Please read the abstract in the section 07back of this document / Dissertation (MSc (Plant Pathology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Thesis (MScAgric)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Phaeomoniella chlamydospora is the main causal organism of Petri disease, which causes severe decline and dieback of young grapevines (1-7 years old) and also predisposes the wood for infection by other pathogens. Knowledge about the epidemiology and especially inoculum sources of this disease is imperative for subsequent development of management strategies. Through isolation studies it was shown that Pa. chlamydospora is mainly distributed through infected propagation material in South Africa. However, the infection pathways and inoculum sources in grapevine nurseries are still unclear. The only existing method to detect this pathogen in various media is by means of isolation onto artificial growth media. This has proven to be problematic since this fungus is extremely slow growing (up to 4 weeks from isolation to identification) and its cultures are often over-grown by co-isolated fungi and bacteria before it can be identified. The aim of this study was (i) to develop a protocol for the molecular detection of Pa. chlamydospora in grapevine wood, and (ii) to use this protocol along with others, to test different samples (water, soil, rootstock and scion cuttings and callusing medium) collected from nurseries in South Africa at different nursery stages for the presence of Pa. chlamydospora. A protocol was developed and validated for the molecular detection of Pa. chlamydospora in grapevine wood. Firstly, several previously published protocols were used to develop a cost-effective and time-efficient DNA extraction method from rootstock pieces of potted grapevines. Subsequently, PCR amplification using species-specific primers (Pch1 and Pch2) was found to be sensitive enough to detect as little as 1 pg of Pa. chlamydospora genomic DNA from grapevine wood. The protocol was validated using various grapevine material from 3 different rootstock cultivars (101-14 Mgt, Ramsey and Richter 99) collected from each of 3 different nurseries, including grapevines that were subjected to hot water treatment. The basal end of the rootstock was parallel analysed for Pa. chlamydospora using isolations onto artificial medium and molecular detection. The identity of PCR products obtained from a subset of samples, that only tested positive for Pa. chlamydospora based on molecular detection, was confirmed to be Pa. chlamydospora specific through restriction digestion with AatII. Molecular detection was found to be considerably more sensitive than isolations, detecting Pa. chlamydospora from samples with positive as well as negative isolations. On average, the molecular technique detected Pa. chlamydospora in 80.9% of the samples, whereas only 24.1% of the samples tested positive for Pa. chlamydospora by means of isolations. Pa. chlamydospora was not isolated from hot water treated samples. The results confirm the importance of hot water treatment for proactive management of Petri disease in grapevine nurseries. However, Pa. chlamydospora DNA was molecularly detected in hot water treated samples in frequencies similar to that detected in non-hot water treated samples. As expected, the DNA in hot water treated plants was not destroyed and could be detected by the developed molecular detection protocol. This is an important consideration when using molecular detection for disease diagnosis or pathogen detection and shows that these methods should be used in conjunction with other diagnostic tools. Most importantly, the DNA extraction protocol was shown to be 10 to 15 times cheaper than commercial DNA extraction kits. Preliminary studies showed that the aforementioned molecular detection technique was not specific and sensitive enough for detection of Pa. chlamydospora in soil and water (unpublished data). Therefore, a one-tube nested-PCR technique was optimised for detecting Pa. chlamydospora in DNA extracted from soil, water, callusing medium and grapevine wood. Rootstock cane sections and soil samples were taking from the mother blocks from several nurseries. Water samples were collected from hydration and fungicide tanks during pre-storage and grafting. Scion and rootstock cuttings were also collected during grafting and soil were collected from the nursery beds prior to planting. The one-tube nested-PCR was sensitive enough to detect as little as 1 fg of Pa. chlamydospora genomic DNA from water and 10 fg from wood, callusing medium and soil. PCR analyses of the different nursery samples revealed the presence of several putative Pa. chlamydospora specific bands (360 bp). Subsequent sequence analyses and/or restriction enzyme digestions of all 360 bp PCR bands confirmed that all bands were Pa. chlamydospora specific, except for five bands obtained from callusing media and one band from water. Considering only Pa. chlamydospora specific PCR bands, the molecular detection technique revealed the presence of Pa. chlamydospora in 25% of rootstock cane sections and 17% of the soil samples collected from mother blocks, 42% of rootstock cuttings collected during grafting, 16% of scion cuttings, 40% of water samples collected after the 12- hour pre-storage hydration period, 67% of water samples collected during grafting and 8% of the callusing medium samples. These media should therefore be considered as potential inoculum sources or infection points of the pathogen during the nursery stages. The results furthermore confirmed previous findings that Pa. chlamydospora is mainly distributed through infected rootstock canes and cuttings. Infected scion cuttings were also shown to be potential carriers of the pathogen. Management strategies should include wound protection of rootstock mother plants, eradicating this pathogen from rootstock-cuttings (e.g. hot water treatment), biological or chemical amendments in the hydration water and callusing medium and wound protection from soil borne infections. / AFRIKAANSE OPSOMMING: Phaeomoniella chlamydospora is die hoof veroorsakende organisme van Petri se siekte wat lei tot die agteruitgang en terugsterwing van jong wingerdplante (1-7 jaar oud) en veroorsaak verhoogde vatbaarheid van hout vir infeksie deur ander patogene. Kennis oor die epidemiologie en veral die inokulumbronne van die siekte is noodsaaklik vir die daaropvolgende ontwikkeling van beheerstrategieë. Isolasies het getoon dat Pa. chlamydospora meestal versprei deur middel van geïnfekteerde voortplantingsmateriaal in Suid-Afrika. Die infeksieweë en inokulumbronne in wingerdkwekerye is egter steeds onbekend. Die enigste bestaande metode vir die opsporing van die patogeen, in verskeie mediums, is deur middel van isolasie op kunsmatige groeimediums. Dit is egter gevind om problematies te wees aangesien die swam uiters stadig groei (dit vat tot 4 weke vanaf isolasie tot identifikasie) en die kulture is telkens oorgroei deur ander organismes voordat identifikasie kan plaasvind. Die doel van die studie was (i) om ‘n protokol te ontwikkel vir die molekulêre opsporing van Pa. chlamydospora in wingerdhout, en (ii) om die protokol te gebruik, saam met ander, om verskillende monsters (water, grond, onderstok- en bostok-ente en kallusmedium) te toets, wat versamel is van kwekerye in Suid- Afrika, tydens verskillende kwekerystadiums, vir die teenwoordigheid van Pa. chlamydospora. ‘n Protokol is ontwikkel en geverifieer vir die molekulêre opsporing van Pa. chlamydospora in wingerdhout. Eerstens is verskeie protokols wat voorheen gepubliseer is, is as grondslag gebruik vir die ontwikkeling van ‘n ekonomiese en tydbesparende DNA ekstraksie protokol. Hierna is PKR (polimerase ketting reaksie) amplifikasie met spesie-spesifieke inleiers (Pch1 en Pch2) gevind om sensitief genoeg te wees om so min as 1 pg van Pa. chlamydospora genomiese DNA van wingerdhout op te spoor. Die protokol is geverifieer deur verskeie wingerdhoutmateriaal van 3 verskillende onderstokkultivars (101-14 Mgt, Ramsey en Richter 99) te gebruik, wat elk versamel is van 3 verskillende kwekerye. ‘n Aantal van die wingerstokke is ook onderwerp aan warmwaterbehandeling. Die basale kant van die onderstok is parallel geanaliseer vir Pa. chlamydospora deur gebruik te maak van isolasies op kunsmatige groeimedium asook molekulêre opsporing. Die identiteit van ‘n submonster van PKR produkte van verskeie monsters, wat slegs positief getoets het vir Pa. chlamydospora met die molekulêre opsporing, is bevestig om Pa. chlamydospora spesifiek te wees. Dit is gedoen deur middel van restriksie ensiem analise met AatII. Molekulêre opsporing is gevind om aansienlik meer sensitief te wees as isolasies, deurdat Pa. chlamydospora opgespoor is van positiewe sowel as negatiewe isolasies. Die molekulêre tegniek het Pa. chlamydospora in ‘n gemiddeld van 80.9% van die monsters opgespoor, terwyl slegs ‘n gemiddeld van 24.1% van die monsters postief getoets het vir Pa. chlamydospora, deur middel van isolasies. Pa. chlamydospora is nie geïsoleer van die monsters wat warmwaterbehandeling ondergaan het nie. Die resultate bevestig hoe belangrik warmwaterbehandeling is vir die proaktiewe beheer van Petri se siekte in wingerdkwekerye. Pa. chlamydospora DNA is met die molekulêre tegniek opgespoor, in warmwaterbehandelde monsters, in getalle wat ooreenstemmend is met die van niewarmwaterbehandelde monsters. Soos verwag, is DNA in warmwaterbehandelde plante nie vernietig nie en kon dit telke male opgespoor word deur die ontwikkelde molekulêre opsporing protokol. Dit is ‘n belangrike feit wat in ag geneem moet word wanneer molekulêre opsporing gebruik word in siekte diagnose en opsporing van patogene en dit is ‘n aanduiding dat die metodes gebruik moet word in samewerking met ander diagnostiese tegnieke. Die DNA ekstraksie protokol het getoon om tot en met 10 tot 15 kere goedkoper te wees as kommersiële DNA ekstraksie pakkette. Voorlopige studies het getoon dat die bogenoemde molekulêre opsporings tegniek nie spesifiek en sensitief genoeg is vir die opsporing van Pa. chlamydospora uit grond en water nie (ongepubliseerde data). Daarom is ‘n enkel-buis geneste-PKR tegniek geoptimiseer vir die opsporing van Pa. chlamydospora DNA wat geëkstraheer is vanaf grond, water, kallusmedium en wingerdhout. Dele van onderstokke en grond monsters is geneem vanaf moederblokke van verskeie kwekerye. Gedurende die voor-opberging en enting periodes is watermonsters versamel vanaf hidrasie en fungisied tenke. Bostok- en onderstokente is ook versamel gedurende enting en grond is versamel vanaf kwekerybeddens net voor uitplanting. Die enkelbuis geneste-PKR was sensitief genoeg om so min as 1 fg van Pa. chlamydospora genomiese DNA vanaf water en 10 fg vanaf hout, kallusmedium en grond op te spoor. PKR analise van die verskillende kwekerymonsters het getoon dat daar ‘n teenwoordigheid is van verskeie putatiewe Pa. chlamydospora spesifieke bande (360 bp). Daaropvolgende analise deur middel van DNA volgordebepaling en restriksie ensiem analise het bevestig dat al die 360 bp PKR bande wel Pa. chlamydospora spesifiek is, behalwe vir vyf bande wat verkry is vanaf kallusmedium en een band verkry vanaf water. As slegs Pa. chlamydospora spesifieke bande in ag geneem word, is daar met molekulêre opsporing die teenwoordigheid van Pa. chlamydospora gevind in 25% van die onderstokke, 17 % van die grond versamel vanaf moederblokke, 42% van die onderstokente versamel tydens enting, 16% van die bostokente, 40% van die watermonsters versamel voor die 12-uur hidrasie periode, 67% van die watermonsters versamel gedurende enting en 8% van die kallusmediummonsters. Hierdie mediums moet dus beskou word as potensiële inokulumbronne of infeksiepunte van die patogeen gedurende die kwekerystadiums. Die resultate bevestig ook verdere bevindinge wat aandui dat Pa. chlamydospora meestal versprei word deur geïnfekteerde onderstokke en ente. Geïnfekteerde bostokente is ook aangedui om potensiële draers van die patogeen te wees. Beheerstrategieë moet dus wondbeskerming van onderstok moederplante insluit, asook uitwissing van die patogeen vanaf onderstokente (bv. warmwaterbehandeling), toediening van biologiese of chemiese stowwe in die hidrasie water en kallusmedium en wondbeskerming teen grondgedraagde infeksies.

Thesis (PhD(Agric))--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: The purpose of this study was to characterize the race and vegetative compatibility of Fusarium oxysporum f. sp. melonis (FOM) isolates collected in the major melon producing areas, to report on their geographical distribution, and their possible relatedness to isolates from other countries. Seventy two FOM isolates obtained from 30 fields in 17 melon producing regions were race-typed using the differential cultivars Topmark (susceptible to all races), Doublon (Fomi), CM 17187 (Fom2) and Perlita (Fom3) and grouped by means of vegetative compatibility. All isolates belonged to vegetative compatibility group 0134, indicating a high degree of genetic homogeneity among the South African FOM population. Fifty four isolates were identified as race 0, eight as race 1, and 10 as race 2. Race 0 occurred in 15 of the regions whereas race 1 was sporadically recovered. Race 2, on the other hand, was obtained only from four fields located in one geographical region. Perlita plants (carrying the gene Fom3) inoculated with local isolates ofrace 0 and race 2 and reference isolates of race 0 became stunted, their leaves turned yellow, and became thickened and brittle. These results suggested that Fom3 in Perlita confers a tolerant reaction compared to the resistant reaction of gene FornI in Doublon. The disease reaction of cultivar Perlita to FOM was therefore reinvestigated. Twenty isolates, including the four FOM races (0, 1, 2, and 1,2) obtained from different countries, were used. The differential cultivars were included to verify virulence of the isolates. Perlita plants inoculated with three isolates of race 2 remained asymptomatic. The remaining race 2 and 0 isolates, induced severe stunting of Perlita plants, but mean percentage stunting values did not differ significantly (P = 0.05) and ranged between 25.1 and 50.0. Leaves of stunted plants were chlorotic, thickened and brittle. Disease reaction of Perlita was verified at a lower inoculum concentration with two race 2 (pipette method) and two race 0 isolates (root dip method). Results proved that Fom3 does not confer similar resistance towards race 0 and some race 2 isolates as FornI in Doublon. Cultivars possessing Fom3, should therefore be considered tolerant to FOM races 0 and 2. The ability of a nit mutant isolate, generated from FOM race 0 which belongs to VCG 0134, to change its virulence during infection of melon plants, was investigated under quarantine. Seedlings of melon cultivars Imperial 45 and Early Sweet (no resistance genes), Amber (Fom2) and Fiata (FomI, Fom2) were consecutively grown in two cement troughs in a gauzehouse. Each planting was terminated when plants had advanced Fusarium wilt or after the fruit were harvested. In the first planting, Imperial 45 seedlings were transplanted and artificially inoculated with the nil mutant isolate. In the consecutive plantings, seeds were sown in the infested soil to enable natural infection. For each crop, representative plants showing Fusarium wilt were selected for isolation. All F. oxysporum isolates recovered were single-spored and their nit mutant and VCG status verified. Virulence of the labelled isolates was determined using differential cultivars. In trough A, all plants of the susceptible cultivars Imperial 45 and Early Sweet crops showed Fusarium wilt. The labelled isolates recovered from the selected plants were all designated race O. In the first crop (planting No.5) of the resistant cultivar Amber, 6.7% of the plants developed Fusarium wilt. In the second Amber crop the disease incidence increased to 56.6%, and to 81.8% in the final crop. Contrary to the susceptible cultivars, only race 2 isolates were obtained from the symptomatic Amber plants. Similar data were found with the susceptible cultivar Imperial 45 and the resistant cultivar Amber in trough B. Planting of Fiata caused a dramatic reduction in Fusarium wilt incidence in trough B. However, 1.2% of plants were affected by Fusarium wilt in the first Fiata crop (planting No.6), whereas 4% of the plants were symptomatic in the final planting. From these symptomatic Fiata plants only race 1,2 isolates were obtained. These findings, and the fact that the symptomatic plants represented a substantial proportion of the first Amber (approximately 7-15%) and Fiata (approximately 2%) crops, provedthat changes in the race structure of this fungal pathogen occurred rapidly when confronted with a resistant cultivar. The potential of RAPD analysis to differentiate between the isolates displaying virulence changes was evaluated. Four F. oxysporum f. sp. niveum isolates were included as an outgroup. A histopathological study was conducted to verify whether these isolates retain their ability to behave as true vascular pathogens. The three primers used clearly distinguished the 12 FOM isolates from the four F. oxysporum f. sp. niveum isolates. However, the primers showed a highly conserved and characteristic banding pattern for the FOM isolates which represented three physiological races (race 0, race 2, race 1,2), indicating that RAPD analysis cannot detect race-specific groupings in FOM. Disease reactions on the three differential cultivars confirmed the virulence of FOM isolates. The histopathological data furthermore proved that the two FOM races (race 2, race 1,2), which derived from the race 0 parent isolate, retained their ability to behave as true vascular pathogens. / AFRIKAANSE OPSOMMING: DIE KARAKTERISERING EN PATOGENESITEIT VAN SUID-AFRIKAANSE ISOLATE VAN FUSARIUMOXYSPORUMF. SP.MELONIS Die doel van die studie was om Fusarium oxysporum f. sp. melonis (FOM) isolate wat in die hoof spanspekproduserende gebiede versamel is, volgens ras en vegetatiewe verenigbaarheid te karakteriseer, en hul geografiese verspreiding en verwantskap met isolate van ander lande aan te dui. Twee en sewentig FOM isolate afkomstig vanaf 30 landerye wat 17 spanspekproduserende areas verteenwoordig, is gebruik. Die differensiële kultivars Topmark (vatbaar vir alle rasse), Doublon (Forni), CM 17187 (Fom2) en Perlita (Fond) is gebruik om die rasbepalings te doen asook om die vegetatiewe verenigbare groepe (VVG) te bepaal. Al die isolate is as VVG 0134 geklassifiseer, wat 'n hoë mate van genetiese homogenesiteit binne die Suid-Afrikaanse populasie aandui. Vier en vyftig isolate is as ras 0, agt as ras 1 en 10 as ras 2 geklassifiseer. Ras 0 is vanaf 15 gebiede afkomstig, terwyl ras 1 sporadies voorgekom het. Ras 2 is vanuit vier landerye binne dieselfde geografiese gebied verkry. Plante van die kultivar Perlita wat met plaaslike isolate van ras 0 en 2, asook verwysings-isolate van ras 0 geïnokuleer is, het verdwerg voorgekom. Die blare van die plante het vergeel, verdik en bros voorgekom. Hierdie siekte reaksie het aangedui dat Fond in Perlita toleransie bewerkstellig in teenstelling met die weerstandbiedende reaksie van geen Fomi in Doublon. Die siekte reaksie van Perlita teenoor FOM is dus verder ondesoek. Hiervoor is 20 isolate wat al vier FOM rasse insluit (0, 1, 2, en 1,2), en van verskillende wêrelddele afkomstig is, gebruik. Die virulensie van die isolate is met die differensiële kultivars bevestig. Drie van die ras 2 isolate het geen siektesimptome op Perlita veroorsaak nie. Die ander ras 2 isolate, en al die ras 0 isolate, het egter die Perlita plante aansienlik verdwerg en die blare vergeel en verdik. Laasgenoemde groep isolate het 'n gemiddelde verdwergingsindeks van tussen 25.1% en 50.0% veroorsaak. Die siekte reaksie by Perlita is verder bevestig deur plante teen 'n laer inokulumdigtheid van twee ras 2 (pipet metode), en twee ras 0 (wortel-doop metode) isolate, te inokuleer. Uit die resultate was dit duidelik dat die weerstand wat Fom3 teenoor ras 0 en sommige ras 2 isolate verskaf, van FornI verskil. Kultivars wat oor die weerstandsgeen Fom3 beskik moet dus as tolerant beskou word. 'n Ondersoek is geloods na die vermoë van 'n nil mutant isolaat, genereer vanaf die wilde ras 0 isolaat van FOM (VVG 0134), om onder kwarantyn sy virulensie gedurende infeksie van spanspekplante te verander. Saailinge van die spanspekkultivars Early Sweet (geen weerstandsgene), Amber (Fom2) en Fiata (FornI, Fom2) is opeenvolgens in twee sement trêe in 'n gaashuis verbou. Die afsonderlike aanplantings is beëindig sodra gevorderde Fusarium-verwelksimptome verkry is, of nadat vrugte ge-oes is. Vir die eerste aanplanting is oorgeplante Imperial 45 saailinge kunsmatig met die nil mutant isolaat geïnokuleer. Tydens die opeenvolgende aanplantings is saad direk in die besmette grond gesaai ten einde natuurlike infeksie te verkry. Met elke aanplanting is isolasies gedoen vanaf verteenwoordigende plante wat Fusarium-verwelksimptome getoon het. Alle F. oxysporum isolate wat verkry is, is ge-enkelspoor en hul nit mutant status en VVG is bevestig. Virulensie van die gemerkte isolate is bepaal deur inokulasie van die differensiële kultivars. Alle plante van die vatbare Imperial 45 en Early Sweet kultivars wat in trog A geplant is, het Fusarium-verwelksimptome getoon. Die gemerkte isolate wat vanaf die verteenwoordigende plante verkry is, is almal as ras 0 geklassifiseer. Tydens die eerste aanplanting van die weerstandbiedende kultivar, Amber (aanplanting No.5), het 6.7% van die plante Fusarium-verwe1ksimptome ontwikkel. Tydens die tweede en derde aanplanting van Amber het die frekwensie van siektevoorkoms verhoog na 56.6% en 81.8 %, onderskeidelik. In teenstelling met die vatbare kultivars, is slegs ras 2 vanuit die Amber plante met siektesimptome verkry. Soortgelyke resultate is met Imperial 45 en Amber in trog B verkry. Aanplanting van kultivar Fiata het egter 'n dramatiese verlaging in die voorkoms van Fusarium-verwelk bewerkstellig. Tydens die eerste Fiata aanplanting (aanplanting No.6) het 1.2% plante Fusarium-verwelksimptome ontwikkel, en 4% tydens die laaste aanplanting. Vanaf hierdie plante is slegs ras 1,2 isolate verkry. Hierdie bevindings, en die feit dat 'n aansienlike hoeveelheid van die Amber (ongeveer 7-15%) en Fiata plante (ongeveer 2%) siektesimptome getoon het, bewys dat FOM vinnig van virulensie verander wanneer die patogeen 'n weerstanbiedende kultivar infekteer. Die vermoë van RAPD analise om tussen isolate wat in virulensie verander het, te onderskei, is ondersoek. Vier isolate van F. oxysporum f. sp. niveum is as 'n buite-groep ingesluit. Om te bevestig dat die isolate wat van ras verander het wel egte vaskulêre patogene is, is 'n histopatologiese ondersoek gedoen. Die drie inleiers wat gebruik is, het die 12 FOM isolate duidelik van die vier F. oxysporum f. sp. niveum isolate onderskei. Die 12 FOM isolate wat drie fiosologiese rasse (ras 0, ras 2, ras 1,2) verteenwoordig het, is egter saam gegroepeer, wat aandui dat hierdie metode nie tussen rasse van FOM kan onderskei nie. Inokulasiestudies met die differensiële kultivars het die virulensie van die isolate bevestig. Die histopatologiese ondersoek het verder bewys dat beide FOM rasse (ras 2, ras 1,2) wat vanaf die wilde tipe ras ° isolaat ontstaan het, hul vermoë behou het om as egte vaskulêre patogene op te tree.

Thesis (MScAgric)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Botrytis cinerea Pers.: Fr., a pathogen of grapevine (Vitis vinifera L.), moves mainly through conidia by air currents in vineyards which are deposited intermittently on the surfaces of leaves, inflorescences and bunches. Little is known about the relationship between the inoculum dosage in air and incidence of Botrytis bunch rot, and how the relationship is influenced by environmental and host factors. To better understand this relationship, information is needed on the period over which conidia have accumulated, the time they are able to survive and remain infectious, time of symptom expression in relation to conidium arrival at the infection court and host surface wetness. The aims of this study were (i) to estimate the amount of viable B. cinerea occurring in air in vineyards, and at different positions on leaves, inflorescences and bunches of grape at different phenological stages, (ii) to determine the relationships between the number of B. cinerea colonies recorded on spore traps placed in the bunch zone of vines and the incidence of B. cinerea recorded from the different tissues, and (iii) to compare the efficacy of fenhexamid on leaves and inflorescences carrying natural B. cinerea inoculum with those inoculated with dry, airborne conidia. Different techniques were used to detect viable Botrytis cinerea in air currents and on plant material obtained from table (cultivars Dauphine and Waltham Cross in Paarl- and Worcester-district) and wine grape (cultivars Chardonnay, Sauvignon Blanc and Merlot in Stellenbosch- and Malmesbury district) vineyards in the Western Cape province during 2001-02 and 2002-03. For four consecutive days during prebloorn, bloom, pea-size, bunch closure, veraison and harvest, sets of Petri dishes with freshly prepared Kerssies' B. cinerea selective medium (spore traps) were left overnight in the bunch zone of vines. Plant material was collected from the vines on the fourth day. Leaves, infloresence and bunches were treated with paraquat to terminate host resistance and to promote the development of the pathogen on the tissues. The B. cinerea inoculum dosage in air, and the incidence at which the pathogen was detected at various positions on leaves and in bunches normally differed between vineyards. However, the various tests revealed that the pathogen generally occurred in a consistent pattern in air in the bunch zone of vines, on leaves and in bunches from all vineyards. The inoculum dosage in air in the bunch zone of the vine was generally highest during prebioom or during bloom, it decreased at pea size and mostly remained at a very low level at the later growth stages. The estimations of viable B. cinerea residing naturally on leaves and in bunches, showed that their amounts depicted levels occurring in air in the bunch zone of the vine. Necrotic leaves occurring early season in vineyards were identified as an important source of secondary inoculum for dispersal to the developing bunches. Latent infections at the various positions in bunches were few at véraison and harvest. However, due to the necrotrophic ability of the pathogen, extensive berry rot (due to berry-to-berry contact) and thus severe bunch rot developed from a single berry that become symptomatic at the base of the pedicel/berry attachment zone. The B. cinerea occupation pattern explains why Botrytis bunch rot develops mostly from the inner bunch and why disease management strategies should concentrate on the bloom to pre-bunch closure stage and on inhibiting B. cinerea development in the inner bunch during the early part of the season. Thus, to effectively reduce B. cinerea in grapevine, preventative applications are recommended to reduce two primary infection events: (a) between budding and pre-bloom to counteract primary leaf infection; (b) during late bloom or early pea size stage, to reduce the amount of the pathogen on leaves and infloresences and to prevent colonisation of floral debris. A third spray can be applied at bunch closure to reduce the amount of B. cinerea at various positions of the inner bunch, especially for cultivars with tight bunches. The efficacy of fenhexamid on leaves and inflorescences carrying natural B. cinerea inoculum was compared with those inoculated with dry, airborne conidia. Shoots were obtained during late bloom from a vineyard (wine grape cultivar Merlot) in the Stellenbosch region. The shoots were divided into two main groups. One group of shoots was left uninoculated, the other shoots were inoculated by dusting with dry B. cinerea conidia in a settling tower. Before inoculation, equal numbers of shoots in each main group was sprayed with fenhexamid, or left unsprayed. Following inoculation and incubation, shoots of each treatment were divided in two equal groups. The one lot of shoots were rinsed in water. The other lot of shoots were immersed in paraquat solution to terminate host resistance and to promote the development of the pathogen from the tissues. For both uninoculated and inoculated shoots, irrespective of fungicide treatment, leaves remained asymptomatic at both the blade and petiole position for the water rinse treatment. No symptom of B. cinerea decay developed at any of the positions on leaves from shoots sprayed with fenhexamid. Spraying of shoots with fenhexamid completely suppressed B. cinerea infection and symptom expression on both uninoculated and inoculated inflorescens. For inoculated shoots, B. cinerea developed from approximately 50% of the laterals in the water rinse treatment. However, inflorescences rinsed in water remained asymptomatic. The laboratory studies showed that fungicides, if applied properly to shoots and bunches under controlled conditions, effectively reduced the amount of B. cinerea at the various positions on leaves and inflorescence, and prevented infection and symptom expression at bloom. However, these goals are not achieved in vineyards where the fungicides are applied by conventional spraying methods. Therefore, more work is needed to evaluate fungicide application techniques by conventional spraying methods for proper fungicide coverage, and the reduction of B. cinerea in bunches. / AFRIKAANSE OPSOMMING: Botrytis cinerea Pers.: Fr., 'n patogeen van druiwe (Vilis vinifera L.), beweeg hoofsaaklik deur middel van konidia in lugstrome deur die wingerd, en word dan afwisselend op die oppervlakte van die blare, bloeiwyses en trosse gedeponeer. Daar is nog min bekend oor die verhouding tussen die hoeveelheid inokulum in die lug en die voorkoms van Botrytis op die trosse, en hoe die verhouding deur omgewings- en gasheerfaktore beïnvloed word. Ten einde hierdie interaksie beter te verstaan, word inligting benodig oor die tydperk waarin die konidia akkumuleer, die tyd wat hulle oorleef en virulent bly, en die tyd van simptoom-uitdrukking in verhouding tot die verspreiding van die konidia by die infeksie-setel en benatbaarheid van die gasheer-oppervlakte. Die doel van hierdie studie was (i) om die hoeveelheid lewensvatbare B. cinerea wat in die lug voorkom, asook by verskeie posisies op blare, bloeiwyses en trosse by verskillende fenologiese stadiums te kwantifiseer, (ii) om die verhouding tussen die aantal aangetekende B. cinerea kolonies op spoorvangers wat in die trossone van die wingerd geplaas is, en die voorkoms van B. cinerea, aangeteken van verskeie weefsels, te bepaal, en (iii) om die effektiwiteit van fenhexamid op blare en bloeiwyses wat natuurlike B. cinerea inokulum dra, te vergelyk met dié wat met droë, luggedraagde konidia geïnokuleer is. Verskillende tegnieke is gebruik om lewensvatbare Botrytis cinerea in lugstrome en op plantmateriaal van tafeldruiwe (kultivars Dauphine en Waltham Cross In Paarl- en Worcester-distrik) en wyndruiwe (kultivar Chardonnay, Sauvignon Blanc en Merlot in Stellenbosch- en Malmesbury distrik) in wingerde van die Wes-Kaap provinsie gedurende 2001-02 en 2002-03 te kwantifiseer. Petri bakkies met vars voorbereide Kerssies medium, selektief vir B. cinerea (spoorvangers), is vir vier agtereenvolgende dae gedurende vóórblom, blom, ertjiekorrel, trostoemaak, kleurbreek en oes, oornag in die trossone van wingerdstokke in betrokke wingerde, gelaat. Plantmateriaal is op die vierde dag versamel. Blare, bloeiwyses en trosse is met paraquat behandel ten einde die gasheerweerstand af te breek en ontwikkeling van die patogeen op die weefsel te bevorder. B. cinerea inokulum in die lug, en die frekwensie waarby die patogeen op verskeie posisies op blare en in die trosse voorgekom het, het normaalweg tussen wingerde verskil. Die verskeie toetse het getoon dat die patogeen normaalweg in 'n vaste patroon in die lug en die trossones van wingerde, asook op blare en in trosse van alle wingerde voorkom. Die inokulumkonsentrasie in die lug in die trossones van wingerdstokke was normaalweg die hoogste gedurende vóórblom of gedurende blom. Die inokulumdruk het by ertjiekorrel verminder en meestal by 'n 'n baie lae vlak tydens die latere groeistadia gebly. Die bepaling van lewensvatbare B. cinerea wat natuurlik op blare en in trosse gedeponeer is, het getoon dat hul hoeveelhede ooreenstem met vlakke wat in die lug in die trossone van die wingerd voorkom. Nekrotiese blare vroeg in die seisoen is 'n belangrike bron van sekondêre inokulum en speel dus 'n belangrike rol by die verspreiding van Botrytis tussen die ontwikkelende trosse. Latente infeksies by die verskeie posisies in trosse was laag by kleurbreek en oes. Weens die saprofitiese vermoëns van die patogeen, kan uitgebreide korrelvrot (a.g.v. korrel-tot-korrel kontak) en dus ernstige trosvrot, ontwikkel. 'n Enkele korrel kan by die basis van die pedisel/korrel vashegtingsone simptomaties raak, en vandaar na aangrensende korrels versprei. Die B. cinerea kolonisasiepatroon verduidelik waarom Botrytis trosvrot meestal vanaf die binneste tros ontwikkel en waarom siektebeheerstrategieë op die vóórblom- tot blomstadium gekonsentreer moet word, en op die inhibering van B. cinerea ontwikkeling in die binneste tros gedurende die vroeë stadia van die seisoen. Dus, om B. cinerea effektief tydens die twee primêre infeksie stadiums in wingerde te verminder, kan voorkomende toedienings aanbeveel word: (a) tussen knopvorming en vóórblom om primêre blaarinfeksie te verhoed; (b) gedurende láátblom en vroeë ertjiekorrel om die hoeveelheid inokulum op die blare en bloeiwyses te verminder, en die kolonisasie van blomdebris te voorkom. 'n Derde toediening kan tydens trostoemaak aangewend word om B. cinerea by verskeie posisies in die binneste tros te verminder, veral by kultivars met digte trosse. Die effektiwitiet van fenhexamid op blare en bloeiwyses waarop natuurlike B. cinerea inokulum voorkom is vergelyk met dié wat met droë, luggedraagde konidia geïnokuleer is. Lote is vanaf 'n wingerd (wyndruif kultivar Merlot) in die Stellenbosch distrik tydens láátblom verkry en in twee hoofgroepe verdeel. Die een groep lote is geïnokuleer deur droë B. cinerea konidia in 'n afsettingstoring te strooi, terwyl die ander groep nie geïnokuleer is nie. Vóór inokulasie, is die helfte van die lote in elke groep met fenhexamid behandel, terwyl die ander helfte onbehandeld gelaat is. Ná inokulasie en inkubasie, is lote van elke behandeling verder in twee eweredige groepe verdeel. Die een groep lote is in water gespoel, terwyl die ander groep lote in 'n paraquatoplossing gedompel is om die gasheerweerstand te verwyder, en die ontwikkeling van die patogeen vanuit die weefsels te bevorder. Vir die waterspoelbehandeling van beide ongeïnokuleerde en geïnokuleerde lote, ongeag van die fungisiedbehandeling, het die blare asimptomaties by beide die bladoppervlakte en blaarsteelposisie gebly. Geen simptome van B. cinerea verrotting het by emge van die blaarposisies van die lote, met fenhexamid gespuit, ontwikkel nie. Die spuit van die lote met fenhexamid het die B. cinerea infeksie en die simptoomontwikkeling op beide die ongeïnokuleerde en geïnokuleerde bloeiwyses heeltemalonderdruk. By die geïnokuleerde lote, het B. cinerea vanaf ongeveer 50% van die laterale in die waterspoelbehandeling ontwikkel, alhoewel, bloeiwyses wat in water afgespoel is, heeltemal asimptomaties gebly het. Laboratoriumstudies het getoon dat fungisiedes, indien korrek toegedien op lote en trosse onder gekontroleerde toestande, tot effektiewe vermindering van B. cinerea getalle by die verskillende posisies op blare en bloeiwyses lei, en infeksie en simptoomuitdrukking tydens blom voorkom. Weens die feit dat die doelwitte nie behaal kan word in wingerde waar die fungisiede deur konvensionele spuitmetodes toegedien is nie, moet meer studies gedoen word om fungisied toedieningstegnieke, by konvensionele spuitmetodes, VIr deeglike fungisiedbedekking en die vermindering van B. cinerea in trosse, te evalueer.

Thesis (PhDAgric)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Barley leaf scald, caused by Rhynchosporium secalis, is the most important disease of barley (Hordeum vulgare) in the Western Cape province of South Africa. The disease was first reported from South Africa in 1937. The present study is the first attempt to characterise the South African R. secalis population. Topics such as pathogenesisrelated proteins, virulence spectra, variability of pathotypes, sources of variation, host resistance, breeding strategies, molecular characterisation and fungicide sensitivity are summarised in Part 1 of this dissertation. In succeeding Parts the focus is on the characteristics of the local R. secalis population regarding virulence spectrum, DNA polymorphisms, in vitro as well as in vivo fungicide sensitivity. These aspects are treated as separate entities, leading to some duplication which is unavoidable. In Part 2 the virulence spectra of 50 R. secalis isolates from a population in the. Western Cape province were determined. Twenty-one races were detected using 17 differential barley cultivars. The two most prevalent races, namely races 4 and 7 had three and four virulence genes respectively. Both race 4 and 7 were virulent on the most susceptible cultivars, namely West China, Steudelli, C.I.8618 and C.I.2226. Considering the resistance genes reported for cultivars Atlas 46, Turk, and C.I.3515 which showed no susceptible cultivar-pathogen interaction, it would appear that the Rh- Rh3-Rh4 complex is primarily involved in conferring resistance to the local R. secalis isolates. A total of 20 races (47 isolates) characterised in Part 2 were selected for further characterisation by means of DNA fingerprinting. In Part 3 an anonymous multilocus DNA probe was used to characterise the genotypic structure of these isolates by means of RFLP analysis. No correlation between any particular fingerprint pattern, race, district, field or lesion was found. The two most prevalent races, 4 and 7, did not share the same genotypes, even when isolated from the same field or lesion. The genotypic diversity of the isolates studied was 46.5% of the theoretical maximum diversity. The high level of genotypic variation observed in the South African R. secalis population resembled the genotypic diversity observed in other cereal pathogens with known sexual structures. Although no teleomorph has yet been observed, these data suggest that sexual recombination may operate within the local population of R. secalis. In South Africa barley scald is primarily controlled by means of fungicides. The continued use of fungicides on cereal crops results in the build-up of fungicide resistance in the population, which could lower the efficacy of these compounds. These aspects were investigated in Part 4, where isolates (collected during 1993 to 1995) were evaluated in vitro for sensitivity to triadimenol, tebuconazole, flusilazole and propiconazole. The sensitivity fluctuated but in 1995 isolates were significantly less sensitive towards triadimenol than in the previous two years. In a second experiment, isolates collected from two fields with a 5-6 year-history of triadimenol seed treatments and tebuconazole applications, were evaluated for their fungicide sensitivity. A significant positive correlation was observed between tebuconazole and triadimenol sensitivity among,R. secalis populations from these fields. However, such a correlation was not found within the R. secalis population collected during 1993-1995 where shorter crop rotation patterns and a range of fungicides was applied. In a third experiment, the fungicide sensitivity of local R. secalis isolates was evaluated towards two new triazole fungicides, namely bromuconazole and triticonazole. Correlation coefficients observed between these new triazoles and those previously applied in South Africa were not significantly positive. The lack of significant cross-resistance has important practical implications regarding the management of fungicide resistance. In Part 5, isolates with different minimum inhibitory concentration (MIC) towards tebuconazole in vitro (1, 3 and 10 ug/ml) were compared in vivo. The aim of this study was to determine how MIC values would influence virulence (leaf area affected) and sporulation. Results indicated that all isolates were equally fit to induce lesions and sporulate in the absence of tebuconazole. Thus no fitness cost was associated with the degree of tebuconazole sensitivity in the present study. All R. secalis isolates were able to induce lesions on tebuconazole treated leaves, but differed significantly with respect to the percentage leaf area affected. Isolates, least sensitive (MIC = 10 ug/rnl) towards tebuconazole were more adapted on tebuconazole treated leaves, being able to repeatedly cause larger lesions than sensitive R. secalis isolates (MIC = 1 ug/rnl), Sporulation was not significantly different between isolates on lesions of untreated or tebuconazole treated leaves. Larger leaf areas affected and adequate sporulation suggest that a less sensitive population would result in more disease in tebuconazole treated fields. In conclusion, this study revealed the variability associated with the South African R. secalis population regarding virulence spectrum and genotypic structure. The data in this study suggest that it is likely that the local population will easily adapt to newly introduced, single gene resistance. For more durable resistance, higher levels of quantitative resistance should be introduced. This type of resistance is, however, more difficult to identify and incorporate than single gene resistance. Consequently, barley scald control will remain dependent on the efficacy of fungicide applications. Furthermore, the lack of cross-resistance and low frequency of resistant isolates indicates a low risk for the development of fungicide resistance in the local R. secalis population. Other factors such as current crop rotation practices and the range of fungicides being ~pplied also contribute to this low risk level. However, the status of these factors can change over time. The in vivo tebuconazole sensitivity study has indicated that a resistant field population of R. secalis may be able to build-up. It is, therefore, necessary to monitor the fungicide sensitivity of R. secalis isolates at timely intervals with view to successful barley cultivation in the future. / AFRIKAANSE OPSOMMING: Blaarvlek op gars (Hordeum vulgare), veroorsaak deur Rhynchosporium secalis, is die belangrikste siekte van gars in die Wes-Kaap provinsie van Suid-Afrika. Die voorkoms van R. secalis op gars is in Suid-Afrika vir die eertse keer in 1937 gerapporteer. Hierdie studie is die eerste poging tot karakterisering van die plaaslike R. secalis-populasie. Aspekte soos proteïene betrokke by patogenese, virulensiespektra, variabiliteit van patotipes, bronne van variasie, gasheerweerstand, teeltprogramme, molekulêre karakterisering en swamdodersensitiwiteit word in Deel I van die tesis opgesom. In die daaropvolgende gedeelte is die fokus op die karakterisering van die R. secalis-populasie en behels DNA karakterisering, virulensiespektrum, en swamdodersensitiwiteit in vitro asook in vivo. .. In Deel 2 is die virulensiespektra van 50 R. secalis isolate van 'n populasie in die. Wes-Kaap geëvalueer teenoor 17 differensiëel weerstandbiedende gars kultivars en hieruit is 21 rasse geïdentifiseer. Die twee mees algemene rasse (rasse 4 en 7), met onderskeidelik drie en vier virulensie gene, het virulent vertoon teenoor die mees vatbare kultivars soos West China, Steudelli, C.I.8618 en C.I.2226. Geen vatbare kultivar-patogeen interaksies is met kultivars Atlas 46, Turk en C.I.3515, wat al drie die Rh-Rh3-Rh4 kompleks dra, gevind nie. Dit wil dus voorkom asof hierdie genekompleks effektiewe gasheerweerstand teen die plaaslike R. secalis isolate kan bied. 'n Totaal van 20 rasse (47 isolate), gekarakteriseer in Deel 2, is geselekteer vir verdere karakterisering met behulp van DNA bandpatrone. In Deel 3 is 'n anonieme multilokus DNA peiler gebruik om deur middel van RFLP analise die genotipiese struktuur van hierdie R. secalis-isolate te bepaal. Geen assosiasie is gevind tussen DNA bandpatroon en ras, distrik, garsland of letsel nie. Die twee rasse (4 en 7) wat mees algemeen voorkom, het nie dieselfde bandpatroon vertoon nie, ook nie dié afkomstig vanuit dieselfde garsland of letsel nie. Die genotipiese diversiteit van isolate was 46.5% van die teoretiese maksimum diversiteit. Die hoë vlak van variasie waargeneem in die R. secalis populasie is soortgelyk aan variasie waargeneem in ander graanpatogene wat oor 'n geslagtelike stadium in die lewenssiklus beskik. Alhoewel geen geslagtelike stadium tot dusver geidentifiseer is nie, dui die vlak van variasie daarop dat geslagtelike rekombinasie moontlik wel plaasvind binne die plaaslike R. secalis populasie. In Suid-Afrika word blaarvlek op gars primêr deur swamdoders beheer. Die toenemende gebruik van swamdoders op graangewasse veroorsaak moontlik 'n opbou van swamdoderweerstand in die populasie. Dit kan die effektiwiteit van swamdoders verlaag. Hierdie veronderstelling is in Deel 4 ondersoek, waar die sensitiwiteit van isolate in vitro teenoor triadimenol, tebukonasool, flusilasool en propikonasool geëvalueer is. Die triasooi sensitiwiteit van R. secalis isolate wat gedurende die 1993- 1995 seisoen versamel is het gewissel en slegs vir triadimenol was daar 'n tendens na meer weerstandbiedenheid. 'n Swamdoder-evaluasie is in 'n aparte eksperiment op isolate gedoen wat versamel is vanaf twee garslande met 'n 5-6 jaar geskiedenis van triadimenol saadbehandelings en tebukonasool bespuitings. 'n Betekenisvolle positiewe korrelasie is waaJ~geneem tussen tebukonasool en triadimenol sensitiwiteit in R. secalis isolate afkomstig vanaf hierdie twee garslande. 'n Soortgelyke korrelasie is egter nie gevind in die populasie wat gedurende die 1993-1995 seisoene versamel IS me. Laasgenoemde kan moontlik toegeskryf word aan korter wisselboupatrone en die toediening van 'n verskeidenheid van swamdoders. In 'n derde eksperiment is die sensitiwiteit van plaaslike R. secalis isolate teenoor twee nuwe triasole, naamlik bromukonasool en tritikonasool getoets. Die korrelasie waargeneem tussen die twee nuwe triasole en triasooi swamdoders reeds voorheen in gebruik in die Wes-Kaap was me betekenisvol positief me. Die gebrek aan betekenisvolle kruisweerstandbiedendheid het belangrike praktiese implikasies vir die bestuur van swamdoder -weerstandbiedendheid. In Deel 5 is isolate met wisselende minimum inhiberende konsentrasies (MIKs) teenoor tebukonasool in vitro (1, 3 en 10 ug/ml) en in vivo vergelyk. Die doel van hierdie studie was om te bepaal hoe wisselende MIK-waardes virulensie (blaaroppervlakte geïnfekteer) en sporulasie sal beïnvloed. Resultate dui daarop dat alle R. secalis isolate in hierdie studie ewe fiks was om, in die afwesigheid van tebukonasool, letsels te induseer en te sporuleer. Die bevinding is dat die verlies in fiksheid nie geassosieer is met die mate van tebukonasool weerstand nie. Alle R. secalis isolate het die vermoë gehad om letsels op tebukonasool-behandelde blare te veroorsaak maar het betekenisvol verskil ten opsigte van die blaaroppervlakte geaffekteer. Isolate wat minder sensitief (MIK = 10 ug/rnl) teenoor tebukonasool in vitro is, het meer aangepastheid op tebukonasool-behandelde blare getoon. Gevolglik het hierdie isolate herhaaldelik meer letsels veroorsaak as sensitiewe isolate (MIK = 1 ug/ml), Sporulasie het nie betekenisvol verskil tussen isolate vanaf letsels op ondehandelde of tebukonsoolbehandelde blare nie. Hierdie resultate dui egter daarop dat 'n minder sensitiewe populasie tot meer siektevoorkoms in tebukonasool-bespuite lande kan lei. Die studie het die veranderlike karakter van die Suid-Afrikaanse R. secalispopulasie aangaande virulensiespektrum en genotipiese struktuur blootgelê. Dit is dus baie moontlik dat die R. secalis-populasie maklik sal aanpas by teelmateriaal met nuwe enkelgeen-weerstand. Vir volgehoue gasheerweerstand is dit egter nodig dat hoër vlakke van kwantitatiewe weerstand ingeteel moet word. In die praktyk is hierdie tipe weerstand egter baie moeiliker om te identifiseer en by nuwe teelmateriaal in te sluit as in die geval van enkelgeen-weerstand, Dit bring mee dat blaarvlekbeheer afhanklik bly van swamdodertoedienings as beheermaatreël. Die resultate van hierdie studie dui daarop dat daar tans 'n lae risiko vir die ontwikkeling van swamdoderweerstand in die plaaslike populasie is, as gevolg van die afwesigheid van kruisweerstandbiedendheid en die lae voorkoms van weerstandbiediende isolate. Ander faktore soos die wisselboustelsels wat toegepas word en die verskeidenheid van swamdoders toegedien dra ook daartoe by. Ten spyte hiervan kan die status van hierdie faktore egter oor tyd verander. Die in vivo tebukonasool studie het daarop gedui dat 'n weerstandbiedende veldpopulasie van R. secalis die potensiaal het om te vermeerder. Gevolglik is die tydige monitering van swamdodersenisitiwiteit van R. secalis isolate noodsaaklik om 'n volhoubare garsproduksie te verseker.

Fusarium circinatum is a fungal pathogen that has had a serious impact on pine production throughout the world. It attacks most Pinus species including Pinus elliottii, Pinus patula and Pinus radiata. Infections in South Africa (SA) are largely on seedlings, and result in fatal seedling wilt. Accurate and quick detection systems suitable for field use are needed to monitor the spread of the disease and optimize fungicide applications. Detection of F. circinatum is currently based on visual observations of typical symptoms. However, symptoms are not unique to the pathogen and can be caused by other biotic and abiotic stress factors. Nucleic acid-based identification techniques using PCR are available for different fungal species. These are sensitive and accurate, but they are expensive and require skilled biotechnologists to conduct the assays. In this study an enzyme-linked immunosorbent assay (ELISA) was developed to identify F. circinatum in infected seedlings. This optimized ELISA is able to discriminate between F. circinatum and two other fungi that frequently affect pine. This method has advantages over other assays because of its ease of operation and sample preparation, sensitivity and the ability to run multiple tests simultaneously. Mycelium-soluble antigens from Diplodia pinea (=Sphaeropsis sapinea), F. circinatum and F. oxysporum were prepared in nutrient broth. Analysis of these antigens on SDS-PAGE indicated the presence of common antigens between the different fungal pathogens. Some antigens were expressed more by some isolates than by others. Separate groups of chickens were immunised with mycelium-soluble antigens from D. pinea, F. circinatum and F. oxysporum and exo-antigen from F. circinatum prepared in nutrient broth. A 34 kDa protein purified from SDS-PAGE specific for D. pinea was also used for immunisation. Five sets of antibodies were obtained including anti-D. pinea, anti-F. circinatum, anti-F. oxysporum, anti-F. circinatumexo and anti-D. pinea 34 kDa antibodies, respectively. Reactivity of these antibodies was evaluated against antigens prepared in nutrient broth using western blotting and ELISA. Western blot analysis indicated that immuno-dominant antigens for F. circinatum were larger than 34 kDa and their reactivity was not the same between different isolates. Each of the antibodies prepared using mycelium-soluble antigens showed increased reactivity when detecting its own specific pathogen, but cross-reactivity was observed. Anti-D.pineaantibodies showed minimal cross-reactivity with antigens from F. circinatum and F. oxysporum. Anti-F. circinatum antibodies cross-reacted with antigens from F. oxysporum but showed little cross-reactivity with D. pinea antigens. Anti-F. oxysporum antibodies showed more cross-reactivity towards antigens from F. circinatum than those from D. pinea. No reactivity was observed when anti-F. circinatum-exo antigen and anti-D. pinea 34 kDa antibodies were used in immuno-blotting analysis. Evaluation of antibody reactivity using indirect ELISA showed patterns similar to those observed on western blotting, where anti-D. pinea, anti-F. circinatum and anti-F. oxysporum antibodies showed the same cross-reactivity relationships. Anti-F. circinatum and anti-F. oxysporumantibodies showed a significant difference when reacting with antigens isolated from other pathogens including D. pinea, F. circinatum, F. oxysporum, F. solani, F. graminearum and F. culmorum (P = 0.001). No significant difference was observed when the antigens were detected with anti-D. pinea antibodies. Reactivity of anti-F. circinatum-exo and anti-D. pinea34 kDa antibodies was mostly similar to that of non-immune antibodies and showed no significant difference between detection of different antigens. Pine seedlings were artificially infected with the three fungal pathogens using a spore concentration of 1 – 1 x 106conidiaml-1.Infection was monitored using scanning electron microscopy. Results showed increased levels of mycelium growth on the stem and roots of the F. circinatum and F. oxysporum infected seedlings and on the leaves and stem in the case of D. pinea infected seedlings. These plant parts were used in ELISA tests for the detection of antigens. Isolation of antigens from the plant materials involved crushing plant parts in buffer and centrifugation of the suspension. The supernatant obtained was directly used in the assay. ELISA tests prepared in this study were sensitive enough to detect infection caused by 1 conidium ml-1at two weeks post inoculation. A positive reaction for detection of F. circinatum and F. oxysporum was indicated by an ELISA reading above an optical density at 405 nm. The plant material used in ELISA tests were further analysed using PCR. Results indicated that there was no cross-infection between seedlings and served as a confirmation of the disease-causing pathogen. This indicated that cross-reactivity observed was due to other factors such as common epitopes on the major antigens. Use of an ELISA dip-stick or ELISA using these antibodies should provide an easy, fast field test to identify infections of pine, discriminating between F. circinatum, F. oxysporum and D. pinea. / M.Sc.Agric. University of KwaZulu-Natal, Pietermaritzburg 2013.

Thesis (MScAgric)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Soilborne diseases of grapevines represent a complex problem with limited information available, both locally and internationally. Previous research in South Africa indicated that Phytophthora and Pythium spp. were the most widespread and devastating pathogens in grapevine nurseries and vineyards in the Western Cape province. The local grapevine industry is currently expanding; new cultivars, methods and agricultural chemicals are being used which can affect soilborne pathogens. It has therefore become necessary to reassess the status of soilborne pathogens in nurseries, since information in this regard is crucial for the development of disease management practices for the expanding local grapevine industry. Soilborne fungal genera associated with roots and crowns of declining nursery grapevines were assessed in surveys conducted at three different grapevine nurseries in the Western Cape province. Cylindrocarpon, Fusarium, Pythium, and Rhizoctonia spp. were consistently isolated from roots and crowns of declining nursery grapevines. Cylindrocladiella spp. and Phytophthora cinnamomi were infrequently isolated from diseased roots, crowns and soil whereas Pythium spp. were abundant in most of the soils. Results suggest that the status of soilborne fungal pathogens in grapevine nurseries in the Western Cape province has changed over the last 30 years. The DNA phylogeny and pathogenicity of the isolates of Cylindrocladiella were determined. Four species of Cylindrocladiella occur on grapevines in South Africa, namely C. lageniformis, C. parva, C. peruviana, as well as a new species, described in this study as C. viticola, which forms part of the C. infestans species complex. Pathogenicity trials were inconclusive. Ten Fusarium spp. were isolated from roots and crowns of declining nursery grapevines, namely F. acuminatum, F. anthophilum, F. chlamydosporum, F. equiseti, F. nygamai, F. oxysporum, F. proliferatum, F. scirpi, F. semitectum and F. solani. The dominant species was F. oxysporum, followed by F. proliferatum and F. solani. In pathogenicity trials F. oxysporum and F. solani significantly reduced root volume, root dry mass, length of new shoots, stem diameter and number of leaves, but increased the percentage of chlorotic leaves and root rot severity. Fusarium proliferatum also caused a significant reduction in new shoot growth, number of leaves and increased root rot severity compared to the controls. Fusarium so/ani seems to be more virulent than F. oxysporum, followed by F. pro/iferatum. This is the first report of F. oxysporum, F. pro/iferatum and F. so/ani as pathogens of grapevines in South Africa, and the first report of F. proliferatum as a pathogen of grapevines in the world. Phytophthora cinnamomi was isolated at low frequencies from declined grapevines, although present in the rhizosphere soil. It is possible that the extensive use of downy mildew chemicals in grapevine nurseries may protect grapevines from infection by P. cinnamomi. The effect of chemicals used to combat downy mildew on Phytophthora root rot of nursery grapevines was evaluated in a glasshouse. There was very little discernable effect of the chemicals tested relative to the control plants for the parameters measured and it was concluded that the inoculation technique needed refinement. However, plants treated with phosphorous acid tended to be taller and have more leaves, greater stem diameter and root volume than controls or plants treated with the other chemicals. The data obtained in this study are not conclusive, but indicated certain trends that more glasshouse trials and field trials would resolve. Results presented in this thesis indicate that a major shift has occurred in the status of soilborne fungi associated with roots and crowns of grapevines in nurseries in the Western Cape since the 1970s when Phytophthora and Pythium were predominant. The prevalence and role of soilborne fungi need to be determined so that new appropriate disease management strategies can be developed to limit losses in grapevine nurseries and ensure the sustainable production of healthy plants for the grapevine industry. / AFRIKAANSE OPSOMMING: 'N ONDERSOEK NA GRONDGEDRAAGDE SWAMME GEASSOSIEER MET WORTELS EN KRONE VAN WINGERD IN KWEKERYE Grondgedraagde siektes van wingerd is 'n komplekse probleem waaroor min inligting, beide plaaslik en internasionaal, beskikbaar is. Vorige navorsing in Suid-Afrika het aangedui dat swamme van die genera Phytophthora en Pythium die mees algemene en vernietigende grondgedraagde patogene in kwekerye en wingerde in die Wes-Kaap provinsie is. Die plaaslike wingerdbedryf brei huidiglik uit; nuwe kultivars, metodes en landbouchemikalieë word gebruik wat 'n invloed kan hê op grondgedraagde patogene. Gevolglik het dit noodsaaklik geword om die status van grondgedraagde patogene in wingerdkwekerye weer te bepaal, aangesien inligting in hierdie verband noodsaaklik is vir die ontwikkeling van siekte bestuurspraktyke vir die ontwikkelende plaaslike wingerdbedryf. Grondgedraagde swamgenera geassosieer met wortels en krone van terugsterwende wingerd in kwekerye is bepaal in opnames wat by drie verskillende wingerdkwekerye in die Wes-Kaap provinsie uitgevoer is. Cylindrocarpon, Fusarium, Pythium, en Rhizoctonia spp. is konstant vanuit wortels en krone van terugsterwende wingerdplante in kwekery geïsoleer, Cylindrocladiella spp. en Phytophthora cinnamomi is ongereeld vanuit siek wortels, krone en grond geïsoleer, terwyl Pythium spp. algemeen in meeste gronde voorgekom het. Resultate dui daarop dat die status van grondgedraagde swampatogene in wingerdkwekerye in die Wes- Kaap provinsie oor die laaste 30 jaar verander het. Die DNA filogenie en patogenisiteit van die isolate van Cylindrocladiella is bepaal. Vier spesies van Cylindrocladiella kom voor op wingerd in Suid-Afrika, naamlik C. lageniformis, C. parva, C. peruviana, sowel as 'n nuwe spesie, wat in hierdie studie as C. viticola aangedui is en wat deel is van die C. infestans spesie kompleks. Patogenisiteits proewe was onvoldoende om die patogeniese status van die swam me te bepaal. Tien Fusarium spp. is vanuit wortels en krone van terugsterwende wingerdplante in kwekery geïsoleer, naamlik F. acuminatum, F. anthophilum, F. chlamydosporum, F. equiseti, F. nygamai, F. oxysporum, F. proliferatum, F. scirpi, F. semitectum en F. solani. Die dominante spesies was F. oxysporum, gevolg deur F. proliferatum en F. solani. In pathogenisteitsproewe het F. oxysporum en F. solani gelei tot 'n betekenisvolle laer wortelvolume, droë massa van wortels, lengte en droë massa van nuwe groei en aantal blare, maar het die persentasie chlorotiese blare en graad van wortelvrot verhoog. Fusarium proliferatum het ook gelei tot 'n betekenisvolle afname in lengte en massa van nuwe groei, aantal blare en 'n verhoogde graad van wortelvrot in vergelyking met die kontrole behandelings. Dit wil voorkom asof Fusarium solani meer virulent is as F. oxysporum, gevolg deur F. proliferatum. Hierdie is die eerste aanmelding van F. oxysporum, F. proliferatum en F. solani as patogene van wingerd in Suid-Afrika, en die eerste aanmelding van F. proliferatum as 'n patogeen van wingerd in die wêreld. Phytophthora cinnamomi is konstant teen lae frekwensies vanuit terugsterwende wingerd in kwekerye geïsoleer, alhoewel dit in risosfeer gronde teenwoordig was. Dit is moontlik dat die ekstensiewe gebruik van chemikalieë teen donsskimmel in wingerdkwekerye die wingerdplante kan beskerm teen infeksie deur P. cinnamomi. Die effek van chemikalieë wat gebruik word teen donsskimmel op Phytophthora wortelverrotting van wingerd in kwekerye, is 'n glashuis geëvalueer. Die chemikalieë wat gestoets is, het vir die gemete parameters, tot baie min onderskeibare effek gelei relatief tot die kontrole plante, en daar is afgelei dat die inokulasie tegniek verbetering benodig. Plante wat met fosforiensuur behandel is, het egter geneig om langer te wees met meer blare, 'n groter stamdeursnee en wortelvolume as kontrole plante of plante behandel met ander chemikalieë. Data verkry vanuit die hierdie studie was onvoldoende, maar sekere neigings is aangedui wat deur verdere glashuis- en veldproewe verklaar sal word. Resultate wat in hierdie tesis weergegee is, het aangedui dat 'n algehele verskuiwing in die status van grondgedraagde swamme geassosieer met wortels en krone van wingerd in kwekerye vanaf die 1970s, toe Phytophthora en Pythium die dominante genera was, plaasgevind het. Die voorkoms en rol van grondgedraagde swamme moet bepaal word, sodat nuwe voldoende siektebestuurspraktyke ontwikkel kan word om verliese in wingerdkwekerye te beperk en sodoende die volhoubare produksie van gesonde plante vir die wingerdbedryf te verseker.

The Sugarcane Industry contributes approximately 400 000 jobs and ZAR 8 billion annually to South Africa’s economy. Due to climate change and the subsequent threat posed by disease, these figures have been on the decline. Brown rust, a contributor to this decline is caused by the basidiomycete Puccinia melanocephala Syd. and P. Syd., which previously resulted in 50% yield losses in susceptible varieties. This highlighted the need for improved screening and breeding techniques which will result in the replacement of susceptible varieties. The objectives of this study were to: a) Adopt and optimise a glasshouse whorl inoculation screening technique applicable for mass screening of large populations. b) Develop a rapid and cost effective rust resistance screening technique using detached leaves. c)Utilise two flanking marker sets (R12H16 and 9O20-F4-PCR primers) for the rust resistance Bru1 gene in a diagnostic polymerase chain reaction (PCR) to identify rust resistant genotypes lacking Bru1 and possessing either quantitative resistance or an alternative major qualitative resistance gene. d) Correlate rust phenotypic data to AFLP marker data for the Linkage Disequilibrium (LD2) mapping population. e) Utilise suppression subtractive hybridization (SSH) profiling on rust challenged genotypes to discover differentially expressed genes between susceptible and resistant (susceptible Bru1 negatives taken away from resistant Bru1 negatives); and resistant genotypes (resistant Bru1 positives taken away from resistant Bru1 negatives). 4 Results from the glasshouse whorl inoculation trials showed the technique could be reliably used to screen large populations, as two independently conducted pot trials showed highly correlated rust ratings. A visually assessed detached leaf assay (DLA) was developed using selected genotypes. Chlorophyll fluorescence and SPAD readings were used in the DLA to determine the leaf photochemical efficiency (PIABS) with relation to chlorophyll content, resulting in reduced assessment time of at least two days. PCR diagnostics revealed 31% of LD2 did not possess either flanking marker, 8% had one or the other marker, and 61% had both markers. The overall rust phenotypic ratings (rating scale of 0-10) and Bru1 status of the genotypes was used to group the population, with the Bru1 negative genotypes containing all three rating categories (resistant 0-3.5; intermediate 3.51-6.5; susceptible 6.51-10); while the Bru1 positive genotypes were all resistant. The phenotypic data was correlated to AFLP data using the Pearson product-moment correlation coefficient and stepwise multiple linear regression employed to build marker based models to use for predicting non-Bru1 mediated resistance. SSH analysis was then subsequently conducted on genotypes selected on the basis of Bru1 status and AFLP correlation data. Two subtraction cDNA libraries were constructed and the cDNA inserted into electro-competent Escherichia coli cells. PCR on transformed cells revealed cDNA inserts ranging from 200- 1300bp. BLAST analysis of the cDNA sequences indicated the presence of high proportions of disease and drought stress related sequences in the libraries. Analysis of the sequences in both libraries showed that the resistant Bru1 negative genotypes contained oxidative stress related sequences which were however absent in the Bru1 positive resistant genotypes. The library comparing the Bru1 negative resistant genotypes against the Bru1 negative intermediate and susceptible genotypes showed a higher proportion of differentially expressed sequences coding for putative disease resistance proteins, highlighting their presence in the resistant genotypes. Both subtraction libraries also contained high proportions of a leucine rich repeat protein coding cDNA which contained a conserved domain homologous to that of a disease resistance protein conferring resistance to Pseudomonas syringae in Arabidopsis thaliana. The outcomes of this study will subsequently enable an improved understanding of sugarcane-rust resistance mechanisms and improved breeding and screening techniques for sugarcane by identifying SSH and AFLP markers linked to rust resistance QTLs or alternative R genes. / Thesis (M.Sc.Agric)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

The first serious outbreak of brown rust of sugarcane caused by Puccinia melanocephala Syd. & P. Syd. was reported in India in 1907. It was first reported in South Africa (SA) in 1941 on the variety Co301 and is now present in almost all the sugarcane growing areas of the world. In SA, it is now described as an important disease of sugarcane, causing yield losses of up to 26% in susceptible varieties. Within the SA sugar industry, rust is controlled through the use of resistant varieties as it is the most economical method of control. However, most of the newer varieties that are being released have an intermediate resistance rating for rust. An integrated management approach for the control of rust is therefore being investigated. Aspects investigated in this study included environmental conditions required for development of the disease i.e. epidemiology, the use of silicon (Si) as a cultural control method against brown rust and identification of gene sequences expressed in response to brown rust infection. For the epidemiology study, inoculated plants were incubated in a dew chamber at different temperatures and leaf wetness periods. The choice of leaf wetness duration and temperature was based on urediniospore germination studies. The optimum temperature for urediniospore germination and disease development at > 98% relative humidity was found to be between 20 and 25°C with nine hours of leaf wetness. Silicon has been shown to reduce the incidence of diseases and pests in a number of crops. The ability of sugarcane to accumulate Si and the location of Si deposition was established using two uptake and deposition trials. Different concentrations of Si were applied to the plant and accumulation in the roots, stalks, old leaves and young leaves was determined using inductively coupled plasma optical emission spectrometry, with accumulation found to be roots > old leaves > stalks > young leaves. Silicon deposition in the leaves was determined using energy dispersive X-ray mapping on freeze dried specimens and significant differences were found between the upper epidermis, lower epidermis and mesophyll with the most Si being deposited in the lower epidermis. For disease severity, plants were naturally infected with rust and rated weekly. A significant decrease in disease severity and area under disease progress curve was noted when the Si concentration increased, indicating that Si has potential in reducing rust incidence. Currently, the most reliable and economical method of managing brown rust is with the use of resistant varieties. Identification of resistance within breeding lines is therefore important. For this part of the study, suppression subtractive hybridization was used as a tool to identify differentially expressed genes between a susceptible and resistant variety and a susceptible and intermediate variety, in response to brown rust infection. Two efficient subtracted cDNA libraries were generated and differentially expressed sequences were identified within each library. The results of this study show potential for the development of molecular markers which could be used for the early identification of brown rust resistance during the breeding process. This study forms a firm basis on which an integrated management strategy, for the management of brown rust in the SA sugar industry, could be designed. The cDNA sequences identified could be further investigated and used to develop molecular markers to select for rust resistant varieties, the epidemiology results together with further field data could be used to develop a disease prediction model and Si has potential in the field to reduce brown rust severity. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Soybeans, Glycine max, are an economically and strategically important crop in South Africa (SA). In order to meet local demands, large imports of soybeans are required, e.g., in the 2005/2006 soybean production period, 842 107 tonnes of oilcake were imported. Due to an increase in soybean production throughout the world, diseases that affect this crop have also increased in incidence and severity. Sclerotinia sclerotiorum, the causal organism of sclerotinia stem rot (SSR), is an important yield limiting disease of soybeans, as well as numerous other crops. The pathogen was first reported in SA in 1979. However, it was only in 2002 that this fungus was considered a major pathogen of soybeans in SA. The research reported in this thesis was conducted to investigate the epidemiology of S. sclerotiorum and examine numerous potential control methods for this pathogen, i.e., resistant cultivars, biocontrol, chemical control and seed treatments. A S. sclerotiorum isolate was obtained from sunflowers in Delmas, Mpumulanga, SA, in the form of sclerotia. This isolate was cultured and sent for identification and deposition in the Plant Protection Research Institute collection. This isolate, in the form of mycelia, was used for the duration of the study. For epidemiology studies, the effect of temperature, leaf wetness duration (LWD) and relative humidity (RH) were examined for their effect on rate of pathogen development. Twenty four combinations of temperature (19°C, 22°C, 25°C and 28°C), LWD (24, 48 and 72 hr) and RH (85 and 95%) were investigated. No interaction between temperature, LWD and RH was found. Temperature alone was the only factor that affected disease development. At 22°C, the rate of pathogen development (0.45 per unit per day) was significantly higher than all other temperatures, indicating that this temperature is optimum for disease development. Thirteen different soybean cultivars, i.e., LS6626RR, LS6710RR, LS666RR, LS555RR, LS6514RR, LS678RR, Prima 2000, Pan 626, AG5601RR, AG5409RR, 95B33, 95B53 and 96B01B, commercially grown in SA were investigated for their reaction to S. sclerotiorum. Prima 2000, 96B01B, 95B33 and AG5409RR were considered to be the least susceptible as they showed a significantly low rate of pathogen development (0.28, 0.28, 0.24, 0.23 per unit per day, respectively) and produced a significantly low number of sclerotia (3.03, 3.42, 3.21, 2.38, respectively). LS6626R and LS666RR may be considered most susceptible because of their significantly high rate of pathogen development (0.45 and 0.42 per unit per day, respectively) and high sclerotia production (8.16 and 7.50, respectively). Regression analysis showed a positive correlation coefficient (R2=0.71) between rate of growth of the pathogen and number of sclerotia produced, indicating that a higher rate is associated with a higher number of sclerotia. In vitro dual culture bioassays were performed to identify the biocontrol mechanisms of the biocontrol agents, EcoT® (a seed treatment) and Eco77® (a foliar treatment), against hyphae and sclerotia of S. sclerotiorum. Ultrastructural studies revealed that mycoparasitism is the probable mode of action as initial signs of hyphae of EcoT® and Eco77® coiling around hyphae of S. sclerotiorum were observed. Surface colonization of sclerotia by hyphae of EcoT® and Eco77® was also observed. In vitro antagonism of EcoT® against S. sclerotiorum on soybean seed was performed to determine pre-emergence and post-emergence disease. There was no significant difference in percentage germination between seeds treated with EcoT® and plated with the pathogen, untreated seeds and no S. sclerotiorum, and the control (i.e. no EcoT® and no pathogen). However, percentage non infected seedlings from seeds not treated with EcoT® was significantly lower, suggesting that EcoT® may be successfully used as a seed treatment for the control of SSR. In vivo trials were performed to investigate the effect of silicon (Si) alone, and in combination with Eco77®, on the effect of the rate of disease development. Plants treated with Eco77® had a significantly lower rate of disease development (0.19 per unit per day for plants treated with Eco77® and S. sclerotiorum and 0.20 per unit per day for plants treated with Eco77®, S. sclerotiorum and Si), compared to plants not treated with Eco77® (0.29 per unit per day for plants treated with S. sclerotiorum and 0.30 per unit per day for plants treated with S. sclerotiorum and Si), regardless of the application of Si. Similarly, plants treated with Eco77® had a significantly lower number of sclerotia (0.46 for plants treated with Eco77® and S. sclerotiorum and 0.91 for plants treated with Eco77®, S. sclerotiorum and Si), compared to plants not treated with Eco77® (3.31 for plants treated with S. sclerotiorum and 3.64 for plants treated with S. sclerotiorum and Si). The significantly lower rate of disease development coupled with a significant reduction in sclerotia showed that Eco77®, and not Si, was responsible for reducing the severity of SSR. A strong positive correlation between rate of disease development and the number of sclerotia produced (R2=0.79) was observed. For the investigation of various fungicides for the control of S. sclerotiorum, in vitro trials to determine the potential of three different fungicides at different rates, i.e., BAS 516 04F (133 g a.i. ha-1), BAS 516 04F (266 g a.i. ha-1), BAS 512 06F (380 g a.i. ha-1) and Sumisclex (760 g a.i. ha-1) were initially conducted. The control (non-amended PDA) had a significantly higher area under mycelial growth curve (243.0) than all fungicides tested. BAS 516 04F (at both concentrations) and BAS 512 06F completely inhibited the mycelial growth of S. sclerotiorum. Sumisclex inhibited the fungus by 89.07%. For in vivo trials, preventative treatments, i.e., BAS 516 04F (133 g a.i. ha-1), BAS 516 04F (266 g a.i. ha-1), BAS 512 06F (380 g a.i. ha-1), curative treatment, i.e. Sumisclex (760 g a.i. ha-1) and a combination preventative/curative treatment, i.e., BAS 512 06F (380 g a.i. ha-1)/Sumisclex (570 g a.i. ha-1) were investigated. No significant difference in disease severity index (DSI) was found between fungicide treatments and the inoculated control. BAS 512 06F and BAS 512 06F/Sumisclex had significantly lower grain yields (6.09 g and 5.96 g, respectively) compared to all other treatments. There was a positive correlation coefficient (R2=0.76), between DSI and grain yield, indicating that a high DSI is correlated with low grain yield. Trials to evaluate the effect of commercially available and currently unregistered seed treatments for the control of S. sclerotiorum on soybean seeds in vivo and in vitro were performed. Seed germination tests were performed to determine if seed treatments had any negative effects on seed germination in vitro. All seed treatments tested, i.e., BAS 516 03F (8, 16 and 32 ml a.i. 100 kg-1 seed), BAS 512 00F (7.5, 15 and 32 ml a.i. 100 kg-1 seed), Celest XL (100, 125, 200 and 250 ml a.i. 100 kg-1 seed), Sumisclex (5 and 10 ml a.i. 100 kg-1 seed), Benomyl (150 g a.i. 100 kg-1 seed), Captan (240 ml a.i. 100 kg-1 seed), Thiulin (180 g a.i. 100 kg-1 seed) and Anchor Red (300 ml a.i. 100 kg-1 seed), showed no negative effect on seed germination. For in vivo trials, BAS 516 03F (16 and 32 ml a.i. 100 kg-1 seed), BAS 512 00F (7.5, 15 and 32 ml a.i. 100 kg-1 seed), Celest XL (100, 125, 200 and 250 ml a.i. 100 kg-1 seed), Sumisclex (5 and 10 ml a.i. 100 kg-1 seed), Benomyl and Anchor Red had significantly similar percent germination and percent seedling survival as the untreated/uninoculated control. These seed treatments should be recommended for the control of S. sclerotiorum, as they protected seed during germination and subsequent seedling development. BAS 516 03F (8 ml a.i. 100 kg-1 seed) should not be recommended for the control of SSR, as it gave the lowest percent germination and percent seedling survival. The results presented in this thesis have helped to identify optimal environmental conditions for the development of S. sclerotiorum, which is important for the development of forecasting models for disease control. The least and most susceptible cultivars of those tested have been identified. Biocontrol using Eco77® as a foliar application showed great potential. The effect of Si needs to be further investigated, including the testing of more frequent applications and higher concentrations. The fungicides tested in this research did not show any potential for the control of SSR. However, the spray programme tested is for the control of soybean rust (Phakopsora pachyrhizi), and was investigated for its potential for the control of SSR. The spray programme, fungicide application and rating scale needs to be modified, to determine the true potential of these fungicides for the control of SSR. Numerous seed treatments have shown potential for the control of seed infection by SSR. Due to difficulties in producing ascospores, which are the primary source of inoculum for this pathogen in the field, all studies in this research were conducted with mycelia and not ascospores. The production, collection and storage of ascospores needs to be thoroughly investigated, and research conducted with ascospores. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.