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)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Historically root diseases have been a production-limiting problem for the strawberry industry worldwide. In the Western Cape Province of South Africa the most serious root disease is black root rot, which causes losses of up to 30%, annually. The aims of this study were to investigate aspects of the etiology and epidemiology of this disease in the Western Cape, and to provide information that can be incorporated in an integrated disease management strategy. In Chapter I a summary of published information on this disease is presented. Disease symptoms include severe stunting of plants, which have black, rotted, reduced root systems. Even though this disease is of great economic importance, the etiology remains unresolved. However, soilborne fungal root pathogens, particularly Pythium and Rhizoctonia spp. have been implicated as major role players. Control of this disease, as well as the other root diseases affecting strawberries, has relied on soil fumigation with broad spectrum chemical fumigants, in particular methyl bromide. However, due to the ozone depleting action of methyl bromide it was decided at the signing of the Montreal Protocol to remove this chemical from the market. This action has caused great demand for alternative measures to control root diseases on many crops including strawberries. Development of integrated disease management strategies is dependent upon a more complete understanding of the etiology, biology and ecology of the disease complex. In Chapter 2 fungal pathogens associated with diseased plants were isolated and Koch's postulates were carried out. The most frequently isolated fungal pathogens were Pythium irregulare, Rhizoctonia spp. and Cylindrocarpon destructans. Two morphotypes of Rhizoctonia were isolated viz. a brown and a white type. Pythium irregulare was isolated more frequently in July than in September, and was not isolated at all in November. Rhizoctonia spp. were present at all sampling dates but were more frequently isolated in November than at the other times. All the fungi that were tested were pathogenic and caused root lesions. Cylindrocarpon destructans and Coniella fragariae did not have a stunting effect on the plants. These results confirm a major role for Pythium and Rhizoctonia in the black root rot complex and suggest that there is a complimentary seasonal variation in occurrence between these two pathogens. The Rhizoctonia species and anastomosis groups of isolates obtained from diseased strawberries in the Western Cape Province were determined, and their pathogenicity and relative virulence assessed. Both binucleate and multinucleate types were recovered from diseased roots and identified as R. fragariae and R. so/ani, respectively. All isolates of R. solani were members of anastomosis group (AG) 6, whereas three AG types were identified among isolates of R. fragariae, viz. AG-A, AG-G and AG-I at a relative occurrence of 69%, 25%, 6% respectively. All Rhizoctonia isolates were pathogenic to strawberry, but R. solani (AG 6) was the most virulent causing severe stunting of plants. This is the first species confirmation and AG type identification of Rhizoctonia taxa causing root rot of strawberries in South Africa. An assessment of the presence and quantity of black root rot pathogens associated with soils prior to fumigation and post fumigation with methyl bromide, was made in Chapter 4. Isolations were also made from nursery plants to determine whether any black root rot pathogens were in the plants before transplanting. Results demonstrated that after fumigation the soil was free of all pathogenic fungi associated with the disease. However, the main pathogens involved in black root rot, viz. Rhizoctonia fragariae, R. solani and Pythium spp. were isolated from nursery plants. The fact that the plants are already infected with these pathogens renders the prospects for control of this disease difficult. Further studies are urgently required to develop production practices that can be included in disease management programmes. In vitro studies were carried out to determine the ECso values of different fungicides for isolates of Pythium irregulare, Rhizoctonia fragariae AG-A, AG-G and AG-I and R. solani AG 6. Benomyl, fludioxonil and tolc1ofos-methyl were used in these tests. Field trials were also conducted using these fungicides. In Chapter 5 it is shown that in general application of fungicides improved the yield and did not affect the survival rate of strawberry plants. Fludioxonil showed potential for short-term use. Applications of fungicides that targeted specific fungal genera were not sufficient to control the disease. Seasonal fluctuation of Pythium and Rhizoctonia spp. became apparent with the occurrence of Pythium being relatively high early in the season but low late in the season. Conversely, the occurrence of Rhizoctonia was low at the beginning of the season but high late in the season. In the short-term there is potential for fungicide applications as part of an integrated disease management strategy, but the economic feasibility of this practice needs to be assessed. In this study the major pathogens causing black root rot were identified in the Western Cape Province of South Africa, and important information regarding the epidemiology of the disease was reported. These results can be incorporated in an integrated management strategy to reduce losses of strawberry production, attJibutable to black root rot. / AFRIKAANSE OPSOMMING: Wortelsiektes is wêreldwyd 'n produksie-beperkende probleem vir die aarbeibedryf. . Swartwortelvrot, wat jaarliks verliese van tot 30% veroorsaak, is die belangrikste wortelsiekte in die Wes-Kaap Provinsie van Suid-Afrika. Die doelwitte van hierdie studie was om aspekte van die etiologie en epidemiologie van die siekte in die Wes- Kaap te ondersoek en inligting wat in geïntegreerde siektebestuurstrategië ingesluit kan word, te voorsien. In Hoofstuk 1 word 'n opsomming van gepubliseerde inligting aangaande die siekte uiteengesit. Siektesimptome sluit ernstige verdwerging van plante met swart verotte en verkleinde wortelstelsels in. Alhoewel die siekte van groot ekonomiese belang is, is die etiologie grootliks onbekend. Grondgedraagde wortelpatogene swamme, spesifiek Pythium en Rhizoctonia spp., is egter as belangrike rolspelers geïdentifiseer. Tot dusver het die beheer van hierdie siekte sowel as ander wortelsiektes van aarbeie berus op grondberoking met breë spektrum chemiese berokingsmiddels, spesifiek metielbromied. As gevolg van die osoonafbrekende aksie van metielbromied is daar egter tydens die ondertekening van die Montreal Protocol besluit om dié middel van die mark te verwyder. Hierdie besluit het 'n groot aanvraag na alternatiewe beheermaatreëls vir wortelsiektes van verskeie gewasse, insluitende aarbeie, veroorsaak. Die ontwikkeling van geïntegreerd siektebestuurstrategieë is egter afhanklik van 'n meer volledige begrip van die etiologie, biologie en ekologie van die siektekompleks. In Hoofstuk 2 is die patogene swamme wat met die siekte geassosieer word, geïsoleer, en is Koch se postulate uitgevoer. Die mees algemeen geïsoleerde patogene swamme was Pythium irregulare, Rhizoctonia spp. en Cylindrocarpon destructans. Twee morfotipes van Rhizoctonia is geïsoleer, nl. 'n bruin tipe en 'n wit tipe. Pythium irregulare is meer dikwels in Julie as in September geïsoleer, maar glad nie in November nie. Rhizoetonia het tydens alle monstertye voorgekom, maar is meer dikwels in November geïsoleer. Al die swamme wat getoets is, was patogenies en het letsels op die wortels veroorsaak. Cylindroearpon des true tans en Coniella fragariae het nie'n verdwergingseffek op plante gehad nie. Hierdie resultate bevestig die dominante rol van Pythium en Rhizoctonia in die swartwortelvrot kompleks en dui op 'n komplementêre seisoenale variasie in die voorkoms van hierdie twee patogene. Die Rhizoctonia spesies en anastomose groepe (AG) van die isolate geisoleer vanaf siek aarbeiplante in die Wes-Kaap Provinsie is bepaal, en die patogenisiteit en relatiewe virulensie is beraam. Sowel tweekernige as multikernige tipes is vanaf siek wortels geïsoleer en respektiewelik as R. fragariae en R. so/ani geïdentifiseer. Alle isolate van R. so/ani was lede van anastomose groep 6, terwyl drie AG tipes, nl. AGA, AG-G en AG-I onder die R. fragariae isolate geïdentifiseer is met relatiewe voorkomste van 69%, 25%, 6% respektiewelik. Alle Rhizoctonia isolate was patogenies op aarbeie, maar R. so/ani (AG 6) was die mees virulente en het ernstige verdwerging van plante veroorsaak. Hierdie is die eerste bevestiging van spesies en identifisering van AG tipes van Rhizoctonia taksa wat wortelvrot van aarbeie in Suid Afrika veroorsaak. In Hoofstuk 4 is 'n beraming van die voorkoms en hoeveelheid swartwortelvrot patogene geassosieer met grond voor, en na beroking met metielbromied, gemaak. Isolasies is ook vanaf kwekeryplante gemaak om te bepaal of enige swartwortelvrot patogene voor oorplanting in die plante teenwoordig was. Die resultate het getoon dat grond na beroking vry was van alle patogeniese swamme geassosieër met die siekte. Die hoof patogene betrokke in die swartwortelvrot kompleks, nl. Rhizoctonia fragariae, R. so/ani en Pythium spp. was egter in die kwekery plante teenwoordig. Die feit dat plante reeds met hierdie patogene geïnfekteer is, maak die vooruitsigte vir die beheer van hierdie siekte moeilik. Verdere studies word dringend benodig vir die ontwikkeling van produksiepraktyke wat by siektebestuursprogramme ingesluit kan word. In vitro studies om die ECso waardes van die isolate van Pythium irregulare, Rhizoctonia fragariae AG-A, AG-G en AG-I en R. so/ani AG 6 vir die fungisiedes benomyl, fludioxonil en tolclofos-metiel te bepaal, is uitgevoer. Hierdie fungisiedes is ook in veldproewe getoets. In Hoofstuk 5 is getoon dat aanwending van fungisiedes die opbrengs verbeter het en nie die oorlewing van aarbeiplante beïnvloed het nie. Fludioxonil het potensiaal vir korttermyn gebruik getoon. Die aanwending van fungisiedes wat spesifieke swamgenera teiken, was nie voldoende om die siekte te beheer nie. Seisoenale fluktuasies van Pythium en Rhizoctonia spp. het duidelik geword met die relatief hoë voorkoms van Pythium vroeg in die seisoen, maar lae voorkoms laat in die seisoen, terwyl die voorkoms van Rhizoctonia laag was aan die begin van die seisoen, maar hoog later in die seisoen. In die korttermyn is daar potensiaal vir fungisiedtoedienings as deel van 'n geïntegreerde siektebestuurstrategie, maar die ekonomiese haalbaarheid van hierdie praktyk moet bepaal word. In hierdie studie is die hoof patogene wat swartwortelvrot van aarbeie in die Wes-Kaap Provinsie van Suid-Afrika veroorsaak geïdentifiseer, en belangrike inligting rakende die epidemiologie van die siekte is aangeteken. Hierdie resultate kan in 'n geïntegreerde bestuurstrategie geïnkorporeer word om verliese van aarbeiproduksie, toeskryfbaar aan swartwortelvrot te, verminder.

Thesis (MScAgric)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The resistance of plants to infection by phytopathogenic microorganisms is the result of multiple defence reactions comprising both constitutive and inducible barriers. While disease is the exception, such exceptions can be costly and even devastating. In particular, fungal diseases remain one of the major factors limiting crop productivity worldwide, with huge losses that need to be weighed up against massive cash inputs for pesticide treatments. Part of the defence reactions of plants is the synthesis of pathogenesis-related proteins, such as the plant hydrolases, glucanases and chitinases. In recent years, attention has been paid to the implementation of these proteins in plant transformation schemes. The rationale for this approach was that these antimicrobial agents not only degrade the main cell wall components of fungi, but also produce glucosidic fragments that act as elicitors of the biosynthesis of defence metabolites by the host. Furthermore, since these active antimicrobial agents are individually encoded by single genes, these defence systems should and have been shown to be highly amenable to manipulation by gene transfer. In this study, yeast glucanases from Saccharomyces cerevisiae were evaluated for their potential as antifungal proteins. The glucanases tested for their antifungal activity against Botrytis cinerea were the yeast EXG1 and BGL2 genes, encoding an exoglucanase and an endoglucanase respectively. An in vitro assay performed on these glucanases indicated that exoglucanase had a more detrimental effect on B. cinerea hyphal development and growth than the endoglucanase; the former caused typical disruption of the cells and leakage of cell material. The yeast exoglucanase was subsequently subcloned into a plant expression cassette containing the strong constitutive 358 promoter, yielding plasm ids pEXG1 and pMJ-EXG1. The pMJ-EXG1 construct targeted the exoglucanase to the apoplastic region with a signal peptide from an antimicrobial peptide from Mirabilis jalapa, Mj-AMP2. The pEXG1 and pMJ-EXG1 constructs were mobilised into Agrobacterium tumefaciens to facilitate the subsequent tobacco transformation, which yielded transgenic tobacco lines designated E and MJE respectively. Transgene integration was confirmed with southern blot and PCR analyses for both the E and MJE lines. The expression and heterologous production of the EXG1-encoded exoglucanase in the E-transgenic lines was shown with northern blots and activity assays respectively. Moreover, the high level of expression of the yeast exoglucanase led to a decrease in susceptibility of the E lines to B. cinerea infection in comparison to the untransformed tobacco controls. An average decrease in disease susceptibility of 40% was observed in an in planta detached leaf assay. Crude protein extracts from the E lines were also analysed in an in vitro quantitive fungal growth assay, inhibiting in vitro fungal growth by average 20%, thus further confirming the antifungal nature of the yeast exoglucanase. 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 MJE lines. These lines also did not show any in vitro antifungal activities or a decrease in susceptibility to B. cinerea infection in the detached leaf assay. It is suspected that this result is possibly linked to gene silencing, a phenomenon quite frequently associated with heterologous and/or overexpression of glucanases in plant hosts. It appears as if the targeted overexpression to the apoplastic space triggered the gene silencing response, since the intracellularly overexpressed product was produced and shown to display activity. The yeast exoglucanase thus joins the list of silenced glucanases in overexpression studies in plants. Overall, this study confirmed the antifungal characteristics of the Saccharomyces exoglucanase and provides valuable information of the possibility of utilising yeast glucanases in a transgenic environment. A decrease in the susceptibility of tobacco to B. cinerea infection, as shown by the overexpressed EXG1-encoded exoglucanases, merits further investigation into the use of this gene in the engineering of disease-resistant crops. / AFRIKAANSE OPSOMMING: Die weerstand van plante teen infeksie deur fitopatogeniese mikroórganismes is die resultaat van verskeie meervoudige verdedigingsreaksies wat beide konstitutiewe en induseerbare versperrings behels. Terwyl siekte die uitsondering eerder as die reël is, kan sulke uitsonderinge duur en selfs verwoestend wees. In die besonder is swamsiektes een van die vernaamste faktore wat gewasproduksie wêreldwyd beperk, met enorme verliese wat teen kontantinsette vir plaagdoders opgeweeg moet word. Deel van die verdedigingsreaksie van plante is die sintese van patogeen-verwante proteïene, soos die planthidrolases, -glukanases en -chitinases. In die onlangse tyd is aandag geskenk aan die implementering van hierdie proteïene in plant transformasieskemas. Die grondrede hiervoor was dat hierdie antimikrobiese agente nie net die hoof selwandkomponente van swamme kan afbreek nie, maar ook glukosidiese fragmente produseer wat as ontlokkers van metabolietbiosintese vir die verdediging van die gasheer kan optree. Aangesien hierdie aktiewe antimikrobiese agente individueel deur enkele gene enkodeer word, blyk hierdie verdedigingsisteme om hoogs ontvanklik vir manipulasie deur geenoordrag te wees. In hierdie studie is die gisglukanase van Saccharomyces cerevisiae vir hul potensiaal as antifungiese proteïene geëvalueer. Die glukanases wat vir hul antifungiese aktiwiteit teen Botrytis cinerea getoets is, was die gis EXG1- en -BGL2-gene, wat onderskeidelik vir "n eksoglukanase en 'n endoglukanase enkodeer. "n In vitro toets wat op hierdie glukanases uitgevoer is, het aangedui dat die eksoglukanase 'n meer skadelike effek op die hife-groei en -ontwikkeling van B. cinerea as die endoglukanase gehad het; eersgenoemde het die tipiese ontwrigting van die selle en die uitlek van selmateriaal tot gevolg gehad. Die gis-eksoglukanase is gevolglik in 'n plant uitdrukkingskasset wat die sterk konstitutiewe 35S promotor bevat, gesubkloneer, wat plamiede pEXG1 en pMJ-EXG1 opgelewer het. Die pMJ-EXG1-konstruk het die eksoglukanase na die apoplastiese gebied geteiken deur 'n seinpeptied vanaf "n antimikrobiese peptied van Mirabilisjalaba, Mj-AMP2. Die pEXG1- en pMJ-EXG1-konstrukte is in Agrobacterium tumefaciens gemobiliseer, wat die gevolglike tabaktransformasies gefasiliteer het wat die E en MJE transgeniese tabaklyne onderskeikelik gelewer het. Transgeen-integrasie is deur suidelike klad- en PKR-analises vir beide die E en MJE lyne bevestig. Die uitdrukking en heteroloë produksie van die EXG1-enkodeerde eksoglukanase is in die transgeniese E lyne deur noordelike klad en aktiwiteitstoetse onderskeidelik aangetoon. Verder het die hoë uitdrukkingsvlak van die gis-eksoglukanase tot 'n vermindering in die vatbaarheid van die E lyne vir B. cinerea-infeksie relatief tot die ongetransformeerde tabakkontroles gelei. 'n Gemiddelde vermindering in siektevatbaarheid van 40% is in 'n in planta verwyderde-blaartoets waargeneem. Ru proteïen-ekstrakte van die E lyne is ook in 'n in vitro kwantitatiewe swamgroeitoets geanaliseer en het in vitro swamgroei met tot gemiddeld 20% geïnhibeer, wat dus verder die antifungiese aard van die gis-eksoglukanase bevestig het. Alhoewel die integrasie van die pMJ-EXG1 uitdrukkingskasset bevestig is, kon geen mRNA-vlakke met die noordelike klad- of RT-peR-analises van die MJE-Iyne waargeneem word nie. Hierdie lyne het ook geen in vitro antifungiese aktiwiteite of 'n vermindering in die vatbaarheid vir B. cinerea-infeksie getoon nie, soos in die verwyderde-blaartoets uitgevoer is nie. Dit word vermoed dat hierdie resultaat moontlik aan geenstilmaking gekoppel is, 'n verskynsel wat gereeld met heteroloë- en/of ooruitdrukking van glukanases in plantgashere gekoppel word. Dit blyk dat die ooruitdrukking wat tot die apoplastiese ruimte geteiken is, tot die geenstilmaking-respons aanleiding gegee het, aangesien die intrasellulêre ooruitgedrukte produk gemaak is en aktiwiteit getoon het. Die gis-eksoglukanase word dus deel van die lys van stilgemaakte glukanases in die ooruitdrukkingstudies van plante. In die algemeen het hierdie studie dus die antifungiese kenmerke van die Saccharomyces eksoglukanase bevestig en waardevolle inligting oor die moontlike gebruik van gis-glukanases in 'n transgeniese omgewing verskaf. 'n Afname in die vatbaarheid van tabak vir infeksie deur B. cinerea, soos deur die ooruitdrukking van EXG1-enkodeerde eksoglukanase getoon is, verdien dus verdere ondersoek van die gebruik van hierdie geen in die skepping van siekteweerstandbiedende gewasse.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Acacia cyclops, an invasive weed in South Africa, was initially imported to stabilize the sand dunes in the southern Cape. The spread of A. cyclops is a major threat to the fragile biodiversity of the strandveld and limestone fynbos vegetation. Acacia cyclops dieback has been observed for some time, although the causative agent, Pseudolagarobasidium acaciicola, has only recently been described. This fungus is nominated for development as a mycoherbicide to control A. cyclops. Although current biological and mechanical control efforts are proving to be partially effective, A. cyclops is still causing major damage to natural ecosystems. The introduction of a mycoherbicide would increase the cost effectiveness of controlling this weed in the long term. The majority of the literature that was reviewed supports the use of mycoherbicides as biocontrol agents, especially when taking into account the decrease in acceptance and availability of chemical control agents. Considering that the Pseudolagarobasidium genus consists of saprobes, opportunistic facultative pathogens and endophytes, P. acaciicola is predicted to have similar biological characteristics. The species is also highly likely to be indigenous, although with a wider distribution range than previously envisaged. Strict precautions should still however be taken to ensure that non-target species will not be threatened. This study consists of a unique risk assessment comprising different sections. A field survey was performed to record disease incidence among indigenous woody plant species around 100 diseased A. cyclops trees. Subsequently, DNA extractions were made from the roots of the diseased indigenous plants and A. cyclops trees to verify the presence of P. acaciicola. Of the 2432 indigenous woody plants observed, 22 (0.9%) were dead or dying, while P. acaciicola was detected in 10 of these (0.4%), representing six species. Pseudolagarobasidium acaciicola was detected in 47% of the A. cyclops trees. Although P. acaciicola could be a weak pathogen in a broad range of indigenous plant species, the extremely low disease incidence is an indication of a low level of risk associated with using P. acaciicola as a mycoherbicide. Additionally, pathogenicity trials on indigenous plant species were conducted to give an indication of host susceptibility. A total of 30 indigenous plant species were wound inoculated at two field sites, and potted plants representing 17 indigenous plant species were wound and soil inoculated in a nursery. The optimum growth temperature for P. acaciicola was determined in order to understand it’s seasonal and landscape preference. Mortality was recorded in five of nine indigenous Fabaceae species, while a single plant each of four other non-Fabaceae species died after inoculation. No plants outside the Fabaceae family died in the field. Only A. cyclops seedlings died following soil inoculation. Longitudinal sections of stem inoculated plants revealed no systemic infection in Fabaceae species that survived inoculation. Infection in susceptible Fabaceae species was generally more extensive than infection in susceptible non-Fabaceae species. The optimum growth rate for P. acaciicola was determined at 35°C, indicating an adaptation to summer conditions. Indigenous Fabaceae species do display greater susceptibility than species from other families, indicating some level of specificity, although susceptible species can not be phylogenetically circumscribed. Aside from being a facultative pathogen on A. cyclops, results from this study suggest that P. acaciicola is primarily a saprophyte and an occasional opportunistic pathogen on some indigenous Fabaceae, possibly only being a weak opportunistic pathogen on some non-Fabaceae species. However, the risk of not effectively managing A. cyclops populations in these threatened vegetation types outweighs the risk associated with using P. acaciicola as a mycoherbicide. Therefore the use of P. acaciicola as a mycoherbicide on A. cyclops would be recommended, provided that sufficient monitoring of treated sites is implemented that primarily focus on the indigenous Fabaceae species. The effective control of A. cyclops could be achieved when P. acaciicola is used to compliment current mechanical, biological and chemical control methods in an integrated management strategy. / AFRIKAANSE OPSOMMING: Acacia cyclops, ook bekend as rooikrans, is ‘n indringerplant in Suid-Afrika wat oorspronklik vanaf Australië ingevoer is om die sandduine in die Kaap te stabiliseer. Die verspreiding van rooikrans bedreig die sensitiewe biodiversiteit van die strandveld en kalksteen fynbos. Rooikrans terugsterwing is al vir ‘n geruime tyd opvallend in die grootste deel van die plant se verspreiding in Suid-Afrika, alhoewel die veroorsakende organisme, Pseudolagarobasidium acaciicola, eers onlangs beskryf is. Hierdie swam is as ‘n geskikte kandidaat vir die ontwikkeling van ‘n biologiese onkruiddoder om rooikrans te beheer, genomineer. Alhoewel die huidige biologiese- en meganiese beheer metodes vir rooikrans gedeeltelik suksesvol is, hou dié indringer steeds ‘n ernstige bedreiging vir die natuurlike ekosisteme in. Die gebruik van ‘n swam-gebaseerde onkruiddoder sal die beheer van rooikrans oor die langtermyn meer koste-effektief maak. Die oorgrote meerderheid van die literatuur wat hersien is, ondersteun die gebruik van swam-gebaseerde onkruiddoders as biologiese beheermiddels, veral as die afname in aanvaarbaarheid en beskikbaarheid van chemiese beheermiddels in ag geneem word. Aangesien die Pseudolagarobasidium genus uit saprofiete, opportunistiese fakultatiewe patogene en endofiete bestaan, word daar voorspel dat P. acaciicola ‘n soortgelyke biologiese karakter sal hê. Dit is hoogs waarskynlik dat hierdie swamspesie inheems is, alhoewel die verspreiding wyer mag wees as wat oorspronklik voorspel is. Streng maatreëls moet egter steeds in plek wees om te verseker dat nie-teiken plantspesies nie bedreig word nie. Hierdie studie bestaan uit ‘n unieke risiko-analise met verkeie onderafdelings. ‘n Veld-opname is uitgevoer om die siekte-voorkoms van die inheemse houtagtige plantspesies rondom ‘n 100 siek rooikrans plante te bepaal. DNA ekstraksies is vervolgens vanuit die wortels van siek inheemse plantspesies en -rooikrans uitgevoer, om uiteindelik die teenwoordigheid van P. acaciicola binne die hout te kon bevestig. Uit ‘n totaal van 2432 inheemse houtagtige plante wat aangeteken is, was 22 (0.9%) siek of dood, terwyl die teenwoordigheid van P. acaciicola in 10 van hierdie plante (0.4%), wat ses spesies teenwoordig, bevestig is. Die teewoordigheid van P. acaciicola is ook in 47% van die rooikrans bevestig. Alhoewel P. acaciicola moontlik ‘n swak opportunistiese patogeen op ‘n verskeidenheid inheemse plantspesies is, dui die lae verhouding van dooie inheemse plante teenoor gesonde plante in die veld op ‘n lae risiko vir die gebruik van P. acaciicola as ‘n biologiese onkruiddoder. Patogenisiteitstoetse is op inheemse plantspesies uitgevoer om ‘n aanduiding van gasheervatbaarheid te verkry. Wond-inokulasies is op ‘n totaal van 30 inheemse plantspesies by twee veldstudie-areas uitgevoer, terwyl wond- en grond-inokulasies op 17 inheemse spesies potplante in die kweekhuis uitgevoer is. Die optimale temperatuur waarby P. acaciicola groei, is bepaal om die swam se seisoenale- en habitatsvoorkeure beter te verstaan. Plante van vyf uit die nege inheemse Fabaceae spesies het doodgegaan, terwyl ‘n enkele plant van vier nie-Fabaceae spesies doodgegaan het. Alle plante buite die Fabaceae familie het oorleef in die veld na inokulasie. Slegs rooikranssaailinge het na grond inokulasie doodgegaan. Lengtedeursnee van die stam en wortels van elke geïnokuleerde plant het bevestig dat daar geen sistemiese infeksie in Fabaceae spesies wat inokulasie oorleef het, plaasgevind het nie. Infeksies in vatbare Fabaceae spesies was oor die algemeen meer ernstig as infeksies in vatbare nie-Fabaceae spesies. Die optimale groei van P. acaciicola het by 35°C plaasgevind, wat aandui op ‘n voorkeur vir somerstoestande. Inheemse Fabaceae spesies het meer vatbaar as vatbare plantspesies van ander families voorgekom. Hierdie verskynsel dui op ‘n sekere vlak van spesifisiteit, alhoewel daar geen duidelike filogenetiese grense vir vatbare spesies bepaal kon word nie. Behalwe vir die feit dat P. acaciicola ‘n fakultatiewe patogeen op rooikrans is, stel resultate van hierdie studie voor dat hierdie swam hoofsaaklik ‘n saprofiet is wat soms ook ‘n opportunisties patogeen op sekere inheemse Fabaceae is en moontlik slegs ‘n swak opportunistiese patogeen op plantspesies buite die Fabaceae familie is. Die swak en oneffektiewe bestuur van rooikrans in hierdie bedreigde plantegroeitipes hou egter ‘n groter bedreiging in as die gebruik van P. acaciicola as ‘n biologiese onkruiddoder. Pseudolagarobasidium acaciicola word daarom aanbeveel vir die beheer van rooikrans, mits voldoende monitering, wat fokus op inheemse Fabaceae spesies, gepaard gaan met die gebruik van hierdie biologiese onkruiddoder. Rooikrans kan effektief beheer word as P. acaciicola ingespan word om huidige meganiese-, biologiese- en chemiese beheermetodes in ‘n geïntegreerde bestuurstrategie te komplimenteer.

Thesis (MScAgric)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Botrytis cinerea Pers. Fr. [teleomorph Botryotinia fuckeliana (de Bary) Whetzel] causes serious losses of over 200 crops worldwide, including rooibos seedlings and pears. This pathogen is characterized by morphological, physiological and genetic diversity. The genetic diversity and population structure have not been investigated for B. cinerea populations in South Africa. Botrytis cinerea collected from rooibos seedlings and in pear orchards in the Western Cape of South Africa were investigated in the present study. The study was done with the aid of microsatellite markers, the amplification of mating type alleles MAT1-1 and MAT1-2 and determination of resistance towards various fungicides. Population dynamics was inferred and a similar picture emerged in both production systems. Botrytis cinerea annually causes severe losses of rooibos seedlings (Aspalathus linearis) in nurseries situated in the Clanwilliam region. Sampling was done in five nurseries and the cryptic species status of the isolates obtained was determined through restriction enzyme digestion of the Bc-hch gene. All but one (206 out of 207) of the isolates belonged to Group II or B. cinerea ‘sensu stricto’. Analysis of the B. cinerea Group II population, using seven microsatellite loci, was performed to assess the genetic population structure. Total gene diversity (H) was high, with a mean of 0.67. Two of the nurseries populations’ sample sizes were severely limited after clone correction, yet 100 genotypes were discerned among the 206 isolates genotyped. The percentage of maximal genotypic diversity (G) ranged between 16 and 68 for the five populations, with a total value of 17 for the 100 genotypes. One genotype, represented by 27 clones, was isolated from four nurseries. Relatively low but significant population differentiation was observed in total between nurseries (mean FST = 0.030, P = 0.001). The distribution of mating types MAT1-1 and MAT1-2 differed significantly from the ratio of 1:1 for the total population plus two of the nurseries’ populations. Three nursery populations had an equal mating type distribution. The index of association (IA) analyses suggests that the populations are asexually reproducing. Analysis of molecular variance (AMOVA) indicated that 97% of the total genetic variation is distributed within subpopulations. Fungicide resistance frequency against iprodione for 198 of the genotyped isolates displayed highly varying levels of resistance amongst the five nurseries. The mean total incidence of resistance towards iprodione was 43%, ranging from 0% to 81% for the five nurseries. Baseline sensitivity towards pyrimethanil yielded an average EC50 value of 0.096 mg/L. Botrytis cinerea isolates were collected from pear blossoms (Pyrus communis) in four orchards. Two orchards in the Ceres area and two in the Grabouw area were sampled from. A total of 181 isolates were collected from the four orchards. Incidence of blossom infection in the orchards ranged from 3% to 17%. Overall, there was a high incidence of isolates that had only the Boty transposable element (74%) compared to those harbouring both (Boty and Flipper), simultaneously (transposa, 24%). One isolate examined had the Flipper element only. Cryptic species status according to restriction enzyme digestion of the Bc-hch gene indicated that all the isolates belonged to Group II or B. cinerea ‘sensu stricto’. Analysis of the Group II population, through the use of seven microsatellite loci, was performed to assess the genetic population structure. Total gene diversity (H) was high, with a mean of 0.69 across all populations. Although two of the subpopulations displayed a high clonal proportion, overall 91 genotypes were discerned among the 181 isolates. The percentage of maximal genotypic diversity (G) ranged between 18 and 33 for the four populations, with a total value of 14 for the 91 genotypes. One genotype, represented by 27 clones, was isolated from all orchards. Moderate, but significant population differentiation was present in total among orchards (mean FST = 0.118, P = 0.001). The distribution of the mating types, MAT1-1 and MAT1-2, did not differ significantly from a 1:1 ratio for the total population as well as the subpopulations. Index of association (IA) analyses, on the other hand, suggests that the populations reproduce asexually. Analysis of molecular variance (AMOVA) indicated that 88% of the total genetic variation is distributed within subpopulations, 9% between subpopulations and only 3% between production areas. Fungicide resistance frequency against fenhexamid, iprodione and benomyl varied, with the highest levels of resistance present against benomyl and low levels of resistance seen towards iprodione and fenhexamid. In conclusion, this study has shown that there exist within the studied populations of B. cinerea, obtained from rooibos nurseries and pear orchards, an adaptive capacity to overcome current means of control. The use of population genetics to further our understanding of how plant pathogens interact and spread throughout a given environment is of cardinal importance in aiding the development of sustainable and integrated management strategies. Knowledge of the dispersal of B. cinerea in the two studied cropping systems has shed light on the inherent risk that it poses, and this together with knowledge of the levels of resistance that occurs should serve as an early warning to help divert possible loss of control in future. / AFRIKAANSE OPSOMMING: Botrytis cinerea Pers. Fr. [teleomorf Botryotinia fuckeliana (de Bary) Whetzel] veroorsaak ernstige verliese van meer as 200 gewasse wêreldwyd, insluitende rooibossaailinge en pere. Hierdie patogeen word deur morfologiese, fisiologiese, asook genetiese diversiteit gekenmerk. Die genetiese diversiteit en populasie-struktuur van B. cinerea populasies wat in Suid-Afrika voorkom, is nog nie ondersoek nie. Botrytis cinerea verkryg vanaf rooibossaailinge en in peerboorde in die Wes-Kaap van Suid-Afrika is ondersoek. Hierdie studie is met behulp van mikrosatellietmerkers, amplifikasie van die twee paringstipe gene (MAT1-1 en MAT1-2), asook die bepaling van weerstandsvlakke teenoor verskeie swamdoders, uitgevoer. Populasie-dinamika is afgelei en ‘n soortgelyke tendens is in beide produksie-sisteme waargeneem. Botrytis cinerea veroorsaak jaarliks ernstige verliese van rooibossaailinge (Aspalathus linearis) in kwekerye in die Clanwilliam-area. Monsters is in vyf kwekerye versamel en die kriptiese spesiestatus van die verkrygde isolate is deur restriksie-ensiemvertering van die Bc-hch geen bepaal. Almal behalwe een (206 uit 207) isolaat het aan Groep II of B. cinerea ‘sensu stricto’ behoort. Analise van die B. cinerea Groep II populasie, deur middel van sewe mikrosatellietmerkers, is uitgevoer om die genetiese populasiestruktuur te bepaal. Totale geendiversiteit (H) was hoog, met ‘n gemiddelde van 0.67. Alhoewel twee van die kwekerye se monstergrootte erg ingeperk is ná kloonverwydering, is daar nogtans 100 genotipes onder die 206 isolate wat geïsoleer is, waargeneem. Die persentasie van maksimale genotipiese diversiteit (G) het tussen 16 en 68, vir die vyf populasies, gewissel, met ‘n totaal van 17 vir die 100 genotipes. Een genotipe, verteenwoordig deur 27 klone, is uit vier kwekerye geïsoleer. Relatief lae dog noemenswaardige populasie-differensiasie is in totaal tussen kwekerye waargeneem (gem. FST = 0.030, P = 0.001). Die verspreiding van die twee paringstipes (MAT1-1 en MAT1-2) het beduidend verskil van ‘n 1:1 verhouding vir die totale populasie, asook twee van die kwekerye se populasies. Die drie oorblywende kwekerye se populasies het egter ‘n gelyke verdeling van die twee paringstipes getoon. Die indeks van assosiasie (IA) analises toon dat die populasies ongeslagtelik voortplant. Analise van molekulêre variasie (AMOVA) het aangedui dat 97% van die totale genetiese variasie binne die subpopulasies versprei is. Hoogs variërende vlakke van weerstand tussen die vyf kwekerye teenoor die swamdoder iprodioon, is vir die 198 isolate wat getoets is, gevind. Die totale gemiddelde frekwensie van weerstand teenoor iprodioon was 43%, wat tussen 0% en 81% vir die vyf kwekerye gevarieer het. Fondasie-vlak-sensitiwiteit vir pyrimethanil het ‘n gemiddelde EC50 waarde van 0.096 mg/L opgelewer. Botrytis cinerea isolate is ook vanuit peerbloeisels (Pyrus communis L.) vanuit vier boorde versamel, twee uit elk van die Ceres- en Grabouw-areas. In totaal is 181 isolate vanuit die vier boorde versamel. Die frekwensie van bloeiselinfeksie het tussen 3% en 17% gewissel. Oor die algemeen was daar ‘n hoë frekwensie van isolate wat slegs die Boty transponeerbare element teenwoordig gehad het (74%) in vergelyking met dié wat tegelykertyd beide (Boty en Flipper) teenwoordig gehad het. Een isolaat het slegs die Flipper element gehad. Bepaling van die kriptiese spesiestatus met behulp van restriksie-ensiemvertering van die Bc-hch geen het aangedui dat alle versamelde isolate tot Groep II of B. cinerea ‘sensu stricto’ behoort het. Analise van die Groep II populasie, deur middel van sewe mikrosatellietmerkers, is uitgevoer om genetiese populasie-struktuur te bepaal. Totale geendiversiteit (H) was hoog, met ‘n gemiddelde van 0.69 oor alle populasies. Alhoewel twee subpopulasies ‘n hoë klonale fraksie getoon het, is 91 genotipes tussen die 181 isolate wat verkry is, onderskei. Die persentasie van maksimale genotipiese diversiteit (G) het tussen 18 en 33 vir die vier populasies gewissel, met ‘n totale waarde van 14 vir die 91 genotipes. Een genotipe, verteenwoordig deur 27 klone, was in al vier boorde teenwoordig. Gematigde dog beduidende populasie differensiasie was in totaal tussen boorde teenwoordig (gem. FST = 0.118, P = 0.001). Die verspreiding van die paringstipes (MAT1-1 en MAT1-2) het nie betekenisvol van ‘n 1:1 verhouding vir die totale populasie, insluitende die subpopulasies, verskil nie. Indeks van assosiasie (IA) analises het egter aangedui dat die populasies ongeslagtelik voortplant. Analise van molekulêre variasie (AMOVA) het aangedui dat 88% van die totale genetiese variasie in subpopulasies te vinde was, 9% tussen subpopulasies en slegs 3% tussen produksie-areas. Frekwensie van swamdoder weerstandbiedendheid vir fenhexamid, iprodioon en benomyl het gewissel, met die hoogste vlakke teenoor benomyl waargeneem, maar baie lae vlakke teenoor fenhexamid en iprodioon. Samevattend het hierdie studie getoon dat die populasies van B. cinerea wat in hierdie twee produksie-sisteme, op rooibossaailinge en in peer boorde, ondersoek is, ‘n aanpasbaarheid toon om huidige metodes van beheer te oorkom. Die gebruik van populasiegenetika as ‘n hulpmiddel om ons kennis van patogeen-interaksies en -verspreiding te verbreed, is van kardinale belang in die ontwikkeling van geïntegreerde en volhoubare beheermaatreëls. Kennis van die verspreiding van B. cinerea in die bestudeerde gewasproduksiestelsels, werp lig op die inherente risiko wat dié patogeen inhou. Dít, tesame met kennis van die weerstandsvlakke wat voorkom, kan as ‘n vroegtydige waarskuwing dien ten einde moontlike verlies van beheer in die toekoms te help teenwerk.