibitors could prevent the degradation of this defense protein by proteases of B. cinerea.The expression of two protease inhibitors, a Potato Inhibitor I (VvPin) and a Kunitz (VvKun), and that of three fungal proteases, an aspartic protease (BcAp8), a glutamic acid protease (BcAcp) and a serine protease (BcSer) were followed during infection of Pinot Noir berry and leaf plantlets. The results obtained show that the expression of IP is induced both in the same time as the serine protease, but after that the two fungal acid proteases. The heterologous production of both IPs and the production of acid and serine proteases from B. cinerea secretoms have shown that VvKun is capable to inhibit serine proteases of the fungus. However, neither IP is capable of inhibiting fungal acid proteases, responsible for the degradation of the chitinase VvChi4D.

Includes bibliographical references (leaves 158-180) Appendix A. Vineyard disease survey maps -- appendix B. Log linear graphs

Includes bibliographical references (leaves 158-180) / xiii, [220] leaves : ill. (col.), maps ; 30 cm / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 2002

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Grapevine powdery mildew, caused by the fungus Uncinula necator Schweinitz Burrill, is a major disease affecting grape yield and quality worldwide. In conventional vineyards, the disease is controlled mainly by regular applications of sulphur and synthetic fungicides, such as demethylation inhibiting fungicides (DMIs), and in organic agriculture by sulphur and canola-based oils. The impending restrictions on the use of sulphur in organic viticulture, the development of resistance to DMls in Australia and elsewhere, and the demand for residue-free grapes create a need for effective alternatives to sulphur and synthetic chemicals. This research has identified potential replacements for synthetic fungicides and sulphur in the control of powdery mildew, such as milk, whey, bicarbonates and canola oil-based sprays. A series of greenhouse experiments was conducted to evaluate 34 potential novel materials and biological agents for efficacy in controlling powdery mildew. The most effective treatments applied were Bacillus subtilis (which reduced disease by 94% compared to the untreated control), Synertrol Horti-Oil® (a canola oil-based product, 92%), milk (70%), whey (64%) and Ecocarb® (potassium bicarbonate, 58%). Milk and whey provided increased control of powdery mildew as the concentration increased. The efficacy of milk tended to decrease as the fat content of the milk was reduced. The materials that were most promising in the greenhouse were then assessed in field trials in commercial vineyards. Applications of milk, whey and mixtures of a canola oil-based product and potassium bicarbonate, applied at rates of 300 L/ha to 1000 L/ha depending on canopy development, reduced the severity of powdery mildew. The severity of powdery mildew on vines sprayed with a 1:10 dilution of milk, 45 g/L whey powder and mixed programs was not significantly different from that on vines sprayed with sulphur (wettable powder, 3 g/L). However, the relative control of powdery mildew by the test materials in field trials was highly dependent on the degree of coverage of the plant surface achieved. In vineyards where coverage was compromised, the degree of control of powdery mildew was reduced, often to commercially unacceptable levels. Electron spin resonance (ESR) and scanning electron microscopy (SEM) were used to investigate the possible mode or modes of action of milk and whey in the control of powdery mildew. The ESR experiments showed that production of oxygen radicals by various components of milk in natural light was associated with reduced severity of powdery mildew. SEM images showed that milk and whey caused the hyphae of U necator to collapse and damaged conidia within 24 h of treatment. Hydrogen peroxide, applied as a source of free radicals, also caused collapse of the hyphae of U necator within 24 h but did not damage conidia, and appeared to stimulate germination. Lactoferrin (an antimicrobial component of milk) ruptured conidia, but damage to hyphae was not evident in lactoferrin-treated samples until 48 h after treatment. The results suggested that fats, free radical production along with the action of lactoferrin, and possibly other proteins, are associated with the control of powdery mildew by milk. Novel soft fungicides, such as milk and oil plus bicarbonate mixtures, were effective alternatives to sulphur and synthetic fungicides in certain South Australian conditions. Biological agents (including B. subtilis, which was highly effective in greenhouse experiments) did not provide acceptable control of powdery mildew in the vineyard. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1116612 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture and Wine, 2004

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Grapevine powdery mildew, caused by the fungus Uncinula necator Schweinitz Burrill, is a major disease affecting grape yield and quality worldwide. In conventional vineyards, the disease is controlled mainly by regular applications of sulphur and synthetic fungicides, such as demethylation inhibiting fungicides (DMIs), and in organic agriculture by sulphur and canola-based oils. The impending restrictions on the use of sulphur in organic viticulture, the development of resistance to DMls in Australia and elsewhere, and the demand for residue-free grapes create a need for effective alternatives to sulphur and synthetic chemicals. This research has identified potential replacements for synthetic fungicides and sulphur in the control of powdery mildew, such as milk, whey, bicarbonates and canola oil-based sprays. A series of greenhouse experiments was conducted to evaluate 34 potential novel materials and biological agents for efficacy in controlling powdery mildew. The most effective treatments applied were Bacillus subtilis (which reduced disease by 94% compared to the untreated control), Synertrol Horti-Oil® (a canola oil-based product, 92%), milk (70%), whey (64%) and Ecocarb® (potassium bicarbonate, 58%). Milk and whey provided increased control of powdery mildew as the concentration increased. The efficacy of milk tended to decrease as the fat content of the milk was reduced. The materials that were most promising in the greenhouse were then assessed in field trials in commercial vineyards. Applications of milk, whey and mixtures of a canola oil-based product and potassium bicarbonate, applied at rates of 300 L/ha to 1000 L/ha depending on canopy development, reduced the severity of powdery mildew. The severity of powdery mildew on vines sprayed with a 1:10 dilution of milk, 45 g/L whey powder and mixed programs was not significantly different from that on vines sprayed with sulphur (wettable powder, 3 g/L). However, the relative control of powdery mildew by the test materials in field trials was highly dependent on the degree of coverage of the plant surface achieved. In vineyards where coverage was compromised, the degree of control of powdery mildew was reduced, often to commercially unacceptable levels. Electron spin resonance (ESR) and scanning electron microscopy (SEM) were used to investigate the possible mode or modes of action of milk and whey in the control of powdery mildew. The ESR experiments showed that production of oxygen radicals by various components of milk in natural light was associated with reduced severity of powdery mildew. SEM images showed that milk and whey caused the hyphae of U necator to collapse and damaged conidia within 24 h of treatment. Hydrogen peroxide, applied as a source of free radicals, also caused collapse of the hyphae of U necator within 24 h but did not damage conidia, and appeared to stimulate germination. Lactoferrin (an antimicrobial component of milk) ruptured conidia, but damage to hyphae was not evident in lactoferrin-treated samples until 48 h after treatment. The results suggested that fats, free radical production along with the action of lactoferrin, and possibly other proteins, are associated with the control of powdery mildew by milk. Novel soft fungicides, such as milk and oil plus bicarbonate mixtures, were effective alternatives to sulphur and synthetic fungicides in certain South Australian conditions. Biological agents (including B. subtilis, which was highly effective in greenhouse experiments) did not provide acceptable control of powdery mildew in the vineyard. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1116612 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture and Wine, 2004

Thesis (Ph. D.)--Oregon State University, 2009. / Printout. Includes bibliographical references (leaves 61-74). Also available on the World Wide Web.

Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Grapevine leafroll is one of the most damaging viral diseases that affect many viticultural regions of the world. Numerous reports over the last few years have associated closterovirus-like particles with leafroll disease. To date, eight serologically distinct closteroviruses have been isolated from leafroll infected vines, of which grapevine leafroll associated closterovirus-3 (GLRaV-3) is the best characterized. Virus resistance in transgenic plants based on the expression of a virusderived gene is known as pathogen-derived resistance. The viral coat protein (CP) gene, which expresses a structural protein responsible for coating the virus particles, was used in the first demonstration of virus-derived resistance. Coat protein-mediated resistance is currently the most feasible and most widely used method to obtain virus resistance in crop plants. The CP gene of a South African isolate of GLRaV-3 infected grapevine was isolated, cloned and sequenced. Double stranded RNA (dsRNA) was extracted from GLRaV-3 infected material and a high molecular weight band, of -18 kb was identified from infected vines. The dsRNA was used as a template in a reverse transcription PCR together with GLRaV-3 CP gene specific primers for the amplification of the GLRaV-3 CP gene (975 bp). The GLRaV-3 CP gene was cloned into the pGem®-T Easy vector. Clones hosting the CP gene in the sense (pLR3CP+) and antisense (pLR3CP-) orientations respectively were obtained. The sequence obtained from these two clones showed 99.26 % similarity to the only other GLRaV-3 CP nucleotide sequence available. The GLRaV-3 CP gene was excised from pLR3CP+ and pLR3CP- and subcloned into a plant expression vector, pCAMBIA 3301 in the sense (pCamBLR3CP+) and antisense (pCamBLR3CP-) orientations respectively, therefore enabling sense and antisense gene expression in transgenic plants. The GLRaV-3 CP gene was also subcloned from pCamBLR3CP+ into another plant expression vector, pCAMBIA 2301 in the sense orientation and designated as pCVSLR3CP+. These three constructs were given to Dr. M. Vivier (Institute for Wine Biotechnology, Stellenbosch) for grapevine transformation experiments. Two of these constructs, pCamBLR3CP+ and pCamBLR3CP- as well as pCAMBIA 3301 were used to transform Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation. Plants were selected for their ability to withstand the herbicide, Basta. This resistance is due to the presence of a plant selectable marker gene on each of these constructs, known as the bar gene. PCR with GLRaV-3 CP gene specific primers showed no amplification of the GLRaV-3 CP gene in the plants transformed with pCamBLR3CP+ and pCamBLR3CP-. Southern blot analysis with the GLRaV-3 CP gene as hybridization probe showed no signal for these plants, thus confirming the PCR results. PCR with bar gene specific primers showed no amplification of the bar gene in the plants infected with pCAMBIA 3301. The plants transformed with pCamBLR3CP+ and pCamBLR3CP- were also screened for the presence of the bar gene. Three of the eight plants tested showed amplification of the -560 bp bar gene. This result suggests that these plants were transformed with pCAMBIA 3301 (vector without the ligated GLRaV-3 CP gene) and not pCamBLR3CP+ or pCamBLR3CP- as had been expected. This project provides preliminary work for the subsequent transformation of grapevine with the GLRaV-3 CP gene, in an attempt to impart virus resistance. / AFRIKAANSE OPSOMMING: Wingerd rolblaar is een van die mees beskadigende virale siektes wat baie wingerd areas in die wêreld aantas. In Aantal verslae oor die afgelope jare het closterovirus partikels met wingerd rolblaar geassosieer. Tot hede, is agt serologiese onderskeibare closterovirusse geïsoleer vanuit geaffekteerde wingerde, waarvan wingerd rolblaar geassosieerde closterovirus-3 (GLRaV-3) die beste gekarakteriseerd is. Virus bestandheid in transgeniese plante gebaseer op die uitdrukking van gene afkomstig vanaf virusse, staan bekend as patogeen-afgeleide weerstand. Die virale kapsule protein (CP) geen vervaardig In strukturele protein wat verantwoordelik is vir die bedekking van die virus partikel. Dié geen was gebruik in die eerste demonstrasie van patogeen-afgeleide weerstand. Kapsuul protein-bemiddelde weerstand is tans die mees praktiese en algemene gebruikte metode om virus weerstand in plant gewasse te verkry. Die CP geen van In Suid Afrikaanse isolaat van GLRaV-3 geïnfekteerde wingerde is geïsoleer, gekloneer en die volgorde is bepaal. Dubbelstring RNA (dsRNA) was uit GLRaV-3 geïnfekteerde materiaal geëkstraheer en In hoë molekulêre gewig band van -18 kb is geïdentifiseer. Die dsRNA is gebruik as In templaat vir In omgekeerde transkripsie PKR saam met GLRaV-3 CP geen spesifieke inleiers vir die amplifikasie van die GLRaV-3 CP geen (975 bp). Die GLRaV-3 CP geen is gekloneer in die pGem®-T Easy vektor. Klone met die CP geen in die sin (pLR3CP+) en teensin (pLR3CP-) oriëntasies respektiewelik is verkry. Die volgorde wat verkry is vanuit hierdie twee klone dui op In 99.26 % ooreenstemming met die enigste ander GLRaV-3 CP geen volgorde wat beskikbaar is. Die GLRaV-3 CP geen is uit pLR3CP+ en pLR3CP- gesny en is gesubkloneer in In plant ekspressie vektor, pCAMBIA 3301 in die sin (pCamBLR3CP+) en teensin (pCamBLR3CP-) oriëntasies respektiewelik, wat die sin en teensin geen ekspressie in transgeniese plante in staat stel. Die GLRaV-3 CP geen was ook gesubkloneer vanaf pCamBLR3CP+ in In ander plant ekspressie vektor, pCAMBIA 2301 in die sin orientasie en is as pCVSLR3CP+ benoem. Hierdie drie konstruksies is aan Dr. M. Vivier (Instituut vir Wyn Biotegnologie, Stellenbosch) gegee vir wingerd transformasie eksperimente. Twee van hierdie konstruksies, pCamBLR3CP+ en pCamBLR3CP- asook pCAMBIA 3301 is gebruik om Nicotiana tabacum deur middel van Agrobacterium tumefaciens-bemiddelde transformasie te transformeer. Plante is geselekteer vir hul vermoë om die onkruiddoder, Basta, te weerstaan. Die teenwoordigheid van die plant selekteerbare merker geen, bar, op elke konstruksie lui tot dié weerstand. Die plante wat getransformeer is met pCamBLR3CP+ en pCamBLR3CP- is deur PKR saam met die GLRaV-3 CP geen spesifieke inleiers getoets, en geen amplifikasie van die GLRaV-3 CP geen is getoon nie. Southern blot analise met die GLRaV-3 CP geen as hibridisasie peiler het geen sein gewys vir hierdie plante nie, wat die PKR resultate bevestig. Die plante wat getransformeer is met pCAMBIA 3301 is deur PKR saam met die bar geen spesifieke inleiers getoets, en geen amplifikasie van die bar geen is getoon nie. Die plante wat getransformeer is met pCamBLR3CP+ en pCamBLR3CP- is ook getoets vir die teenwoordigheid vir die bar geen. Drie van die agt plante wat getoets is, het amplifikasie van die -560 bp bar geen getoon. Hierdie onverwagte resultate stel voor dat dié plante met pCAMBIA 3301 (vektor sonder die geligeerde GLRaV-3 CP geen) en nie met pCamBLR3CP+ en pCamBLR3CPgetransformeer is nie. Hierdie projek verskaf voorlopige werk vir die daaropvolgende transformasie van wingerd met die GLRaV-3 CP geen in 'n poging om virus bestandheid te verskaf.