Spine title: Systemic insecticides in grape vines. Typescript (copy). Includes bibliographical references (leaf. 82-84).

" 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

" 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

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 (MScAgric (Plant Pathology))--Stellenbosch University, 2009. / ENGLISH ABSTRACT: Sufficient fungicide deposition on the target site is an essential requirement for effective chemical management of fruit- and foliar diseases such as grey mould of grapevines. Control failure is often attributed to insufficient quantitative deposition on susceptible grapevine tissue. However, in high disease pressure situations control failure might also be attributed to poor qualitative deposition. The primary objective of spray technology is to optimise deposition, of which the plant surface is a critical component in the spray application process, specifically in the retention of spray droplets. Adjuvant technology is reported to improve the wettability and spread of droplets by surface-acting-agents on the target surface and thereby improve deposition and retention of the fungicide active ingredient. However, this relatively new spray technology on viticulture and horticultural crops, and possible effects of adjuvants on epicuticular wax affecting plant disease development, needs to be investigated. Moreover, the development of useful prescriptions for adjuvants by determining water volumes and adjuvant dosages is required for different pesticide tank mixes. The aims of this study were, firstly to determine the effect of selected adjuvants on quantitative and qualitative spray deposition on grapevine leaves and subsequent biological efficacy of a fungicide, and secondly to evaluate selected adjuvants under field conditions and determine the effects of adjuvant dosage and spray volume on deposition. Leaves were sprayed under similar laboratory conditions to pre-run-off with 1 mL of a mixture of fenhexamid (Teldor® 500 SC, Bayer) at recommended dose, a fluorescent pigment (SARDI Fluorescent Pigment, 400 g/L EC; South Australian Research and Development Institute) at 0.2 L/100 L, as well as 15 selected commercial adjuvants to manipulate the deposition quality of a given quantity of deposited spray. Spray deposition on leaves was illuminated under black light (UV-A light in the 365 nm region) and visualised under a stereo microscope (Nikon SMZ800) at 10× magnification. Photos of sprayed leaf surfaces were taken with a digital camera (Nikon DMX 1200). Digital images were quantitatively and qualitatively analysed with Image-Pro Discovery version 6.2 for Windows (Media Cybernetics) software, to determine spray deposition. The sprayed leaves were inoculated with 5 mg dry airborne conidia of Botrytis cinerea in a spore settling tower and incubated for 24 h at high relative humidity (≥ 93%). Leaf discs were isolated onto Petri dishes with paraquat-amended water agar and rated 11 days later for development of B. cinerea from isolated leaf discs. B. cinerea incidence on the upper and lower surfaces of water sprayed leaves averaged 90.4% and 95.8%, respectively. Despite full spray cover of leaves, applications with fenhexamid alone did not completely prevent infection and resulted in 34.6% and 40.8% B. cinerea incidence on the upper and lower surfaces of leaves, respectively. Through the addition of certain adjuvants, B. cinerea incidences were significantly lower (2.9-17.1% and 10.0-30.8%, respectively), while some adjuvants did not differ from the fungicide-only treatment, even though they might have improved spray deposition. The effects of Hydrosilicote and Solitaire alone and in combination with fenhexamid on germinating Botrytis conidia on leaf surfaces were studied in a histopathology study using epifluorescence microscopy. Distinct differences were observed in conidium mortality, germination and germ tube lengths between adjuvants alone and in combination with the fungicide, which might be attributed to indirect effects of the adjuvant mode of action on B. cinerea. The laboratory study clearly demonstrated the potential of adjuvants to improve the bio-efficacy of a fungicide directly through improved deposition on grapevine leaf surfaces. For the vineyard evaluations, the same fluorometry, photomicrography and digital image analysis protocol were used to assess quantitative and qualitative spray deposits under varying adjuvant dosage and volume applications. The Furness visual droplet-rating technique was initially included to determine optimum spray volume with a STIHL SR400 motorised backpack mistblower by assessment of pigment deposition on Chardonnay leaves under illuminated black light. Both assessment protocols showed that quantitative spray deposition increased with increasing spray volume applications of 40 L/ha to 750 L/ha, but decreased at 900 L/ha, possibly due to run-off. The addition of selected adjuvants at recommended dosage and at 600 L/ha demonstrated the potential of adjuvants to increase quantitative and qualitative deposition significantly on upper and lower leaf surfaces. Agral 90, BB5, Nu-film-P, and Solitaire significantly improved deposition on upper and lower leaf surfaces compared with the fenhexamid only and water sprayed control. Break-thru S 240 and Villa 51 did not improve quantitative deposition, although remarkably better qualitative deposition was obtained. An adjuvant dosage effect (within the registered dosage range) was evident, especially those retained on the upper leaf surfaces. Agral 90 and Nu-film-P affected significant improvement of spray deposition at the higher, but not at the lower dosage tested. Solitaire improved deposition at the lower dosage tested, whereas reduced deposition at the higher dosage was attributed to excessive spray run-off. No significant improvement of spray deposition was observed for both dosages tested with Villa 51. Spray mixtures with adjuvants Agral 90 and Solitaire yielded similar deposition values at 600 L/ha compared with the fenhexamid only control at 900 L/ha, but reduced deposition at the higher spray volume, possibly due to spray run-off. This study clearly demonstrated the potential of adjuvants to improve quantitative and qualitative deposition, but highlights the necessity to match adjuvant dosages and application volumes on the spray target to achieve maximum spray deposition. / AFRIKAANSE OPSOMMING: Effektiewe beheer van vrug- en blaarsiektes soos vaalvrot op wingerde benodig voldoende deponering van die swamdoder op die teikenoppervlak. Verlies aan beheer word gewoonlik aan onvoldoende kwantitatiewe deponering op vatbare wingerddele toegeskryf. Onder ‟n hoë siektedruk kan mislukte beheer ook moontlik toegeskryf word aan swak kwalitatiewe deponering. Die primêre doelwit van spuittegnologie is om deponering te optimaliseer met die plantoppervlak as ‟n belangrike komponent in die spuittoedieningsproses, spesifiek in die retensie van spuitdruppels. Byvoemiddel tegnologie het bewys dat oppervlak-aktiewe-agente verbeterde benatting en verspreiding van druppels op die teiken oppervlakte tot gevolg kan hê, en verder ook die deponering en retensie van die aktiewe fungisied bestanddele kan verbeter. Hierdie relatiewe nuwe spuittegnologie op wingerd- en hortologiese verbouing, asook die moontlike effekte van byvoegmiddels op epikutikulêre waks om siekte ontwikkeling te beïnvloed, moet ondersoek word. Verder word nuttige aanbevelings benodig vir byvoegmiddel toedienings by verskillende spuitvolumes en dosisse van die betrokke spuitmengsel. Die doelwit van hierdie studie was, eerstens om die effek van sekere byvoegmiddels op kwantitatiewe en kwalitatiewe spuitbedekking van wingerdblare te bepaal en dan te vergelyk met die biologiese effektiwiteit van ‟n fungisied, en tweedens om van die byvoegmiddels onder veldtoestande te evalueer, asook die effek van byvoegmiddel dosisse en spuitvolumes te bepaal. Blare is onder dieselfde laboratorium toestande tot net voor-afloop met 1 mL van ‟n spuitmengsel, bestaande uit fenhexamied (Teldor® 500 SC, Bayer) teen die aanbevole dosis, ‟n fluoreserende pigment (400 g/L EC; Suid Australiese Navorsing en Ontwikkeling Instituut) teen 0.2 L/100 L, sowel as 15 geselekteerde kommersiële byvoegmiddels gespuit om die kwalitatiewe deponering, vir ‟n gegewe kwantiteit van spuitdeponering, te manipuleer. Die fluoreserende pigment is op die blaaroppervlak belig met ‟n swart lig (UV-A ligbron in die 365 nm golflengte) en deponering is onder ‟n stereo mikroskoop (Nikon SMZ800) teen 10× vergroting waargeneem. Die gespuite blaaroppervlaktes is op die manier met ‟n digitale kamera afgeneem (Nikon DMX 1200), waarna die digitale foto‟s kwantitatief en kwalitatief deur die gebruik van „Image-Pro Discovery version 6.2 for Windows (Media Cybernetics)‟ sagteware geanaliseer is om spuitbedekking te bepaal. Na elke blaarspuit is die blare met 5 mg droë konidia van B. cinerea in ‟n inokulasietoring geïnokuleer en daarna vir 24 h onder hoë relatiewe humiditeit (≥ 93%) geïnkubeer. ‟n Aantal skyfies vanuit elke blaar is op Petri bakkies met paraquat medium geïsoleer en 11 dae later is die persentasie van B. cinerea ontkieming bepaal. Die gemiddelde voorkoms van B. cinerea op die blare wat slegs met water gespuit is, was 90.4% op die boonste en 95.8% op die onderste blaaroppervlaktes. Spuitbehandelings met slegs fenhexamied, ongeag goeie blaarspuitbedekking, kon nie die B. cinerea infeksie ten volle voorkom nie, en infeksie van gemiddeld 34.6% en 40.8% is onderskeidelik op die boonste- en op die onderste blaaroppervlaktes waargeneem. Met die byvoeging van sekere byvoegmiddels het die voorkoms van B. cinerea betekenisvol verminder (2.9-17.1% en 10.0-30.8%, onderskeidelik), terwyl ander byvoegmiddels nie van die fenhexamied behandeling verskil het nie, hoewel hierdie middels meestal wel spuitdeponering verbeter het. Die effek van slegs Hydrosilicote en Solitaire, en in kombinasie met fenhexamied op ontkiemende Botrytis conidia, is bestudeer in ‟n histopatologiese studie deur middel van die gebruik van epifluoresensie mikroskopie op die blaaroppervlak. Duidelike verskille in die aantal dooie konidia, ontkiemingpersentasies en kiembuislengtes is tussen die byvoegmiddels en in kombinasie met fenhexamied waargeneem, waar sommige waarnemings moontlik aan die indirekte effek van die byvoegmiddel op B. cinerea toegeskryf kan word. Hierdie laboratoriumstudie wys duidelik dat byvoegmiddels oor goeie potensiaal beskik om die bio-effektiwiteit van die fungisied te verbeter deur die direkte verbetering van deponering op die wingerdblaaroppervlak. Dieselfde fluorometrie, fotomikrografie en digitale foto-analise protokol is in ‟n wingerd evaluasie om die kwantitatiewe en kwalitatiewe spuitdeponering van verskillende byvoegmidel dosisse and spuitvolumes te bepaal, gebruik. Die Furness visuele druppel meting tegniek is aanvanklik ingesluit om die optimale spuit volume met ‟n „STIHL SR400 motorised backpack mistblower‟ te bepaal deur visuele meetings van gedeponeerde pigment op Chardonnay blare onder ‟n swart ligbron. Beide protokolle wys dat kwantitatiewe spuitbedekking met ‟n toename in spuit volumes 40 L/ha tot 750 L/ha verbeter het, maar afgeneem het teen 900 L/ha, moontlik as gevolg van druppel-afloop. Die byvoeging van ‟n byvoegmiddel teen die aanbevole dosis en 600 L/ha wys uitstekende potensiaal om kwantitatiewe en kwalitatiewe deponering betekenisvol op boonste en onderste blaaroppervlaktes te verbeter. Agral 90, BB5, Nu-film-P, en Solitaire het deponering betekenisvol op boonste en onderste blare in vergelyking met die fenhexamied alleen en die water kontrole verbeter. Break-thru S 240 en Villa 51 het nie kwantitatiewe deponering verbeter nie, alhoewel verbeterde kwalitatiewe bedekking met hierdie produkte waargeneem is. ‟n Byvoegmiddel dosis effek (binne die registreerde dosis reeks) was duidelik waarneembaar, veral vir druppel retensie op die boonste oppervlak van blare. Agral 90 and Nu-film-P verbeter die spuit deponering betekenisvol met die hoër getoetste dosis, maar nie teen die lae dosis nie. Solitaire verbeter egter die deponering teen die laer dosis, maar minder deponering teen ‟n hoër dosis kan moontlik toegeskryf word aan oormatige druppel-afloop. In die geval van Villa 51 was geen betekenisvolle verbetering van spuitdeponering vir beide die behandelingsdosisse waargeneem nie. Spuitmengsels met byvoegmiddels, Agral 90 en Solitaire, het soortgelyke deponerings gelewer teen 600 L/ha in vergelyking met die fenhexamied kontrole teen 900 L/ha, maar deponering neem af teen hoër spuitvolumes met byvoegmiddels moontlik as gevolg van druppel-afloop. Hierdie studie wys duidelik die uitstekende potensiaal van Byvoegmiddels om kwantitatiewe en kwalitatiewe deponering te verbeter, maar beklemtoon die noodsaaklikheid van die korrekte gebruik van byvoegmiddel dosis en volume om die maksimum spuitdeponering op die teiken te verkry.

Thesis (MScAgric)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Several attempts have been made to reduce Botrytis cinerea grey mould in vineyards and in storage by means of biological control. However, the so called "silver bullet" approach in utilising a single antagonist, has its limitations when compared with synthetic fungicides. Often the antagonist has a limited spectrum of activity and the duration of its effectiveness is less than that provided by synthetic fungicides. Furthermore, antagonists are more likely to be effective in preventing initial infection rather than resumption of latent infection. Therefore, due to the various infection sites in grape bunches utilised by B. cinerea and the fact that the pathogen can remain latent in the grapevine tissue, it may be possible to obtain effective control of the pathogen by integrating fungicides and different biological control agents each aimed at a different site in grape bunches, protecting the bunch at the various phenological stages of growth and under different micro climatic conditions. In this study the potential of three fungal antagonists (Glioc/adium roseum, Uloc/adium atrum and Trichoderma harzianum) and one yeast (Trichosporon pullulans) to colonise different sites in grape bunches, and to reduce B. cinerea infection, was investigated in commercial vineyards. As the biological control agents were used in an integrated system, the effect of various fungicides frequently applied to local vineyards on the organisms was also investigated. Fungicide trials were conducted taking into account two possible scenarios. Firstly, the possible effect of fungicides applied to the vineyard after an application of the biological control agent or shortly before the application of the biocontrol agent. This entailed exposing the biocontrol agents to relatively low concentrations of the active ingredient of the fungicides, similar to the residue levels to which these organisms would be exposed under field conditions. Secondly, the possibility of applying the organisms and the fungicides at the same time by making use of spray tank mixtures. This meant exposing the biocontrol agents to relatively high doses of the active ingredient of the various fungicides. Mycelial growth and germination tests were performed on agar in Petri dishes to determine the effect of fungicides. It was assumed that if the fungicide effectively inhibits the antagonist at 2.5 !-lg a.Uml, the fungicide and antagonist can not be used in an integrated programme. Based on this criterium, T harzianum can not be applied to vineyards with penconazole, mancozeb/metalaxyl, pyrifenox or mancozeb. In addition T harzianum can not be applied as tank mixtures with iprodione. However, T harzianum can be used in conjunction with pyrimethanil, folpan, iprodione, fosetyl-Al and copperhydroxide, provided the chemicals and the antagonist are applied alternately. Gliocladium roseum can not be applied in a tank mixture with pyrimethanil and penconazole, but can be used on grapevine in conjunction with penconazole, pyrifenox, pyrimethanil, iprodione and fosetyl-Al. Ulocladium atrum can not be applied with pyrimethanil and iprodione. Ulocladium atrum can be applied in conjunction with penconazole, pyrifenox, pyrimethanil, iprodione, fosetyl-Al and mancozeb. The fungus can be applied in a tank mixture with penconazole and pyrifenox. The antagonists were applied as conidial suspensions to bunches at various phenological stages in commercial vineyards planted with the wine grape cultivar Chardonnay in the Stellenbosch region, or the table grape cultivar Dauphine planted in Paarl region. Bunches were collected 2 wk after application, surface-sterilised and used for determining antagonist colonisation and B. cinerea infection at specific sites in the bunches. In Chardonnay, the antagonists colonised the different sites, but colonisation during the three seasons was inconsistent and sporadic. Ulocladium atrum and G. roseum colonised floral debris to a degree in the 1996 season. However, in the 1997 season these two antagonists did not develop from floral debris. Trichoderma harzianum colonised floral debris extensively in the 1996 season. In the 1997 season colonisation by T harzianum dropped, but unlike G. roseum and U atrum, T harzianum occurred at a low level in flowers. Ulocladium atrum only colonised bunches during bloom, and was not found in bunches monitored from pea-size stage to véraison. This finding suggests that the saprophyte colonised moribund and dead flower parts occurring in bunches during full bloom to the pre-pea size stage, and is not likely to be found in living tissue. Gliocladium roseum colonised grape berries and pedicels to some degree and T harzianum colonised these grape parts extensively. Botrytis cinerea occurred inconsistently and at low frequencies in the different sites in bunches. It was therefore not possible to comment on the effectivity of the various antagonists in the three seasons during which the trials were performed. However, it was noted that, during the peasize stage in 1996, when high levels of B. cinerea were recorded, T harzianum controlled these infections in the pedicels more effectively than any other treatment. / AFRIKAANSE OPSOMMING: ONDERDRUKKING VAN BOTRYTIS CINEREA DEUR ANTAGONISTE IN LEWENDE, AFSTERWENDE EN DOOIE WINGERDWEEFSEL Die benadering om Botrytis cinerea verrotting van wingerd met behulp van 'n enkele biologiese beheeragent in plaas van met sintetiese fungisiede te beheer, het sekere beperkinge. Antagoniste het dikwels 'n beperkte spektrum van aktiwiteit, en die duur van hul effektiwiteit is minder as dié van fungisiede. Antagoniste is gewoonlik ook minder effektief in die beheer van latente infeksie. Die patogeen het verder die opsie om druiwetrosse deur verskillende infeksieweë te koloniseer. Fungisiede kan druiwetrosse beter teen infeksie deur veelvuldige infeksieweë beskerm as 'n enkele antagonis. In die lig hiervan is die beheer van die patogeen deur 'n kombinasie van fungisiede en verskillende biologiese beheeragente, wat elk gemik is om 'n ander infeksiepunt in die druiwe te beskerm, ondersoek. Drie swamagtige antagoniste (Glioc/adium roseum, Uloc/adium atrum en Trichoderma harzianum) en een gis (Trichosporon pullulans) is in die ondersoek gebruik. Voorloper ondersoeke, waar twee moontlike scenarios in ag geneem is, is met fungisiede uitgevoer. In die eerste scenario is die effek van fungisiede, aangewend op wingerd kort vóór aanwending van die biologiese beheeragent, of kort ná aanwending, ondersoek. Hierdie proef het die blootstelling van die biologiese beheeragent aan relatief lae konsentrasies van die aktiewe bestanddeel van die fungisied, vergelykbaar met residuvlakke waaraan die organismes onder veldtoestande blootgestel sou word, behels. Tweedens is die moontlikheid om antagoniste en fungisiede gelyktydig as spuitpompmengsels toe te dien, ondersoek. In hierdie proef is die biologiese beheeragente aan relatief hoë dosisse van die aktiewe bestanddeel van verskillende fungisiede blootgestel. Miseliumgroei en ontkiemingstoetse is op agar in Petribakkies uitgevoer om die effek van die fungisiede te bepaal. As kriterium is aanvaar dat indien 'n fungisied die antagonis effektief by 2.5J..lglml aktiewe bestanddeel inhibeer, die fungisied en antagonis nie in 'n geïntegreerde program gebruik kan word nie. Gebaseer op hierdie kriterium kan T harnzianum nie aangewend word in 'n wingerd wat met penconazole, mancozeb/metalaxyl, pyrifenox of mancozeb behandel is nie. Ook kan T harzianum nie in 'n spuitpompmengsel met iprodione aangewend word nie. Trichoderma harzianum kan egter saam met pyrimethanil, folpan, iprodione en fosetyl-Al gebruik word, mits dié chemikalieë en die antagonis afwisselend aangewend word. Glioc/adium roseum kan nie in 'n spuitpompmengsel met pyrimethanil en penconazole aangewend word nie, maar kan saam met penconazole, pyrifenox, pyrimethanil, iprodione en fosetyl-Al gebruik word. Uloc/adium atrum kan nie saam met pyrimethanil, iprodione en fosetyl-Al gebruik word nie. Die swam kan wel in 'n spuitpompmengselmet penconazole en pyrifenox aangewend word. In verdere proewe is die antagoniste as spoorsuspensies op trosse op verskillende groeistadia in kommersiële wingerde, wat met die wyndruitkultivar Chardonnay of die tafeldruifkultivar Dauphine aangeplant is, ondersoek. Trossies is twee weke na toediening versamel, oppervlakkig gesteriliseer en gebruik om vlakke van antagoniskolonisasie en B. cinerea infeksie op spesifieke nisse in die trosse te bepaal. In die geval van Chardonnay het die antagoniste die verskillende nisse gekoloniseer, maar die kolonisasie was sporadies en nie konstant gedurende die drie seisoene van ondersoek nie. Uloc/adium atrum en G. roseum het blomdeeltjies tot 'n beperkte mate in die 1996 seisoen gekoloniseer, maar nie in die daaropvolgende seisoen nie. Daarteenoor het T. harzianum blomdeeltjies ekstensief in die 1996 seisoen gekoloniseer, en in 'n beperkte mate in die daaropvolgende seisoen. Uloc/adium atrum kon nie trosse van ertjiekorrelgrootte tot deurslaan vestig nie. Hierdie bevinding dui daarop dat die saprofiet afsterwende en dooie blomdeeltjies, wat van volblom tot ertjiekorrelstadium in die trosse voorkom, koloniseer, maar dat dit nie in lewende weefsel voorkom nie. Daarteenoor het T. harzianum die verskillende trosdele ekstensief gekoloniseer. Botrytis cinerea het gedurende die drie seisoene wisselvallig en teen lae frekwensies in die verskillende nisse in die trosse voorgekom. Dit was gevolglik nie moontlik om 'n konkrete afleiding oor die effektiwiteit van die verskillende antagoniste as biobeheeragente van B. cinerea te maak nie. In die geval van Dauphine was die onderskeie organismes swak koloniseerders van blomdeeltjies. Trichoderma harizanum kon egter die lewende trosdele koloniseer. Kolonisasievlakke was laag en was nooit meer as 50% nie. In beide seisoene het die kolonisasievermoë van T. harzianum drasties ná trostoemaak gedaal. Daarteenoor het beide G. roseum en U atrum tydens al die ontwikkelingstadia die lewende trosdele swak gekoloniseer. Botrytis cinerea het ook uiters sporadies en teen baie lae vlakke voorgekom. Die bevindinge het getoon dat klimaatsomstandighede wat in tafeldruifwingerde in die Wes-Kaap heers, nie geskik is vir die vestiging van die biologiese beheeragente wat in die studie ondersoek is nie.

Thesis (MScAgric)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: The evaluation of fungicide efficacy in commercial vineyards can be influenced by the sporadic occurrence of Botrytis cinerea at various positions on vines, differences in bunch structure during bunch development and the phenomenon that symptom expression in shoots and bunches is governed by the resistance reaction of the various shoot and bunch parts. It has been postulated that, following air and water dispersal, infection by solitary conidia should playa prominent role in the epidemiology of B. cinerea on grapevine. The aim of this study was to determine (i) infection and (ii) fungicide efficacy at specific sites on shoots of vinelets and bunches (table grape cultivar Dauphine and the wine grape cultivar Merlot) inoculated with dry, airborne conidia of B. cinerea. Vinelets, prepared from cuttings, and bunches obtained from the vineyards at full bloom, pea size, bunch closure, véraison and harvest stages, were sprayed in a spray chamber at the recommended dosages with iprodione, pyrimethanil, cyprodinil/fludioxonil and fenhexamid or were left unsprayed. After 24 h the vinelets or bunches were dusted with dry conidia of Botrytis cinerea in a settling tower and incubated for 24 h at a high relative humidity (±93%). Following incubation, both the vinelets or bunches were divided into three groups. Vinelets and bunches of the one group were surface-sterilised, the others were left unsterile. Vinelets and bunches of one unsterile group were placed in dry chambers, kept for 14 days at 22°C with a 12 h photoperiod daily and monitored for symptom expression and the development of B. cinerea. Vinelets and bunches of the sterile group, and from one unsterile group were used for isolation. From each of these vinelets leaf blades, leaf petioles, shoots and inflorescences were removed. Sites used for isolation in bunch parts were rachises, laterals and pedicels, and sites on berries were the pedicel-end, cheek and style-end. The different parts and segments were placed in Petri dishes on Kerssies' B. cinerea selective medium, or on water agar medium supplemented with paraquat and incubated for 14 days at 22°C with a 12 h photoperiod daily. Infection and fungicide efficacy was determined by observing intact vinelets and bunches for symptom expression, and by estimating the amount of B. cinerea at the various sites on the vinelets and bunches with isolation studies. No symptoms of B. cinerea decay developed on sprayed and unsprayed vinelets that were kept in dry chambers during the 2 wk observation period. The isolation and incubation studies showed that the different fungicides were highly and nearly equally efficient in reducing superficial B. cinerea inoculum and latent infection. .In the case of leaf blades, which showed a high amount of B. cinerea on unsprayed vinelets under the two sterility regimes, decay was significantly reduced by each fungicide on both cultivars. This was not the case for the other parts, which yielded B. cinerea at low incidences under the two sterility regimes. The study with bunches showed that dry, airborne conidia, and the fungicide sprays, penetrated loose and tight clustered bunches from bloom to harvest and evenly landed on the various bunch parts. At full bloom, the amount of B. cinerea in unsprayed bunches was high on the laterals and pedicels, but low on the embryos. Unsprayed intact bunches at full bloom were highly susceptible to B. cinerea and developed symptoms of grey mould. The fungicides inhibited symptom expression at full bloom, but could not prevent infection. Unsprayed bunches inoculated at the other stages remained asymptomatic. The amount of B. cinerea was generally high in the rachises and laterals at pea size and bunch closure stages, and in the pedicel end of berries at harvest. Infection was constantly low in the berry cheek. The fungicides had a differential effect on infection at the various sites. In the case of rachises, the amount of B. cinerea was at each growth stage drastically reduced by each fungicide. In laterals, it was effectively reduced at pea size and bunch closure. However, at these two sites, significant differences were found between the fungicides in efficacy at stages when the amount of B. cinerea was high. This study showed that if these fungicides are applied properly to vine in commercial vineyards between budding and prebloom, during flowering, and at bunch closure, they should effectively prevent infection and symptom expression and thus the development of B. cinerea epiphytotics. / AFRIKAANSE OPSOMMING: INFEKSIE DEUR DROË, LUGGEDRAAGDE BOTRYTIS CINEREA KONIDIA EN DIE EFFEK VAN FUNGISlEDE OP VERSKILLENDE SETELS BINNE WINGERDTROSSE EN OP LOTE: Evaluering van fungisieddoeltreffendheid in kommersiële wingerde word beïnvloed deur die sporadiese voorkoms van Botrytis cinerea op verskeie posisies van wingerddele, verskille in trosstruktuur tydens trosontwikkeling, en die feit dat simptoomuitdrukking in lote en trosse deur die weerstandsaksie van die verskillende morfologiese dele van lote en trosse beheer word. In die natuur speel infeksie deur enkel konidia 'n prominente rol in die epidemiologie van B. cinerea van wingerd. Die doel van hierdie studie was om (i) infeksie en (ii) die effek van fungisiede op verskillende posisies op lote en trosse (tafeldruif kultivar Dauphine, wyndruif kultivar Merlot), wat met droë, luggedraagde konidia van B. cinerea geïnokuleer is, te bepaal. Lote, verkry vanaf steggies, en trosse versamel vanuit die wingerde tydens blom-, ertjiekorrel-, trostoemaak-, deurslaan- en oesstadium, is teen aanbevole dosisse met iprodione, pyrimethanil, cyprodinillfludioxonil of fenhexamid in 'n spuitkas bespuit, of is onbehandeld gelaat. Na 24 h is die lote en trosse met droë konidia van B. cinerea in 'n inokulasietoring geïnokuleer en daarna vir 24 h onder hoë humiditeit [±93% RH] geïnkubeer. Na inkubasie is die lote en trosse in drie groepe verdeel. Die een groep lote en trosse is oppervlakkig gesteriliseer om die patogeen op die oppervlakte te elimineer, en die ander twee groepe is onbehandeld gelaat. Die lote en trosse van een nie-steriele groep is vir 14 dae in droë voghokke by 22°C met 'n 12 uur daaglikse fotoperiode geplaas, en daagliks vir siekteuitdrukking en die ontwikkeling van B. cinerea gemonitor. Lote en trosse van die ander twee groepe is vir isolasiestudies gebruik. Vanaf elke loot is blaarskywe, blaarstele, internodes en ongeopende blomtrossies verwyder. Vanaftrosse is ragisse, laterale en korreisteie verwyder, en vanaf korrels is skilsegmente aangrensend aan die korrelsteel, die stempel-end, en die wang verwyder. Die dele en segmente is op B. cinerea selektiewe medium, en op paraquat medium in Petri bakkies geplaas en vir 14 dae by 22°C met 'n 12 uur daaglikse fotoperiode geïnkubeer. Infeksie en die fungisiedeffek is bepaal deur die intakte lote en trosse vir siekte- uitdrukking te monitor, en deur die hoeveelheid B. cinerea op verskeie posisies op lote en trosse te bepaal. Geen simptome het op enige posisie op bespuite en onbespuite lote, wat in droë hokke gehou is, ontwikkel nie. Die isolasie- en inkubasiestudies het getoon dat die verskillende fungisiede hoogs effektief op lote was, en inokulumvlakke van die patogeen doeltreffend verlaag het. In die geval van blaarskywe, wat hoë vlakke van B. cinerea op onbespuite steggies onder die twee steriliteitskondisies getoon het, is verrotting op beide kultivars betekenisvol deur die fungisiedes verlaag. Dit het egter nie vir die ander dele, waarop daar 'n lae voorkoms van B. cinerea onder die twee steriliteitskondisies was, gegeld me. Die studie met trosse het getoon dat droë, luggedraagde konidia en fungisiednewels beide oop en kompakte trosse vanaf blomstadium tot oes penetreer en eweredig op die verskillende dele land. Met blomstadium was die hoeveelheid B. cinerea in onbespuite trosse hoog op laterale en korrelstele, maar laag op die embrios. Onbespuite, intakte trosse was hoogs vatbaar vir B. cinerea by blomstadium en het simptome van vaalvrot ontwikkel. Die fungisiede het siekte-uitdrukking by blomstadium voorkom, maar kon nie infeksie voorkom me. Onbespuite trosse wat op ander stadia geïnokuleer is, het geen siekte-uitdrukking getoon me. Die hoeveelheid B. cinerea was hoër in die ragi, asook in laterale by ertjiekorrel- en trostoemaak stadium, en hoër in korreisteie by oesstadium. Infeksie was konstant laag in die korrelskil. Die fungisiede het 'n differensiële effek op infeksie by die verskillende posisies gehad. In die geval van ragi was die hoeveelheid B. cinerea drasties deur elke fungisied by alle groeistadia verlaag. In laterale was dit effektief by ertjiekorrel- en trostoemaakstadium verminder. By hierdie twee posisies waar die hoeveelheid B. cinerea hoog was, is daar egter betekenisvolle verskille in die doeltreffendheid van fungisiedes gevind. Hierdie studie toon dat as fungisiede behoorlik in kommersiële wingerde tussen botvorming en blomstadium, en tydens blom- en trostoemaakstadium toegedien word, infeksie en siekte-uitdrukking, en dus ook die epifitotiese ontwikkeling van B. cinerea, voorkom behoort te word.

Thesis (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Grapevine leafroll-associated virus-3 is one of ten members of the C/osteroviridae that are known to infect grapevine. Nine of these viruses are associated with grapevine leafroll disease, of which GLRaV-1 and GLRaV-3 are the most important and widespread. Members of the C/osteroviridae are unique amongst the viruses, as it is the only known family whose members encode a heat shock protein 70 kOa homolog (Hsp70h). The Hsp70h is a movement protein (MP) that is required for the active translocation of the virion structure through the plasmodesmata into adjacent cells. Broad-spectrum resistance to unrelated viruses can be obtained by a pathogen-derived resistance (POR) strategy that is based on the expression of a dysfunctional MP in plants. The Hsp70h has two distinct domains. The N-terminal two thirds of the protein is an ATPase domain and shares high homology with the ATPase domains of all Hsp70h proteins from the C/osteroviridae and Hsp70 proteins from the prokaryote and eukaryote kingdoms. Conserved amino acids are found in the ATPase domain and are required for the positioning of the ATP at the catalytic site for ATP hydrolysis. The C-terminal domain is variable and the function of this domain in the Closteroviridae is not known. In prokaryote and eukaryote Hsp70 proteins, the C-terminal domain is required for protein-protein interactions. The American NY-1 isolate of GLRaV-3 has been sequenced and POR strategies have been attempted with the coat protein, divergent coat protein and replicase genes, but not with a dysfunctional form of the hsp70h gene. In this study, double-stranded RNA was isolated from a commercial vineyard with unknown virus status, but with distinct grapevine leafroll symptoms, and from two grapevine sources of known virus status, one with mild and one with severe symptoms. The GLRaV-3 hsp70h gene was amplified by RT-PCR from the dsRNA and the gene sequence was analysed. The hsp70h gene from the three virus sources contained more than 94% nucleotide sequence homology to the NY-1 isolate and the conserved amino acids required for ATPase activity were present. The hsp70h gene isolated from GLRaV-3 from a commercial Stellenbosch vineyard showing clear leafroll symptoms was selected for further work and was subjected to site-directed mutagenesis to engineer four point mutations in the gene. These four mutations resulted in the substitution of Asn for Asp", Gly for Thr1O, Lys for Glu 174 and Asn for Asp 197. The wild type (WT) and mutated (Mut) forms of the hsp 70h genes were cloned into a bacterial expression vector. Expression of both the WT- and Mut-Hsp proteins was achieved, and the protein was expressed in the insoluble inclusion bodies. All attempts to refold and isolate active proteins from the inclusion bodies were unsuccessful. Attempts to increase the concentration of soluble protein within the expressing bacteria were unsuccessful. Due to the lack of active protein, biochemical tests on the ATPase activity of the WT- and Mut-Hsp proteins could not be conducted. The wt- and mut-hsp genes were cloned into a plant expression vector for transformation into tobacco plants. These transformations were successful and gave rise to 22 Km' and 18 Km' plants from the WT- and Mut-Hsp constructs respectively. Two plant lines, M5 and M10, transformed with the mut-hsp transgene construct, appeared to have a high level of resistance to the challenging potato X potexvirus, whereas all the other tested plants were susceptible to the challenging virus. It was thus shown that a dysfunctional form of the GLRaV-3 Hsp70h could provide resistance to an unrelated virus in tobacco. / AFRIKAANSE OPSOMMING: Wingerdrolblaar-geassosieerde virus 3 (GLRaV-3) is een van 10 lede van die Closteroviridae wat wingerd kan infekteer. Nege van die virusse is met wingerdrolblaar geassosieer. Die GLRaV-1 en GLRaV-3 is die belangrikste en mees wyd verspreide lede van die rolblaar-geassosieerde Closteroviridae. Lede van die Closteroviridae is uniek in die opsig dat die virusse vir 'n 70 kDa-homoloë hitteresponsproteïen (Hsp70h) kodeer. Die Hsp70 is 'n bewegingsproteïen (MP) wat belangrik is vir die translokasie van die virus deur die plasmodesmata na die naasliggende sel. Breë-spektrum weerstand teen onverwante virusse kan behaal word deur 'n patogeen-afgeleide weerstandstrategie (POR), wat op die uitdrukking van 'n disfunksionele MP wat in plante uitgedruk word, gebaseer is. Die Hsp70hproteïen het twee gebiede. Die N-terminale gebied is In ATPase-gebied en toon hoë homologie met ander ATPase-gebiede van Hsp70h-proteïene van die Closteroviridae, asook die prokariotiese en eukariotiese koninkryke. Gekonserveerde aminosure wat belangrik is vir die posisionering van ATP in die katalitiese domein vir ATP-hidrolise is in die ATPase-gebied gevind. Die C-terminale gebied is variërend en die funksie van die gebied in die Closteroviridae is onbekend. In prokariotiese en eukariotiese Hsp70h-proteïene is die C-terminale gebied belangrik vir proteïenproteïen interaksies. Die nukleotiedvolgorde van die Amerikaanse NY-1-isolaat van GLRaV-3 is al bepaal en POR-strategieë is ook op die kapsiedproteïen, uiteenlopende kapsiedproteïen en die replikasie-proteïen uitgevoer, maar nog nie op 'n disfunksionele vorm van die Hsp70h-geen nie. In hierdie studie is dubbelstring-RNA (dsRNA) van 'n kommersiële wingerd met onbekende virusstatus wat rolblaarsimptome toon, geïsoleer, asook van twee wingerde met 'n bekende virusstatus, een met ligte en een met strawwe simptome. Die GLRaV-3 hsp70h-geen is met hulp van die polimerasekettingreaksie-metode (PKR) vanaf die dsRNA geamplifiseer en die geen se nukleotiedvolgorde is bepaal. Die hsp 70-gene van drie verskillende wingerde het meer as 94% homologie met die NY-1-isolaat getoon. Die gekonserveerde aminosure wat vir ATPase-aktiwiteit belangrik is, was teenwoordig. Die hsp70h-geen van GLRaV-3, wat uit 'n kommersiële wingerd met duidelike rolblaarsimptome in die Stellenbosch-gebied geïsoleer is, is vir verdere navorsing gekies en dit is aan setel-gerigte mutagenese blootgestelom vier mutasies van die geen te bewerkstellig. Die gevolg van hierdie vier mutasies was die verandering van Asn na Asp", Gly na Thr1o, Lys na Glu174 en Asn na Asp197. Die wilde (WT) en veranderde (Mut) vorms van die hsp-gene is in 'n bakteriese uitdrukkingsvektor gekloneer. Uitdrukking van beide die WT- en die Mut-Hspproteïene is behaal, maar die proteïene was in die onoplosbare fraksie geleë. Pogings om die onoplosbare proteïene te isoleer en in 'n aktiewe oplosbare vorm te verkry, was onsuksesvol. Verdere pogings om die proteïene in die oplosbare fraksie van die bakteriese ekspressiesisteem uit te druk, was ook onsuksesvol. As gevolg van die gebrek aan aktiewe proteïen kon biochemiese toetse nie op die ATPaseaktiwiteit van die WT- en Mut-Hsp proteïne gedoen word nie. Die wt- en mut-hsp-gene is ook in In plantekspressievektor gekloneer vir transformasie in tabakplante. Hierdie transformasies was suksesvol en het aanleiding gegee tot 22 kanamisienbestande (Km') en 18 Km' plante vanaf die WT- en Mut-Hspkonstrukte onderskeidelik. Twee plantlyne, M5 en M10, wat met die mut-hsptransgene getransformeer is, het 'n hoë vlak van weerstand teen die infekterende aartappelvirus X getoon in vergelyking met ander plante wat met die virus geïnfekteer is. Daar is dus bewys gelewer dat 'n disfunksionele vorm van die GLRaV-3 Hsp70h weerstand kan bied teen 'n onverwante virus in tabak.

Thesis (MScAgric)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Colonies of Planococcus ficus (Signoret) were reared from three different areas, Hex River Valley, Robertson and Stellenbosch. An insectary colony and a table grape colony from Nietvoorbij experimental farm were also included in the study. A range of concentrations of chlorpyrifos was applied topically to individuals from the different colonies. The Stellenbosch population had the lowest LDso, although it was not significantly different from that of the insectary and Robertson colonies. The Hex River Valley and table grape colonies had a significantly higher LDso than the Robertson, Stellenbosch and insectary colonies, although the relative tolerance was 1.5, which would probably not result in significant control failure in the field. However, this does indicate that there is potential for the development of resistance to chlorpyrifos in the vine mealybug in South Africa. / AFRIKAANSE OPSOMMING: Kolonies van Planococcus ficus (Signoret), is versamel en geteel uit drie verskillende areas, Hex.riviervallei, Robertson en Stellenbosch. 'n Bestaande insektarium kolonie van die Lanbou Navorsings Raad en 'n tafeldruif kolonie vanaf Nietvoorbij proefplaas is ook ingesluit in die studie. 'n Reeks konsentrasies van chlorpyrifos is topikaal aangewend aan individue van die verskillende kolonies. Die Stellenbosch populasie het die laagste LDso getoon alhoewel dit nie betekenisvol verskil het van die LDso van die insektarium - en Robertson kolonies nie. Die Hexriviervallei en tafeldruif kolonies se LDso was betekenisvol hoër as die Robertson, Stellenbosch and insektarium kolonies. Alhoewel die relatiewe weerstand 1.5 was, sal dit waarskynlik nie tot 'n aansienlike beheermislukking in die veld lei nie. Nogtans dui dit op die potensiaal vir moontlike ontwikkeling van weerstand teen chlorpyrifos in die wingerdwitluis.

No description available.