Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Seed germination is the most vulnerable time in a plant's life cycle, since the thick protective seed coat ruptures and the moist and humid soil environment not only favours seed germination, but also the growth and development of plant pathogens. Infection of plant seeds during germination, however, is the exception rather than the rule. Plant seeds have - - -developed a--cemplex preformed defense mechanism that includes anttfungal agents thatdiffuse into the surrounding environment to form a protective layer around the seed. This protective layer prevents fungal and bacterial pathogens from infecting the young seedling. Over the last decade, scientists have studied the defense mechanisms of different seeds in an effort to understand and ultimately to introduce and/or manipulate these mechanisms in plants as part of the plant's endogenous disease resistance to pathogens. Various chemical compounds, peptides and proteins that showed strong in vitro activities against various fungi were isolated in these efforts. The mere demonstration of in vitro activity alone, however, is not sufficient to assign a defense role to these antifungal agents. Typically, mutant plants that have lost the ability to produce the antifungal agent, or mutants that are overproducing the agent, have been used to correlate the mutant phenotype to either a decline or increase in disease resistance respectively. Genetic transformation and the subsequent development of transgenic plants have made an unprecedented impact in this regard, specifically in understanding the role of specific defense-related proteins and their interaction with plant pathogens. In this study, the antifungal peptide, Hs-AFP1, from Heuchera sanguinea, a plant defensin, was evaluated in a heterologous in planta environment as a defense protein with potential for engineering disease resistant crops. The in vitro assays performed with Hs-AFP1 against Botrytis cinerea showed antifungal activities of 88% growth inhibition at a concentration of 8 J,lg/ml of the purified peptide, while inducing a characteristic hyperbranching effect on the Botrytis hyphae. Tobacco was subsequently transformed with a construct, pFAJ3068, expressing Hs-AFP1 under the strong constitutive 35S promoter. The peptide was targeted to the apoplastic region with the signal peptide from Mj-AMP2, an antimicrobial peptide from Mirabilis jalapa. Due to reports of peptide instability in transgenic plant systems, two additional constructs were prepared and transformed into tobacco to anticipate possible Hs-AFP1 instability in the heterologous tobacco environment. A putative peptide stabilization construct, pHs-EXG1, consisted of a fusion between Hs-AFP1 and the antifungal exo-glucanase (encoded by EXG1) from Saccharomyces cerevisiae. A control construct, pMj-EXG1, expressing EXG1 targeted to the apoplastic region with the Mj-AMP2 signal peptide, was also prepared and transformed into tobacco to normalize the background antifungal activity as a result of the exoglucanase in the fusion construct lines. Tobacco was successfully transformed with pFAJ3068, pHs-EXG1 and pMj-EXG1, resulting in transgenic tobacco lines designated THs, THE and TME respectively. Transgene expression was confirmed for the THs and THE transgenic lines. The translation of these transcripts into proteins was also confirmed with Western blot analysis. Moreover, the heterologous production of Hs-AFP1 in tobacco led to an increase in disease resistance to B. cinerea in the THs lines in comparison with the untransformed tobacco controls. An increase of up to 42% in disease resistance was observed in an in planta detached leaf assay. Crude protein extracts from the THs lines were also analyzed in an in vitro quantitative fungal growth assay. This assay confirmed the results obtained with the disease resistance assay, with crude protein extracts exhibiting up to 40% fungal growth inhibition. The incubation of B. cinerea in the presence of crude protein extracts from THs lines resulted in hyperbranching of the fungal hyphae, which is characteristic of Hs-AFP1 activity. From these analyses it was clear that the heterologously expressed Hs-AFP1 was quite stable in the transgenic environment. The fusion between Hs-AFP1 and EXG1 did not increase the stability of Hs-AFP1, but rather led to a loss of the Hs-AFP1 activity. All the analyses performed showed the THE lines to be reduced in their ability to inhibit fungal infection in comparison to the THs line. Also, microscopic analysis of the effects of the crude THE extracts on B. cinerea growth showed no hyperbranching activity, again confirming the loss of peptide activity due to the fusion to EXG1. This is in agreement with previous work, in which sarcotoxin 1A was fused to a reporter gene and also lost activity. Although integration of the Mj-EXG1 expression cassette was confirmed, no mRNA levels could be detected with Northern blot or RT-PCR analysis of the TME lines. These lines also did not show any in vitro antifungal activities, probably indicating post-transcriptional gene silencing. This silencing was overcome in the fusion constructs that were expressed in the THE plant lines. These lines also showed EXG1 protein activity, as measured by ~-glucosidase assays. Although the THE lines did not serve the functions originally envisaged, they fortuitously showed that a fusion strategy might stabilize glucanase expression in a transgenic environment. A variety of glucanases have been shown to be prone to gene silencing when overexpressed in a plant environment and the yeast glucanase can now be added to that list if it is not present as a fusion protein. Overall, this study confirmed that Hs-AFP1 is involved in plant defense systems and provided valuable information on the stability of small peptides in a heterologous environment. The positive results obtained with overexpressed Hs-AFP1 on fungal inhibition in this study merits further investigations into the use of this peptide in the engineering of disease-resistant crops. / AFRIKAANSE OPSOMMING: Saadontkieming is die mees vatbare tyd vir siekteontwikkeling gedurende 'n plant se lewenssiklus. Die saadhuid bars en die vogtige grondkondisies bevoordeel nie net saadontkieming nie, maar ook die groei en ontwikkeling van plantpatogene. Infeksie van plantsade tydens ontkieming is egter die uitsondering eerder as die reël. Plantsade besit komplekse -veraeaigingsfueganfsmes-reen moontlike - patoqeeninteksies. Die meqanismes sluit die produksie van antifungiese agense, wat tydens saadontkieming na die omliggende omgewing diffundeer om 'n beskermende sone om die ontkiemende saad te vorm, in. Die gevolglike antifungiese sone beskerm die saad teen infeksie deur bakterieë en swamme. Gedurende die laaste dekade het navorsers baie aandag aan die bestudering van plantsaadverdedigingsmeganismes gegee. Dié kennis word gebruik om die verdedigingsmeganismes beter te verstaan, asook om dié meganismes te manipuleer en/of oor te dra aan plantspesies met inherente swak weerstandsmeganismes wat gereeld aan plantpatogeeninfeksies onderhewig is. Navorsing op plantsade het tot die isolasie van verskeie chemiese agense, peptiede en proteïene, wat sterk in vitro aktiwiteite teen 'n wye reeks swampatogene vertoon, gelei. Die vermoë van dié agense om swamme in 'n in vitro omgewing te inhibeer, is alleen egter nie 'n bewys dat hulle 'n rol in plantverdeging speel nie. Studies waar mutante gebruik word, is gewens om addisionele bewys te lewer dat die substanse 'n rol in plantverdediging vervul. Sodanige mutante sluit plantlyne, waarin die geen van belang gemuteer is of ooruitgedruk word om so die rol van die geen in 'n in planta omgewing te bepaal in. In hierdie toepassings het genetiese transformasie en die daarstelling van transgeniese plante 'n ongeëwenaarde bydrae gelewer. In dié studie is die antifungiese peptied, Hs-AFP1, wat aan die peptiedgroep van plant- "defensins" behoort en van Heuchera sanguine a afkomstig is, in 'n heteroloë in planta omgewing geëvalueer as 'n verdedigingspeptied met die potensiaal om in die generering van transgeniese siektebestande gewasse gebruik te word. Die antifungiese aktiwiteit van Hs-AFP1 is teen Botrytis cinerea in 'n in vitro reaksie geëvalueer, waar die toediening van 8 ,",g/mlgesuiwerde Hs-AFP1 peptied aanleiding gegee het tot 'n 88% afname in hifegroei van B. cinerea. Hipervertakkings van swamhifes, 'n kenmerkende eienskap van Hs-AFP1 aktiwiteit, kon duidelik waargeneem word. Tabakplante is voorts getransformeer met 'n konstruk, pFAJ3068, wat die koderende geen van Hs-AFP1 onder die sterk konstitutiewe CaMV 35S promotor bevat het. Die peptied is met behulp van die seinpeptied wat afkomstig is van die Mirabilis jalapa antimikrobiese peptied, Mj-AMP2, na die apoplastiese omgewing geteiken. Voorheen is gerapporteer dat transgeniese peptiede in die heteroloë omgewing soms onstabiel is. Dit het gelei tot die generering van twee addisionele konstrukte om die moontlikheid van peptiedonstabiliteit te ondervang. 'n Stabiliseringskonstruk, pHs-EXG1, bestaande uit In versmelting tussen Hs-AFP1 en In antifungiese eksoglukanase van Saccharomyces cerevisiae, gekodeer deur EXG1, is in tabakplante getransformeer. In Kontrolekonstruk, pMj-EXG1, met die EXG1-geen saam met die Mj-AMP2-seinpeptied, is ook voorberei en in tabakplante getransformeer. Dit is gebruik om die antifungiese aktiwiteit van die eksoglukanase in die antifungiese aktiwiteitstoetse van die stabiliseringskonstruk te kwantifiseer en te normaliseer. Tabak is suksesvol met pFAJ3068, pHs-EXG1 en pMj-EXG1 getransformeer, wat onderskeidelik gelei het tot die sogenaamde THs, THE en TME transgeniese tabaklyne. Transgeentranskripsie en -translasie in die THs en THE tabaklyne is onderskeidelik deur Noordelike- en Westelike-kladanalises bevestig. Die aktiewe uitdrukking van Hs-AFP1 het die vermoë van tabakplante om B. cinerea infeksies te weerstaan, met tot 42% verhoog in vergelyking met ongetransformeerde kontrole tabakplante tydens 'n in planta siekteweerstandstoets. Totale proteïenekstrakte van THs tabaklyne is voorts ook in In in vitro inhibisietoets geëvalueer, wat gelei het tot resultate wat goed met dié van die in planta toetse ooreenstem. Die totale proteïenekstrakte het swamgroei met 40% geïnhibeer en die kenmerkende hipervertakking van Hs-AFP1-aktiwiteit is ook mikroskopies waargeneem. Resultate wat verkry is vanaf al die analises wat op die transgeniese THs tabaklyne uitgevoer is, het aangedui dat Hs-AFP1 baie stabiel in die heteroloë tabakomgewing is en peptiedstabiliteit was dus nie In probleem, soos verwag is nie. Die fusie tussen Hs-AFP1 en EXG1 het dus nie die stabiliteit van die reeds stabiele Hs-AFP1 peptied verder verbeter nie, maar het wel tot die verlies van Hs-AFP1 aktiwiteit gelei. Die antifungiese analises van die THE tabaklyne het verder bevestig dat dié lyne selfs swakker inhibisie van B. cinereainfeksies tot gevolg gehad het, as ongetransformeerde tabakplante. Mikroskopiese analises van totale THE proteïenekstrakte het voorts ook geen kenmerkende hipervertakkings in die swamhifes vertoon nie, wat alles daarop dui dat die Hs-AFP1-deel van die fusieproteïen as gevolg van die fusie met EXG1 geïnaktiveer is. Dié resultaat is in lyn met vorige navorsing, wat getoon het dat In ander peptied, sarcotoxin 1A, sy antifungiese aktiwiteit verloor indien dit met In verklikkergeen versmelt word. Alhoewel integrasie van die pMj-EXG1-konstruk in die TME-tabaklyne bevestig is, kon geen mRNA met Noordelike-klad- of trutranskriptase-PKR (RT-PKR)-analises waargeneem word nie. Die TME plant het ook geen antifungiese aktiwiteit in in vitro toetse getoon nie en dit het geblyk dat die pMj-EXG1-konstruk aan geenafskakeling in die heteroloë tabakomgewing onderworpe was. Dié afskakelingseffek is egter in die THE plante oorkom, aangesien laasgenoemde sterk EXG1 proteïenaktiwiteit met J3-glukosidase aktiwiteitstoetse vertoon het. Alhoewel die THE plante nie die stabiliteit van Hs-AFP1 verbeter het nie, het dit onwerwags tot die stabilisering van EXG1 in In heteroloë omgewing gelei. Versmeltingstegnologie kan dus moontlik gebruik word as 'n strategie om ander glukanases, wat bekend is vir geenafskakeling in transgeniese omgewings, heteroloog uit te druk. In die geheel gesien, het dié studie getoon dat Hs-AFP1 'n onbetwiste rol in plantverdedigingsmeganismes speel en daar is voorts ook meer kennis oor die stabiliteit van peptiede in 'n heteraloë plantomgewing ingewin. Die positiewe resultate t.o.v. die verhoogde siekteweerstand in die transgeniese THs plantlyne regverdig ook die verdere bestudering van dié peptied om transgeniese siekteweerstand in gewasse te bewerkstellig.

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)--Stellenbosch University, 1998. / ENGLISH ABSTRACT: Eyespot is an important disease of spring wheat (Triticum aestivum L.). Four species of Ramulispora are associated with this disease, of which Tapesia yallundae and T. acuformis. are common. This thesis investigates the broader subjects of genetic variability, reproductive dynamics and fungicide resistance in Tapesia yallundae. Each of the chapters treats specific but related topics. T. yallundae, which is the only species thus far reported from South Africa, has been associated with yield losses of up to 50%. To enable the implementation of more accurate and effective control measures, understanding the dynamics of reproduction and the genetics of the pathogen is of utmost importance. Of the many plant disease control measures such as cultural practices, sanitation, biological control, etc., fungicide application is the most commonly resorted to measure in eyespot control. This thesis investigates the broader subjects of genetic variability, reproductive dynamics and fungicide resistance of Tapesia yallzll7dae. Fungicide application, however, is not without problems. The pathogen can build up resistance to fungicides. The most commonly used fungicides in eyespot control include the benzimidazole carbendazim, triazoles such as flusilazole, tebuconazole, propiconazole, bromuconazole, flutriafol, fenbuconazole, triademinol, and the imidazole, prochloraz. Cases of resistance to the groups listed above have been reported. Frequent monitoring for resistance is thus crucial to prevent wastage of fungicide and unnecessary impregnantation of the environment with potentially ineffective chemicals. In chapter 2 of this thesis 300 isolates of T. yallundae from 15 fields were evaluated for resistance against carbendazim, flusilazole, tebuconazole, propiconazole, bromuconazole, flutriafol and fenbuconazole. These results indicated that to some triazoles, such as fenbuconazole, a high level of resistance was already present in field populations. In a sexually reproducing fungus such as T. yallundae, knowledge pertaining to its ability to pass resistance factors to offspring is equally important. Mating studies were, therefore, also conducted with parental strains that showed signs of triazole resistance. Three generations were subsequently tested for resistance to five triazoles, namely flusilazole, tebuconazole, propiconazole, bromuconazole and flutriafol. Results of this study showed variable sensitivity in progeny, which indicated quantitative inheritance of resistance to triazoles. Although the sexual stage has not yet been observed in the field in South Africa, this knowledge lays the foundation for the long-term understanding of the population dynamics of the fungus. The ability of a heterothallic ascomycete population to reproduce sexually is dependent on the availability of its two mating types, MATI-I and MATI-2, their distribution, and female fertility amongst other factors. In the UK. the teleomorph is commonly observed in the field, which is in contrast to the situation in South Africa, where it has only been induced in the laboratory. A comparative study between the South African and the UK. populations was therefore undertaken. Isolates representative of the two populations were mated with tester strains as both sperm recipients and as sperm donors. This allowed the percentage of hermaphrodites to be determined. No difference in terms of female fertility was observed between the South African and the UK. populations, with both populations showing low effective population numbers. These data suggested, therefore, that the teleomorph would also occur more frequently in South Africa if the climate was more indusive to its development. The overall results of this study indicated that eyes pot could still be controlled by means of fungicide application in South Africa. Although a shift in sensitivity was observed towards fenbuconazole and flusilazole, no resistance was detected towards carbendazim. The latter might be due to the absen<.:eof the sexual stage in the field, coupled by the monocyclic nature of the pathogen and sensible fungicide regimes. The absence of T. acujormis makes the disease situation less complicated in terms of fungicide application and management. Continuous surveys will have to be conducted, however, to monitor this situation in future. / AFRIKAANSE OPSOMMING: Hierdie studie ondersoek die genetiese variasie, reproduksie dinamika en fungisied weerstand in Tapesia yallundae. Elke hoofstuk handel oor spesifieke maar verwante onderwerpe. Oogvlek is 'n belangrike siekte van lentekoring (Triticum aestivum L.). Vier spesies van Ramulispora word geassosieer met die siekte, waarvan Tapesia yallundae en T. acuformis mees algemeen voorkom. T. yallundae, wat tans die enigste spesie is wat in Suid-Afrika aangeteken is, het al verliese van tot 50% veroorsaak. Om meer akkurate en effektiewe beheermaatreels te implementeer, is dit noodsaaklik om die oorlewingsdinamika van die patogeen te verstaan. Van al die siektebeheermaatreels soos kulturele praktyke, sanitasie, biologiese beheer ens., bly fungisiedbehandeling die mees algemene maatreel vir die beheer van oogvlek. Fungisiedtoediening het egter ook verskeie probleme. Die patogeen kan weerstand opbou teen die fungisied. Die mees algemene fungisiedes wat vir oogvlekbeheer aangewend word sluit onder meer die benzimidasool karbendazim in, triasole soos flusilasool, tebukonasool, propikonasool, bromukonasool, flutriafol, fenbukonasool, triadimenol, en die imidasool, prochloraz. Weerstand is egter reeds teen hierdie middels bekend. Gedurige monitering vir weerstand is dus krities om die vermorsing van fungisied en besoedeling van die omgewing met oneffektiewe middels te beperk. In hoofstuk 2 van hierdie manuskrip word 300 isolate van T. yallundae van 15 lande geevalueer vir weerstand teenoor karbendazim, flusilasool, tebukonasool, propikonasool, bromukonasool, flutriafol en fenbukonasool. Resultate dui daarop dat teen sommige van hierdie triasole, soos bv. fenbukonasool, daar reeds 'n hoe vlak van weerstand teenwoordig was in veldpopulasies. In 'n seksueel reproduserende fungus soos T. yalluJ1dae, is dit noodsaaklik om te bepaal wat sy vermoe is om weerstandbiedenheid aan die nageslag oor te dra. Om die rede is paringstudies ook op ouers wat tekens van weerstand teenoor triasole getoon het uitgevoer. Drie generasies was gevolglik getoets vir weerstand teenoor vyf triasole, naamlik flusilasool, tebuconasool, propikonasool, brumukonasool en flutriafol. Resultate van die studie het 'n variasie in sensitiwiteit van die nageslag getoon, wat op 'n kwantitatiewe oorerwing van weerstand teen £riasole dui. Alhoewel die teleomorf nog nie in lande in Suid-Afrika opgemerk is nie, Ie hierdie kennis die fondament vir die langtermyn vertolking van die populasie dinamika van hierdie fungus. Die vermoe van 'n heterotalliese askomiseet populasie om seksueel voort te plant is afhanklik van die beskikbaarheid van sy twee paringstipes, MATI-I en MATl-2, hul verpreiding, vroulike vrugbaarheid en ander faktore. Alhoewel die teleomorf algemeen in lande in die Verenigde Koninkryk opgemerk word, is dit in kontras met die situasie in Suid-Afrika, waar hierdie stadium nog slegs in die laboratorium gelnduseer kon word. 'n Studie is dus onderneem om die Suid-Afrikaanse en V.K. populasies met mekaar te vergelyk. Isolate van die twee populasies is dus gepaar met paringsisolate as beide sperm ontvangers en sperm donors. Hierdie prosedure het dit moontlik gemaak om die persentasie hermafrodiete te bepaal. Geen verskille in vroulike fertiliteit is tussen die Suid-Afrikaanse en V.K. populasies bespeur nie, en beide populasies het ook 'n lae effektiewe populasie getal getoon. Hierdie data het dus voorgestel dat die teleomorf ook meer algemeen in Suid-Afrika sou voorkom as die klimaat meer geskik was vir teleomorf vormmg. Die resultate van hierdie studie het tot die slotsom gelei dat oogvlek steeds deur fungisiedbehandeling in Suid-Afrika beheer kan word. Alhoewel daar 'n merkbare verskuiwing in sensitiwiteit teenoor fenbukonasool en flusilasool was, was geen weerstand teenoor karbendazim waargeneem nie. Laasgenoemde kan dalk toegeskryf word aan die afwesigheid van die teleomorf in die veld, gekombineer met die monosikliese natuur van die patogeen en gebruik van alternerende fungisiedes. Die afwesigheid van T. acuformis maak die plaaslike siektetoestand minder gekompliseerd in terme van fungisied aanwending en bestuur. Voortdurende opnames sal egter uitgevoer moet word om hierdie situasie ook in die toekoms te monitor.