Septoria ampelina causes a disease of grapes known as septoria leaf spot. This study was done to determined which of the fungicides currently used to control the various diseases of grapes, plus one experimental fungicide, is the most effective in controlling septoria leaf spot. Both in vitro and in vivo methods were used. In vivo studies examined the systemic and/or protectant activities of the fungicides. The systemic and protectant fungicides included Bayleton, Benlate, Elite (an experimental fungicide), Nova, Rovral and Rubigan. The protectant only fungicides included Captan, Dithane and Kocide. In vitro tests to determine the minimum inhibitory concentration (MIC) for each fungicide (e.g., the concentration of the fungicide that prevents the fungus from forming colonies on the PEA-fungicide medium), indicate that Benlate (MIC = 0.1 ppm) and Elite (MIC = 1.0 ppm) have the greatest potential'to control septoria leaf spot of grape. These are followed by Dithane, Nova and Rubigan (MIC = 2.0), which in turn are followed by Bayleton and Captan (MIC = 50.0 ppm). Kocide and Rovral did not inhibit fungal growth at concentrations up through 100 ppm. Although all the fungicides tested significantly reduced the incidence of septoria leaf spot in vivo, Benlate and Elite were the most effective fungicides (both in systemic and protectant application). / Department of Biology

Includes bibliography: (p. 96-104) / vii, 104 p. : ill. (some col.) ; 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, 1998

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

" August 2002." / Bibliography: leaves 218-235. / viii, 235 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The pathogenicity of Phomopsis taxon 1 is examined in relation to symptom expression and bud loss on grapevines. Phomopsis taxon 1-specific DNA probe, pT1P180, and taxon 2-specific probe, pT1P25, were used to detect Phomopsis taxon 1 and Phomopsis taxon 2 in infected buds, canes and shoots in glasshouse and field experiments. Experiments confirm the isolates of taxon 1 examined did not cause leaf or shoot symptoms associated with Phomopsis cane and leaf spot, and that taxon 2 is more virulent than taxon 1. Suggests that taxon 1 (Diaporthe) is an endophyte which does not cause harm to the grapevine and that chemical control is not warranted for control of taxon 1 on grapevine. / Thesis (Ph.D.)--University of Adelaide, Dept. of Applied and Molecular Ecology, 2002

[Introduction] In the Western Cape Province the mealybug, Planococcus citri (Risso), was first reported on vines in 1930 by Joubert (1943a). By 1935 this mealybug had spread to the Hex River Valley, and subsequently to all the main table grape producing areas of the Western Cape Province. At present Pl. citri is the most important insect pest of the table grape industry and can, if not effectively controlled, result in a loss of at least five per cent of the export table grape crop (Kriegler, 1954). Some preliminary work on the natural enemies of Pl. citri on vines was carried out by Stubbings in 1948, but no further work of this nature has been undertaken in this area since then. The fact that the natural enemies can be an effective check to this mealybug on vines in the Western Cape Province has been known for a number of years (Potgieter, 1937; Hattingh, 1943; Joubert, 1943a; Myburgh, 1951). The present work is an attempt to obtain some basic knowlege of the population fluctuations of the insects concerned in this biological control. Surveys undertaken have shown that there is a complex of primary, secondary and possibly tertiary Hynenopterous parasites associated with Pl. citri. The presence of hyperparasites reduces the efficiency of the primary parasitic Hymenoptera. The usefulness of these primary parasites is further reduced as they only occur in effective numbers for a short period of the year. On the other hand, although attacked by some parasites, the numerous coccinellids found preying on Pl. citri are of more importance in reducing the mealybug populations, as they are present on the vines in effective numbers for the greater part of the year.

Mealybugs (Hemiptera: Pseudococcidae) cause severe damage to many commercial crops, including grapevine. This is largely because of their ability to transmit various grapevine viral diseases, in particular grapevine leafroll-associated viruses (GLRaVs). Grapevine leafroll is one of the most wide-spread grapevine diseases worldwide. Managing the field-spread of grapevine leafroll disease requires, amongst others, stringent mealybug control. Mealybug monitoring and control methods rely on timely and accurate identification of the species present. However, proper identification of mealybug species is problematic, time-consuming and requires an expert taxonomist. In most cases, only adult females can be reliably identified morphologically. Immature insects, males and damaged specimens cannot be assigned to species. In this study, a molecular method was developed to rapidly and accurately distinguish three mealybug species associated with grapevine, namely the vine mealybug Planococcus ficus (Signoret), the citrus mealybug Planococcus citri (Risso) and the longtailed mealybug Pseudococcus longispinus (Targioni-Tozzetti). During the development of this identification method, a number of tasks were undertaken. Firstly, rapid and reliable DNA extraction methods were tested for mealybug DNA. Two rapid extraction methods were adapted and tested, namely the direct buffer method and the spot-PCR method. These methods reliably extracted DNA even from very small or damaged individuals, and could be performed in 15-20 minutes and three hours, respectively. Secondly, mealybug mitochondrial DNA from the cytochrome c oxidase subunit 1 (CO I) gene was amplified and sequenced. It was found that DNA from the 3’-end of CO I showed minimal intraspecific variation (<1%), but sufficient interspecific variation (7-12%) to clearly delineate species. This region was then used to develop three species-specific forward primers, which were used in conjunction with a common universal reverse primer. These primers were all used in a multiplex PCR to differentially amplify DNA from each of the three species. The primers were designed such that each yielded a DNA product of different length which could be separated by electrophoresis on an agarose gel. In this manner the identity of the species could be determined. The entire identification protocol (including extraction, PCR and electrophoresis) could be completed in approximately four hours. All amplified specimens in a blind trial were correctly identified, regardless of size or condition of the specimen. The protocol is simple enough to be implemented in any molecular laboratory. This represents a considerable improvement over currently available techniques for mealybug identification, and is certain to be of great use in diagnostic identification of mealybugs in vineyards and export consignments. / Dissertation (Magister Scientiae)--University of Pretoria, 2007. / Zoology and Entomology / unrestricted

Thesis (MScAgric.)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Phomopsis cane and leaf spot disease of grapevine is an economically important disease in many of the vine-growing areas of the world. Four different Phomopsis spp. have previously been associated with this disease. The present study investigates the taxonomic significance of the different taxa found on grapevines in South Africa, as well as the endophytic growth and fungicide sensitivity of Phomopsis viticola isolates. The thesis is compiled of several different parts, which deal with specific, but related topics, and hence some duplication has been unavoidable. Understanding the epidemiology of a disease is important for the correct timing of disease control. To investigate the endophytic growth of P. viticola, asymptomatic shoots were collected at eight different growth stages. Nodes, internodes, leaf petioles, leaves, tendrils and bunch peduncles were investigated. Two Phomopsis spp., taxon 1 and 2 were identified in this study. The Phomopsis viticola-complex had a relative importance of 9% and accounted for 3% of the isolations. P. viticola (taxon 2) is mainly isolated from the nodes and internodes. Inoculations of healthy, young vine tissue confirmed taxon 2 to be a virulent pathogen, suggesting that it is a latent pathogen rather than an endophyte. In contrast, taxon 1 appeared to be a true endophyte, and did not seem to be an important pathogen on vines. The true identity of the causal organism of Phomopsis cane and leaf spot disease was investigated by collecting samples from 58 different vineyards in the grapevine growing areas of the Western Cape. P. viiicola occurred in grapevine material collected from Lutzville to Swellendam, but was not found in the Oudtshoorn and Orange River grapevine areas. Diaporthe perjuncta (taxon 1), P. vutcola (taxon 2), taxon 3 and a Phomopsis species commonly associated with shoot blight of peaches in the U.S.A., P. amygdali, were identified among the South African grapevine isolates. Examination of the Australian culture designated as taxon 4 found it to be a species of Libertella, thus excluding it from the P. viticola-complex. An Italian isolate was found to represent a species of Phomopsis not previously known from grapevines, and this was subsequently described as taxon 5. Species delimitation was based on morphological and cultural characteristics, stem inoculations and the formation of the teleomorph in vitro. The identity of each morphological taxon was confirmed by means of phylogenetic analyses of the nuclear ribosomal DNA internal transcribed spacers (ITS 1 and ITS2) and the 5' end partial sequence of the mitochondrial small subunit (mtSSU). P. amygdali, associated with peach shoot blight in the U.S.A., was isolated once only and appeared to be of lesser importance in this disease complex. Furthermore, taxa 1 (Diaporthe perjuncta) and 3 were also rarely encountered and proved to be non-pathogenic, indicating their non-functional role in Phomopsis cane and leaf spot disease. Taxon 2 (Phomopsis viticolas was common and widely distributed in diseased vineyards. This taxon was associated with the typical disease symptoms and proved to be pathogenic. Morphologically taxon 2 corresponded best with P. viticola, which was also neotypified in this study. Taxon 2 was mostly isolated from buds and nodes, indicating that these are important sites in which the fungus survives during winter. Molecular data indicated that taxon 3 and P. amygdali were not host specific to grapevine. The currently used foliar fungicides were compared to the new strobilurin fungicides. The effects of nine fungicides (azoxystrobin, flusilazole, folpet, fosetyl- Al+mancozeb, kresoxim-methyl, mancozeb, penconazole, spiroxamine and trifloxystrobin) were tested in vitro on inhibition of mycelial growth. The following EC50 (ug/ml) values were obtained: azoxystrobin (0.350), flusilazole (0.007), folpet (4.489), fosetyl-Al+mancozeb (3.925), kresoxim-methyl (1.665), mancozeb (2.891), penconazole (0.023), spiroxamine (0.321) and trifloxystrobin (0.051). Additionally, azoxystrobin, folpet, kresoxim-methyl, mancozeb, propineb and trifloxystrobin were tested for their ability to inhibit spore germination in vitro. The subsequent EC50 (ug/ml) values were obtained: azoxystrobin 0.123), folpet (0.510), kresoxim-methyl (0.0037), mancozeb (0.250), propineb (0.156) and trifloxystrobin (0.003). The results reported in part 4 showed that the strobilurin fungicides inhibited the mycelial growth and spore germination of P. viticola. However, further trials need to be conducted to verify these findings under field conditions. In the present study taxa 1, 3 and P. amygdali were infrequently isolated, suggesting that they played a less prominent role in the P. viticolacomplex. / AFRIKAANSE OPSOMMING: Streepvleksiekte van wingerd is 'n ekonomies belangrike siekte wat in die meeste wingerdproduserende gebiede van die wêreld voorkom. Vier Phomopsis spesies is in die verlede met dié siekte geassosieer. Hierdie studie ondersoek die taksonomiese belangrikheid van die verskillende taksa wat op wingerd in Suid Afrika gevind word, asook die endofietiese groei en fungisiedsensitiwiteit van die Phomopsis vitico/a isolate. Hierdie tesis bestaan uit verskeie dele met spesifieke, maar verwante onderwerpe wat tot onafwendbare duplisering lei. Dit is belangrik om die epidemiologie van 'n siekte te verstaan sodat korrekte en tydsberekende siektebeheer toegepas kan word. Die endofietiese groei van P. vitico/a is ondersoek deur simptoomlose lote by agt verskillende groei stadiums te versamel. Nodusse, internodusse, blaarstele, blare, rankies en trosstele is ondersoek. Twee Phomopsis spp., takson 1 en 2 is geïdentifiseer. Die Phomopsis vitico/a-kompleks het 3% van die isolasies uitgemaak en 'n relatiewe belangrikheid van 9% getoon. P. vitico/a (takson 2) is meestal uit die nodus en internodus geïsoleer. lnokulasies van gesonde, jong wingerdweefsel het bevestig dat takson 2 'n virulente patogeen is en dat die takson eerder 'n latente patogeen as 'n endofiet is. In teenstelling hiermee is takson 1 'n ware endofiet en 'n onbelangrike patogeen op wingerd. Die ware identiteit van die veroorsakende organisme van streepvlek is ondersoek deur plantmateriaal vanaf 58 verskillende wingerde in die wingerproduserende gebiede van die Wes-Kaap te versamel. P. vitico/a is in wingerdmateriaal vanaf Lutzville tot Swellendam aangetref, maar nie in die Oudtshoorn en Oranjerivier wingerd produserende gebiede nie. Diaporthe perjuncta (takson 1), P. vitico/a (takson 2), takson 3 en P. amygdali is in die Suid Afrikaanse wingerdisolate geïdentifiseer. P. amygdali word met lootverskroeiing van perske bome in die V.S.A. geassosieer. Die Australiese isolaat wat benoem is as takson 4, is met die huidige ondersoek gevind om 'n spesie van Libertella te wees. Takson 4 is daarvolgens uit die P. vitico/a-kompleks gelaat. 'n Italiaanse isolaat het 'n nuwe spesie van Phomopsis op wingerd verteenwoordig en is vervolgens as takson 5 beskryf. Spesie-onderskeiding is op morfologiese en kulturele eienskappe, staminokulasies en die vorming van die teleomorf in vitro gebaseer. Die identiteit vanelke morfologiese takson is met behulp van filogenetiese analises van die nukleêre ribosomale DNS intern transkriberende spasieerders (ITS 1 en ITS2) en die 5' punt gedeeltelike nukleotied volgorde van die mitochondriale klein subeenheid (mtSSU) bevestig. P. amygdali is slegs een keer geïsoleer en blyk van minder belang in die siektekompleks te wees. Takson 1 (Diaporthe perjuneta) en takson 3 het ook min voorgekom en is nie-patogenies, wat hul nie-funksionele rol in streepvleksiekte aandui. Takson 2 (P. viticola) is algemeen geïsoleer en kom wyd verspreid voor. Hierdie takson is geassosieer met die tipiese siektesimptome en is ook patogenies. Morfologies stem takson 2 met P. viiicola ooreen en is ook geneotipifiseer in hierdie studie. Takson 2 is meestal vanaf die ogies en nodusse geïsoleer, wat daarop dui dat hierdie belangrike setels is waar die swam tydens die winter oorleef. Die molekulêre data toon aan dat takson 3 en P. amygdali nie gasheerspesifiek tot wingerd is nie. Die swamdoders wat tans teen streepvlek gebruik word, is met die nuwe strobilurin swamdoders vergelyk. Die effek van nege swamdoders (azoksistrobin, flusilasool, folpet, fosetyl-Al + mancozeb, kresoxirn-metiel, mankozeb, penconasool, spiroksamien en trifloksistrobin) is in vitro op die inhibisie van miseliumgroei getoets. Die volgende EKso-waardes (g/ml) is verkry: azoxystrobin (0.350), flusilasool (0.007), folpet (4.489), fosetiel-Al + mankozeb (3.925), kresoxirn-metiel (l.665), mankozeb (2.891), penkonasool (0.023), spiroksamien (0.321) en trifloxystrobin (0.051). Azoxystrobin, folpet, kresoxim-rnetiel, mankozeb, propineb en trifloksistrobin is ook in vitro getoets vir hul inhibisie op spoorontkieming. Die volgende EKso-waardes is verkry: azoxystrobin (0.123), folpet (0.510), kresoxim-metiel (0.0037), mankozeb (0.250), propineb (0.156) en trifloxystrobin (0.003). Die resultate vervat in deel 4 toon dat die strobilurin swamdoders die miseliumgroei en spoorontkieming van P. viticola inhibeer. Toetsing in die veld word egter benodig om die effektiwiteit van die middels te bevestig. In hierdie studie is taksa I, 3 en P. amygdali selde geïsoleer, wat aangedui het dat hierdie taksa 'n minder belangrike rol in die P. viticola-kompleks speel.

Thesis (MScAgric)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Phaeomoniella chlamydospora, Eutypa lata, Phomopsis, Phaeoacremonium, and Botryosphaeria spp. are important trunk disease pathogens that cause premature decline and dieback of grapevine. Previous research has focused primarily on wine grapes and the incidence and symptomatology of these pathogens on table grapes were largely unknown. A survey was therefore conducted to determine the status and distribution of these pathogens and associated symptoms in climatically diverse table grape growing regions. Fifteen farms were identified in the winter rainfall (De Doorns, Paarl and Trawal) and summer rainfall (Upington and Groblersdal) areas. Samples were taken in July and August 2004 from Dan-ben-Hannah vineyards that were 8 years and older. Distal ends of arms were removed from 20 randomly selected plants in each vineyard. These sections were dissected and isolations were made from each of the various symptom types observed: brown or black vascular streaking, brown internal necrosis, wedge-shaped necrosis, watery necrosis, esca-like brown and yellow soft wood rot, as well as asymptomatic wood. Fungal isolates were identified using molecular and morphological techniques. Pa. chlamydospora was most frequently isolated (46.0%), followed by Phaeoacremonium aleophilum (10.0%), Phomopsis viticola (3.0%), Botryosphaeria obtusa (3.0%), B. rhodina (2.2%), B. parva (2.0%), Fusicoccum vitifusiforme (0.6%), B. australis, B. dothidea and an undescribed Diplodia sp. (0.2% each), while E. lata was not found. Most of these pathogens were isolated from a variety of symptom types, indicating that disease diagnosis can not be based on symptomatology alone. Pa. chlamydospora was isolated from all areas sampled, although most frequently from the winter rainfall region. Pm. aleophilum was found predominantly in Paarl, while P. viticola only occurred in this area. Although B. obtusa was not isolated from samples taken in De Doorns and Groblersdal, it was the most commonly isolated Botryosphaeria sp., being isolated from Upington, Paarl and Trawal. B. rhodina occurred only in Groblersdal and B. parva in Paarl, Trawal and Groblersdal, while B. australis was isolated from Paarl only. The rest of the isolates (33%) consisted of sterile cultures, Exochalara, Cephalosporium, Wangiella, Scytalidium, Penicillium spp. and two unidentified basidiomycetes, which were isolated from five samples with yellow esca-like symptoms from the Paarl area. These findings clearly illustrate that grapevine trunk diseases are caused by a complex of fungal pathogens, which has serious implications for disease diagnosis and management. Protection of wounds against infection by any of these trunk disease pathogens is the most efficient and cost-effective means to prevent grapevine trunk diseases. However, previous research on the effectiveness of chemical pruning wound protectants has mostly focused on the control of Eutypa dieback only. Fungicide sensitivity studies have been conducted for Pa. chlamydospora, P. viticola and Eutypa lata, but no such studies have been conducted for the pathogenic Botryosphaeria species from grapevine in South Africa. Ten fungicides were therefore tested in vitro for their efficacy on mycelial inhibition of the four most common and/or pathogenic Botryosphaeria species in South Africa, B. australis, B. obtusa, B. parva and B. rhodina. Iprodione, pyrimethanil, copper ammonium acetate, kresoxim-methyl and boscalid were ineffective in inhibiting the mycelial growth at the highest concentration tested (5 μg/ml; 20 μg/ml for copper ammonium acetate). Benomyl, tebuconazole, prochloraz manganese chloride and flusilazole were the most effective fungicides with EC50 values for the different species ranging from 0.36-0.55, 0.07-0.17, 0.07-1.15 and 0.04-0.36 μg/ml, respectively. These fungicides, except prochloraz manganese chloride, are registered on grapes in South Africa and were also reported to be effective against Pa. chlamydospora, P. viticola and E. lata. Results from bioassays on 1-year-old Chenin Blanc grapevine shoots indicated that benomyl, tebuconazole and prochloraz manganese chloride were most effective in limiting lesion length in pruning wounds that were inoculated with the Botryosphaeria spp after fungicide treatment. The bioassay findings were, however, inconclusive due to low and varied re-isolation data of the inoculated lesions. Benomyl, tebuconazole, prochloraz manganese chloride and flusilazole can nonetheless be identified as fungicides to be evaluated as pruning wound protectants in additional bioassays and vineyard trials against Botryosphaeria spp. as well as the other grapevine trunk disease pathogens. / AFRIKAANSE OPSOMMING: Phaeomoniella chlamydospora, Eutypa lata, Phomopsis, Phaeoacremonium, en Botryosphaeria spesies is die mees belangrikste stamsiekte patogene wat agteruitgang en vroeë terugsterwing van wingerd veroorsaak. Voorafgaande navorsing het hoofsaaklik gefokus op wyndruiwe en die voorkoms en simptomatologie van hierdie patogene op tafeldruiwe is dus grootliks onbekend. ‘n Opname is gevolglik gedoen in verskillende klimaaatsareas waar tafeldruiwe verbou word om die voorkoms en verspreiding, asook die simptome geassosieer met hierdie patogene, te bepaal. Vyftien plase is geïdentifiseer in die winter- (De Doorns, Paarl en Trawal) en somer-reënval (Upington en Groblersdal) streke. Wingerde (8 jaar en ouer) met die kultivar Dan-ben-Hannah is gekies vir opname en monsters is gedurende Julie en Augustus 2004 geneem. Die distale deel van ‘n arm is verwyder vanaf 20 lukraak gekose plante in elke wingerd. Hierdie dele is ontleed en isolasies is gemaak vanuit elke simptoomtipe wat beskryf is, naamlik bruin en swart vaskulêre verkleuring, bruin interne nekrose, wig-vormige nekrose, waterige nekrose, esca-geassosieerde bruin en geel sagte houtverrotting en asimptomatiese hout. Identifikasie van die swamagtige isolate is gedoen op grond van morfologiese eienskappe en molekulêre tegnieke. Pa. chlamydospora is die meeste geïsoleer (46.0%), gevolg deur Phaeoacremonium aleophilum (10.0%), Phomopsis viticola (3.0%), Botryosphaeria obtusa (3.0%), B. rhodina (2.2%), B. parva (2.0%), Fusicoccum vitifusiforme (0.6%), B. australis, B. dothidea en ‘n onbeskryfde Diplodia sp. (0.2% elk), terwyl E. lata nie geïsoleer is nie. Hierdie patogene is elk geïsoleer vanuit ‘n verskeidenheid simptoomtipes, wat daarop dui dat siektediagnose nie alleenlik op simptomatologie gebaseer kan word nie. Pa. chlamydospora is geïsoleer vanuit al die gebiede, alhoewel die patogeen opmerklik meer voorgekom het in die winter-reënval area. Pm. aleophilum het hoofsaaklik voorgekom in Paarl, terwyl P. viticola slegs in hierdie area voorgekom het. Alhoewel B. obtusa nie voorgekom het in die De Doorns en Groblersdal areas nie, was dit die mees algemeen geïsoleerde Botryosphaeria sp. en het in Upington, Paarl en Trawal voorgekom. B. rhodina het slegs in Groblersdal voorgekom, B. parva in Paarl, Groblersdal en Trawal en B. australis het slegs in Paarl voorgekom. Die res van die isolate (33%) het bestaan uit steriele kulture, Exochalara, Cephalosporium, Wangiella, Scytalidium, en Penicillium spesies asook twee onbekende basidiomycete isolate, geïsoleer vanuit vyf monsters met geel eska-geassosieerde simptome vanuit die Paarl area. Hierdie resultate illustreer dus die feit dat wingerdstamsiektes deur ‘n kompleks van swampatogene veroorsaak word, wat belangrike implikasies het vir die bestuur en diagnose van hierdie siektes. Wondbeskerming teen infeksie van enige van hierdie stamsiekte patogene is die mees doeltreffende en koste-effektiewe manier om wingerdstamsiektes te voorkom. Vorige navorsing aangaande die effektiwiteit van chemiese wondbeskermingsmiddels het egter slegs gefokus op die beheer van Eutypa terugsterwing. In vitro swamdoder sensitiwiteitstoetse is gedoen vir Pa. chlamydospora, P. viticola en Eutypa lata, maar geen studies is al gedoen ten opsigte van die patogeniese Botryosphaeria spesies op wingerd in Suid-Afrika nie. Tien swamdoders is dus getoets vir inhibisie van in vitro miseliumgroei van die vier mees algemene en/of patogeniese Botryosphaeria spesies wat in Suid-Afrika voorkom, naamlik B. australis, B. obtusa, B. parva en B. rhodina. Iprodione, pyrimethanil, koper ammonium asetaat, kresoxim-metiel en boscalid was oneffektief by die hoogste konsentrasies getoets (5 μg/ml; 20 μg/ml vir koper ammonium asetaat). Benomyl, tebuconasool, prochloraz mangaan chloried en flusilasool was die mees effektiewe swamdoders met EC50 waardes tussen 0.36-0.55, 0.07-0.17, 0.07-1.15 en 0.04-0.36 μg/ml, onderskeidelik vir die verskillende spesies. Hierdie fungisiedes, behalwe prochloraz mangaan chloried, is geregistreer op druiwe in Suid-Afrika en is ook effektief gevind teenoor Pa. chlamydospora, P. viticola en E. lata. Resultate van biotoetse op 1-jaar-oue Chenin Blanc wingerd lote het getoon dat benomyl, tebuconasool en prochloraz mangaan chloried die effektiefste was om die lengte van letsels in snoeiwonde, geinokuleer met Botryosphaeria spesies na die aanwending van swamdoder behandelings, te verminder. Die bevindinge was egter onbeslis as gevolg van die lae en variërende her-isolerings data. Benomyl, tebuconasool, prochloraz mangaan chloried en flusilasool kan egter geïdentifiseer word as swamdoders wat verder geevalueer kan word as snoeiwond beskermingsmiddels teen Botryosphaeria spesies asook ander wingerd stamsiekte patogene in verdere biotoetse en wingerdproewe.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: South Africa is regarded as one of the top wine producing countries in the world. One of the threats to the sustainability of the wine industry is viral diseases of which Grapevine leafroll-associated virus 3 (GLRaV-3) and Grapevine virus A (GVA) are considered to be the most important and wide spread. Scion material is regularly tested for viruses; however scion material is often grafted onto rootstocks that have questionable phytosanitary status. Virus detection in rootstocks is challenging due to low and varying titres, but is imperative as a viral control mechanism. An additional viral control mechanism is the use of transgenic grapevine material which offers resistance to grapevine infection. The objective of this project was to establish a detection system using real time PCR (qPCR) techniques, to accurately and routinely detect GLRaV-3 and GVA in rootstock propagation material. qPCR would furthermore be used to perform molecular characterisation of transgenic plants containing a GLRaV-3 antiviral ΔHSP-Mut construct. A severely infected vineyard (Nietvoorbij farm) in the Stellenbosch area was screened throughout the grapevine growing season to investigate virus prevalence throughout the season and to determine the optimal time for sensitive virus detection. A large scale screening of nursery propagation material for GLRaV-3 infection was also conducted. The qRT-PCR results were compared to DAS-ELISA results to compare the efficacy and sensitivity of the two techniques. For the severely infected vineyard, the ability to detect GLRaV-3 increased as the season progressed towards winter. qRT-PCR was more sensitive and accurate in detecting GLRaV-3 than DASELISA, as the latter technique delivered numerous false positive results later in the season. The best time to screen for GLRaV-3 in the Western Cape region was from the end of July to September. For the nursery screenings, our qRT-PCR results were compared to the results of the DAS-ELISA performed by the specific nurseries. No GLRaV-3 infection was detected in the specific samples received from the two different nurseries. The results for all the samples correlated between the two techniques. This confirms that the propagation material of these nurseries has a healthy phytosanitary status with regards to GLRaV-3. However, the detection of GVA in the severely infected vineyard yielded inconsistent results. Detection ability fluctuated throughout the season and no specific trend in seasonal variation and virus titre fluctuation could be established. The highest percentage of GVA infected samples were detected during September, April and the end of July. Previously published universal primers were used for the detection of GVA, but further investigation indicated that they might not be suitable for sensitive detection of specific GVA variants present in South Africa. Vitis vinifera was transformed with a GLRaV-3 antiviral construct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) and qRT-PCR were utilised as alternative methods for molecular characterisation of transgenic plants. The qPCR and Southern blot results correlated for 76.5% of the samples. This illustrated the ability of qPCR to accurately estimate transgene copy numbers. Various samples were identified during qRT-PCR amplification that exhibited high mRNA expression levels of the transgene. These samples are ideal for further viral resistance studies. This study illustrated that the versatility of real time PCR renders it a valuable tool for accurate virus detection as well as copy number determination. / AFRIKAANSE OPSOMMING: Suid Afrika word geag as een van die top wyn produserende lande ter wereld. Die volhoubaarheid van die wynbedryf word onder andere bedreig deur virus-infeksies. Grapevine leafroll associated virus 3 (GLRaV-3) en Grapevine virus A (GVA) is van die mees belangrike virusse wat siektes veroorsaak in Suid-Afrikaanse wingerde. Wingerd bo-stok materiaal word gereeld getoets vir hierdie virusse, maar hierdie materiaal word meestal geënt op onderstokmateriaal waarvan die virus status onbekend is. Virus opsporing in onderstokke word egter gekompliseer deur baie lae en variërende virus konsentrasies, maar opsporing in voortplantingsmateriaal is ‘n noodsaaklike beheermeganisme vir virus-infeksie. Die doel van die projek was om ‘n opsporingsisteem te ontwikkel via kwantitatiewe PCR (qPCR) tegnieke vir akkurate en gereelde toetsing van GLRaV-3 en GVA in onderstokmateriaal. qPCR sal ook verder gebruik word vir molekulêre karakterisering van transgeniese plante wat ‘n GLRaV-3 antivirale ΔHSP-Mut konstruk bevat. ‘n Hoogs geïnfekteerde wingerd was regdeur die seisoen getoets om seisoenale fluktuasies in viruskonsentrasie te ondersoek en om die optimale tydstip vir sensitiewe virus opsporing te bepaal. ‘n Grootskaalse toetsing van kwekery voortplantingsmateriaal vir GLRaV-3 infeksie was ook uitgevoer. Die qRT-PCR resultate is met die DAS-ELISA resultate vergelyk om die effektiwiteit en sensitiwiteit van die twee tegnieke te vergelyk. Vir die hoogs geïnfekteerde wingerd het die GLRaV-3 opsporing toegeneem met die verloop van die seisoen tot en met winter. qRT-PCR was meer sensitief en akkuraat as DAS-ELISA in die opsporing van GLRaV-3, weens verskeie vals positiewe resultate wat later in die seisoen deur die laasgenoemde tegniek verkry is. Die beste tyd om vir GLRaV-3 te toets is vanaf einde Julie tot September. Tydens die kwekery toetsings was qRT-PCR resultate met die DAS-ELISA resultate van die spesifieke kwekerye vergelyk. Geen GLRaV-3 infeksie was waargeneem in die spesifieke monsters wat vanaf die kwekerye ontvang is nie. Die resultate van die twee tegnieke het ooreengestem vir al die monsters wat v getoets is. Dit het bevestig dat die voortplantingsmateriaal van hierdie kwekerye gesonde fitosanitêre status met betrekking tot GLRaV-3 gehad het. Die opsporing van GVA in die geïnfekteerde wingerd het egter wisselvallige resultate gelewer. Opsporing van die virus het ook regdeur die seisoen gefluktueer en geen spesifieke neiging in seisoenale opsporingsvermoë kon gemaak word nie. Die hoogste persentasie GVA geïnfekteerde monsters was waargeneem tydens September, April en die einde van Julie. Voorheen gepubliseerde universele inleiers was gebruik vir die opsporing van GVA, maar verdere ondersoeke het getoon dat hierdie inleiers nie noodwendig geskik is vir sensitiewe opsporing van GVA variante wat teenwoordig is in Suid-Afrika nie. Vitis vinifera was getransformeer met ‘n GLRaV-3 antivirale konstruct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) en qRT-PCR was ingespan as alternatiewe metodes vir molekulêre karaterisering van transgeniese plante. Die qPCR en Southern-klad resultate het ooreengestem vir 76.5% van die monsters. Dit illustreer die vermoë van qPCR om akkurate kopie-getalle van transgene te bepaal. Verskeie plante is geïdentifiseer tydens qRT-PCR amplifisering wat hoë vlakke van transgeen mRNA uitdrukking getoon het. Hierdie monsters is ideaal vir verdere virus weerstandbiedendheids studies. Hierdie studie het die veelsydigheid van real time PCR bewys en getoon dat dit ‘n kosbare tegniek is vir akkurate virus opsporing sowel as kopie-getal bepaling.