No description available.

The state of leaf health of intact Penneagle creeping bentgrass leaves into which Curvularia lunata was able to ingress was determined by reducing cuticle/wax formation with trichloroacetic acid (TCA) and stressing plants with high air temperatures. Plants were grown until the third leaf had fully expanded and the fourth leaf had not yet emerged from the whorl. Plants were then treated with TCA for 6 times on alternate days. Half of the plants were high air temperature stressed at 38°C for 18 hr before plants were inoculated. Leaf health was estimated throughout the growing period of the plants by extracting chlorophylls and then regressing the values with respect to time. In this manner, leaves at each nodal position could be classified as either juvenile, mature or senescent. Also, selected leaves were examined by scanning electron microscopy. The addition of TCA to plants decreased leaf and plant life, increased tillering and reduced the deposition of leaf surface waxes. High air temperature stressing the plants caused a rapid entry of the leaf into senescence and higher levels of TCA accelerated this process. Plants were inoculated with either C. lunata, C. lunata var. aerea or Drechslera sorokiniana. Histochemical techniques were used to determine if penetration of the fungus into plant tissue had occurred. Inoculation with D. sorokiniana resulted in lesion formation within 2 days. Symptoms commonly attributed to Curvularia blight were present on plants treated with 0.047 or 0.14 mM TCA and then high air temperature stressed and inoculated with C. lunata. Histochemical procedures failed to show the presence bf mycelium Qf C. lunata· within the cells of Curvularia blight symptom areas. In a separate study, plants were grown, clipped and maintained at 2.0 cm and grown until 30 or 120 days of age. Plants were high air temperature stressed or not and clipped 128, 64, 32, 16, 8, 2, 1 or 0 hr before inoculation with C. lunata. Results again showed that C. lunata had the ability to colonize heat stressed and/or old leaves but did not have the ability to infect and colonize juvenile or mature tissues. The amount of the turf foliage that is susceptible to thinning by C. lunata depends on the physiologic age of the leaf tissue. As stresses of high air temperatures are placed on the leaf tissue, a greater percentage of the leaf blades are forced into advanced senescence, thereby increasing their susceptibility to infection and colonization by C. lunata. / Ph. D.

Colonization of Pine Christmas trees by Verticicladiella procera Kendr. causes Procera root disease. Little is presently known regarding the pattern and effects of fungal development within colonized trees. The present studies were undertaken to elucidate the developmental pattern of the fungus in colonized trees, to gather information on possible mechanisms and physiological effects of disease development, and to explore the relationship between V. procera and other, well documented bluestain fungi. The presence of cellulose was demonstrated in the cell walls of X. procera, indicating the probable genetic relatedness of this fungus with Ophiostoma (Ceratocystis) bluestain fungi. Inoculation studies revealed that the fungus could penetrate wounded sapwood, and that colonized seedlings had lower water potentials than uncolonized seedlings. In addition, it was found that the fungus could persist in resinous stem lesions for 22 months without foliar symptoms, and resinous stem lesions with the fungus were significantly longer and deeper than wound lesions. An intensive isolation study revealed that the initial point of colonization in a tree is apparently at the root collar, progressing acropetally in both directions. Analysis of radial growth from increment cores showed that colonized trees had grown more slowly for the preceding three years than uncolonized trees. The sapwood moisture content of these cores was also significantly reduced in the colonized trees, indicating that the stem was drying out as symptoms developed. Histological examination of colonized sapwood showed that U fungal colonization of tissues progressed along rays and resin ducts, in a fashion similar to that of bluestain fungi. Permeability measurements demonstrated that symptomatic sapwood, either resin-soaked or black-stained, had significantly reduced water movement relative to asymptomatic sapwood. / Ph. D.

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

Thesis (MSc (Plant Pathology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Esca is a disease affecting grapevines and is potentially devastating as there are economic losses due to a decrease in yield, wine quality and berry quality. Vineyards also need to be replaced earlier and therefore esca has a great impact on the wine, table grape and raisin industries. The disease is known to affect vineyards worldwide and has been studied extensively in Europe, but not in South Africa. Esca diseased grapevines were observed for the first time prior to 1981 in South African vineyards. The disease is primarily caused by Phaeoacremonium aleophilum, Phaeomoniella chlamydospora (both causing brown and black wood streaking) and white rot basidiomycete species such as Fomitiporia mediterranea which cause wood rot in the trunks and arms of generally older grapevines. Species of the Botryosphaeriaceae and Phomopsis (mainly Phomopsis viticola) and Eutypa lata have also been isolated from esca diseased vines, but their association with esca is unclear. Some of the symptoms associated with the disease on most grapevine cultivars include ‘tiger-stripe’ foliar symptoms, apoplexy and berry symptoms such as shriveling, discoloration and ‘black measles’. These external symptoms as well as internal symptoms are thought to be a result of toxin and enzyme production by the fungi involved. Symptom expression is erratic and varies from year to year making investigations into the causal fungi and the toxins and enzymes secreted in planta difficult. Vines with internal or external symptoms of esca were sampled in this study from table and wine grape cultivars in 37 towns in the Western Cape, Northern Cape and Limpopo provinces. The majority of sampled vines were over ten years of age, but vines as young as two to three years were also found to be infected. The external symptoms included dieback, tiger striped leaves, berry symptoms (shriveling, insufficient colouring and black spots) and apoplexy. These symptoms resembled those found on grapevines in Europe, Australia and the USA. The internal symptoms found were also similar to European symptoms and included white rot, black and brown wood streaking, brown necrosis within white rot, sectorial brown necrosis and central brown/ red/ black margin. The fungi mostly isolated from the white rot were the basidiomycetes. Black and brown wood streaking was primarily caused by Phaeomoniella chlamydospora. Brown necrosis within the white rot was caused by Phaeomoniella chlamydospora and less frequently by Phaeoacremonium spp., Eutypa lata, Botryosphaeriaceae and Pleurostomophora richardsiae. The sectorial brown necrosis and the central/ brown/ red/ black margin were dominated by Phaeomoniella chlamydospora. The fruiting bodies of the basidiomycetes were found on only a few grapevines. The fungal species associated with the internal wood symptoms were characterized on cultural growth patterns, morphology as well as phylogenetic inference. The gene areas sequenced included the internal transcribed spacers and the 5.8S rRNA gene for the basidiomycetes and Phomopsis isolates, the partial b-tubulin gene for Phaeoacremonium isolates and the partial translation elongation-1a gene for the Botryosphaeriaceae isolates. The basidiomycete isolates fell into ten taxa within the Hymenochaetales of which two could be linked to known genera, namely Fomitiporia and Phellinus. The ten basidiomycete taxa do not correspond to any published sequences. Eutypa lata, Diaporthe ambigua, Diplodia seriata, Neofusicoccum australe, Neofusicoccum parvum, Phomopsis viticola, Phomopsis sp. 1, Phaeomoniella chlamydospora and six species of Phaeoacremonium including P. aleophilum, P. alvesii, P. parasiticum, P. iranianum, P. mortoniae and P. sicilianum were also isolated of which the latter three are reported for the first time in South Africa. To understand the role of the basidiomycetes in the complex, toxin and enzyme analyses was determined for these fungi. Selected basidiomycete isolates were grown up in liquid broth and extractions performed to test for the presence of 4-hydroxy-benzaldehyde. All of the basidiomycete isolates were able to produce this toxin which is known to be phytotoxic. The basidiomycetes were then tested for the presence of certain wood degrading enzymes. All of the taxa were able to produce manganese peroxidase. Laccase was produced by all taxa, except Taxon 8. Lignin peroxidase was produced by Taxa 1, 2, 7, Fomitiporia sp. and the Phellinus sp. All the basidiomycete isolates were able to produce cellulose and none were able to produce xylanase. These enzyme tests showed that the basidiomycetes produce a wide variety of enzymes which are able to degrade cellulase and lignin which are both structural components of wood. Given the wide distribution of esca in the grape growing regions investigated in South Africa and the diverse amount of species found, this disease must surely be seen as a limiting factor to the productive lifespan of vineyards and quality of produce. Preventative measures such as sanitation and pruning wound protection contribute to the management of the disease, but many questions still remain about the synergy of the causal fungi, epidemiology and management of esca. / AFRIKAANSE OPSOMMING: Esca is ‘n wingerd siekte wat potensieel skade kan aanrig as gevolg van ekonomiese verliese weens verlaagde opbrengs, wyn kwaliteit en vrug kwaliteit. Wingerde moet ook vroeër vervang word en daarom het esca ’n groot impak op die wyn, tafeldryf en rosyne industrieë. Esca word wêreldwyd gevind op wingerd en is al intensief nagevors in Europa, maar nog nie in Suid-Afrika. Esca is vir die eerste keer in die 1980’s in Suid-Afrikaanse wingerde gerapporteer. Die primêre veroorsaakende organismes van esca is Phaeoacremonium aleophilum, Phaeomoniella chlamydospora wat bruin en swart vaatweefsel verkleuring veroorsaak en basidiomycete spesies soos Fomitiporia mediterranea wat wit verotting veroorsaak in die stam en arms van ouer wingerd. Spesies van die Botryosphaeriaceae en Phomopsis (hoofsaaklik Phomopsis viticola) en Eutypa lata is ook al vanaf esca simptome geïsoleer, maar hul assosiasie met die siekte is nie duidelik nie. Algemene simptome wat voorkom op die meeste wingerd kultivars met esca sluit in ‘tiger-stripe’ blaar simptome, apopleksie en vrug simptome soos verdroging, verkleuring en spikkels (black measles). Interne en eksterne simptome kan wees as gevolg van toksiene en ensiem produksie van die swamme wat betrokke is by esca. Eksterne simptoom uitdrukking is wisselvallig en varieer van jaar tot jaar. Dit bemoelik die bestudering van die swamme en die toksiene en ensieme wat afgeskei word in planta. Wingerd monsters met eksterne en interne simptome is versamel van tafel en wyndruif kultivars in 37 dorpe in die Wes-Kaap, Noord-Kaap en Limpopo provinsies. Die meerderheid monsters was ouer as tien jaar maar wingerde wat twee tot drie jaar oud was, was ook gevind. Die eksterne simptome wat op hierdie kultivars gevind is het terugsterwing, ‘tiger striped’ blare, vrug simptome (verkrimping en onvoldoende verkleuring) en apopleksie ingesluit. Hierdie simptome stem ooreen met soortgelyke simptome gevind op wingerd in Europa, Australië en die VSA. Interne simptome was ooreenstemmend met simptome wat gevind word in Europa. Die interne simptome het wit verotting, bruin en swart streepvorming, bruin nekrose met wit verotting, sektoriale bruin nekrose en sentrale bruin/ rooi/ swart kante ingesluit. Basidiomycete swamme is meestal uit die wit verotting gedeeltes geïsoleer. Swart en bruin hout streepvorming was meestal deur Phaeomoniella chlamydospora veroorsaak. Bruin nekrose binne die wit verotting was meestal deur Phaeomoniella chlamydospora veroorsaak en in ‘n mindere mate deur Phaeoacremonium spp., Eutypa lata, Botryosphaeriaceae en Pleurostomophora richardsiae. Phaeomoniella chlamydospora was die hoof veroorsakende organisme van sektoriale bruin nekrose en die sentrale bruin/ rooi/ swart kante. Vrugliggame van die basiodiomycete is op enkele wingerde gevind. Swam soorte wat geassosieer word met die interne hout simptome was verder gekarakteriseer op kultuur groei, morfologiese eienskappe, en filogenetiese analise. Die geen areas waarvan die basis paar volgorde bepaal was sluit in die interne getranskribeerde spasies en die 5.8S rRNA geen vir die basidiomycete en Phomopsis isolate, die gedeeltelike btubulien geen vir Phaeoacremonium isolate en die gedeeltelike translasie velenging-1a geen vir die Botryosphaericeae isolate. Die basidiomycete isolate was versprei oor tien taksons binne die Hymenochaetales waarvan twee genusse gekoppel kon word aan die genera Fomitiporia en Phellinus. Die tien basidiomycete taksons kom nie ooreen met enige gepubliseerde DNS volgordes. Eutypa lata, Phomopsis viticola, Phomopsis sp. 1, Diaporthe ambigua, Diplodia seriata, Neofusicoccum parvum, Neofusicoccum australe, Phaeomoniella chlamydospora en ses spesies van Phaeoacremonium insluitend P. aleophilum, P. alvesii, P. parasiticum, P. iranianum, P. mortoniae en P. sicilianum is ook geïsoleer. Hierdie is die eerste keer dat P. iranianum, P. mortoniae en P. sicilianum in Suid-Afrika gerapporteer word. Om die rol wat die basidiomycete in die siekte-kompleks speel beter te verstaan is toksien en ensiem analises uitgevoer. Geselekteerde basidiomycete isolate is gekweek in vloeibare groei medium en ekstraksies uitgevoer om te toets vir die teenwoordigheid van 4- hydroxy-benzaldehyde. Al die basidiomycete isolate kon 4-hydroxy-benzaldehyde, wat bekend is om fitotoksies te wees, produseer. Die basidiomycete isolate was verder getoets vir die produksie van spesifieke hout afbrekende ensieme. Al die basidiomycete taksons kon mangaan-peroksidase produseer. Lakkase was geproduseer deur al die taksons, uitsluitend Takson 8. Lignien-peroksidase was geproduseer deur Taksons 1, 2, 7, Fomitiporia sp. en die Phellinus sp. Al die basidiomycete isolate kon sellulose produseer, maar geen kon xilanase produseer. Die ensiem analises het gewys dat die basidiomycete wat moontlik betrokke is by esca ‘n wye reeks van ensieme kan produseer wat sellulose en lignien kan degradeer. Sellulose en lignien is beide strukturele komponente van hout. Weens die wye verspeiding van esca geaffekteerde wingerde in Suid Afrika en die wye reeks van spesies wat betrokke is by die siekte kompleks moet esca sekerlik gesien word as een van die beperkende faktore op die produktiewe leeftyd van wingerde en die kwaliteit van druiwe wat geproduseer word. Sanitasie en snoeiwond beskerming is voorkomende maatreëls wat ingestel kan word om die effek en verspreiding van esca te beperk maar daar is nog baie vrae wat antwoorde benodig oor die sinergie van die veroorsakende swamme, epidemiologie en bestuur van esca.

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

Thesis (MScAgric) -- University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Fungi are an extremely diverse group of organisms and, by acting as pathogens, they can colonise various other organisms, including humans, plants and animals. The effect of this is usually detrimental, not only to agricultural crops and livestock, but also to human well-being. The extensive farming of crops and livestock requires persistent control of fungal populations, commonly through the use of chemical fungicides. However, the exclusive use of fungicides is no longer a sustainable practice, as a result of serious problems, such as increasing fungicide resistance in pathogen strains, the high costs of fungicides, as well as concern about the environment. The search by producers and scientists for alternative control measures is an ongoing process. The fungal cell wall consists of polysaccharides that not only playa role in protection of the fungi, but also in relaying signals for the invasion and infection of susceptible hosts. Chitin, a polysaccharide composed of N-acteylglucosamine (GleNAc) residues linked by P-1,4 glucosidic linkages, is one of the major components of the fungal cell wall, where it plays an important role in the apical growth of the vegetative hyphae. Chitinases (EC 3.2.1.14) are abundant proteins produced by a variety of microorganisms and plants and are necessary for the hydrolysis of the chitin polymer. During the invasion of many plant species by a pathogen, the production of a specific group of proteins, designated pathogenesis-related (PR) proteins that include chitinases, is induced as part of their defence response. Due to the facts that pathogenic fungi contain chitin in their cell walls and that plant chitinases are induced upon pathogen attack, chitinases have been confirmed as an integral and crucial part of the plant's natural defence response. Chitinases have increasingly been targeted to upregulate plants' endogenous disease resistance mechanisms through transgenic overexpression in a variety of hosts. Several species of fungi, including various Trichoderma spp., are potent biocontrol agents of plant pathogenic fungi and insects. The antagonistic activities of these biological control agents towards phytopathogens are based on the secretion of extracellular hydrolytic enzymes, such as cell wall-degrading chitinase enzymes. However, biological control is not restricted to naturally occurring biocontrol agents. Through the process of genetic transformation, other fungal or yeast species can be enhanced to produce their own chitinases or other antimicrobial substances more effectively in order to yield potent biocontrol agents. Various types of chitinases have been applied in the production of fungal resistant plants and some research has been done on the application of chitinases, from a variety of microorganisms, as biological control agents. In contrast, very little is known about the antifungal activity of the Saccharomyces cerevisiae chitinase enzyme, encoded by the CTS1-2 gene. The CTS1-2 gene was utilised in this study as a candidate for overexpression in both yeast and plant expression systems to analyse the ability of the encoding chitinase to inhibit fungal growth. The first objective of this study involved the high level expression and optimisation of the secretion of the CTS1-2 gene in S. cerevisiae to render recombinant yeast with enhanced antifungal abilities and with possible applications as a biocontrol agent to control plant pathogenic fungi. It was hypothesised that high-level expression and efficient secretion would be prerequisites in a biocontrol yeast strain. To this end, two strong promoters and terminators were included in the study and the secretion of the chitinase gene was evaluated by testing three different secretion signals. The secretion signals included: the native CTS1-2 secretion signal, the S. cerevisiae mating pheromone a-factor (MFa1) secretion signal, as well as the Trichoderma reesei f3-xylanase 2 (XYN2) secretion signal. The phosphoglycerate kinase 1 (PGK1) and alcohol dehydrogenase 2 (ADH2) promoters and terminators were employed to achieve high-level expression. The results obtained from the analysis of the recombinant yeasts showed that the PGK1 promoter-terminator constructs yielded high level CTS1-2-expressing and chitinase-producing strains of S. cerevisiae PRY488. The ability of the different secretion signals to efficiently secrete the overexpressed chitinase was analysed and it was found that the non-native secretion signals delivered significantly more protein to the extracellular environment. It was thus evident that the performance of the MFa1 and XYN2 secretion signals was superior to that of the native secretion signal. The antifungal activities of the recombinant chitinases produced by these constructs were tested in in vitro assays against Botrytis cinerea. The enzymes led to a significant reduction in hyphal development, caused by extreme structural damage to the hyphal tips, the hyphal cell walls as well as the ability of the fungus to form reproductive and survival structures, thereby confirming the antifungal abilities of this enzyme. The ADH2 promoter-terminator constructs yielded CTS1-2 transcripts, but no chitinase activity could be detected with any of these strains. The reasons for this still remain unclear. The second objective of this study was to assess the potential of the yeast chitinase gene to upregulate defence against fungal infection in planta. In order to elucidate this, the CTS1-2 gene was constitutively overexpressed in tobacco plants, targeting the chitinase both to the intra- and the extracellular environment. The results obtained showed that the transgenic tobacco lines regenerated in this study stably integrated the transgene, exhibiting transgene expression as well as the production of a biologically active yeast chitinase enzyme. The F, progeny were rigorously tested for resistance to B. cinerea, and both in vitro and in planta assays confirmed that the yeast chitinase increased the plant's tolerance to fungal infection; some of the lines showed disease resistance of 65 and 70%. The plants expressing an extracellularly targeted chitinase gene are still under evaluation. Interesting results are expected relating to the effect of the chitinase on the plant surface with regards to disease resistance to fungal pathogens. In conclusion, the combined set of results from both the yeast and plant overexpression studies has confirmed the strong antifungal effect of yeast chitinases. The yeast CTS1-2 chitinase could be instrumental in the development of a new generation of yeast strains with improved antifungal capabilities. This enzyme could also play an important role in genetic transformation technologies aimed at enhanced disease resistance. / AFRIKAANSE OPSOMMING: Swamme omsluit 'n uiterste diverse groep organismes wat mense, plante en diere deur patogeniese aksie kan koloniseer. Die uitkoms hiervan op landbougewasse, die veebedryf en menslike gesondheid is gewoonlik skadelik. Uitgebreide gewas- en veeboerderye benodig voortdurende beheer van fungiese populasies, tipies deur van chemiese swamdoders gebruik te maak. Die uitsluitlike gebruik van swamdoders is egter nie meer 'n lewensvatbare praktyk nie, hoofsaaklik as gevolg van probleme soos die opbou van weerstand van patogeniese rasse teen swamdoders, die hoë kostes van die middels, asook besorgheid oor die omgewing. Die soektog na alternatiewe beheermaatreëls deur produsente en wetenskaplikes bly 'n aaneenlopende proses. Die swamselwand bestaan uit polisakkariede wat nie net In rol in die beskerming van die swam speel nie, maar ook betrokke is in die oordrag van aanvals- en infeksieverwante seine in 'n vatbare gasheer. Chitien, 'n polisakkaried bestaande uit N-asetielglukosamien (GlcNAc) residu's gekoppel deur 13-1,4glukosidiese bindings, is een van die hoofkomponente van die swamselwand, waar dit 'n belangrike rol in die apikale groei van vegetatiewe hifes speel. Chitinases (EC 3.2.1.14) is proteïene wat oorvloedig deur 'n verskeidenheid van mikroërganismes en plante geproduseer word, waar hulle vir die hidrolise van die chitien polimeer noodsaaklik is. Tydens die infeksie van verskeie plantspesies deur In patogeen, word die produksie van 'n spesifieke groep proteïene, die sogenaamde patogeen-verwante (PR) proteïene wat chitinases insluit, as deel van die plant se verdedigingsreaksie geïnduseer. Die feit dat patogeniese swamselwande chitien bevat en dat plantchitinases tydens infeksie geïnduseer word, het daartoe gelei dat dit bevestig is dat chitinases In integrale en kritiese deel van die plant se natuurlike verdedigingsreaksie uitmaak. Chitinases word toenemend geteiken in pogings om die plant se intrinsieke siekteweerstandsmeganismes te verbeter deur transgeniese ooruitdrukking daarvan in 'n verskeidenheid van gashere. Verskeie swamspesies, insluitend verskillende Trichodenna-spesies, is kragtige bio-antagoniste van plantpatogeniese swamme. Die antagonistiese aksies van hierdie biologiese beheeragente teenoor fitopatogene is gebaseer op die uitskeiding van ekstrasellulêre hidrolitiese ensieme, soos die selwandverterende chitinase ensieme. Nietemin is biologiese beheer nie net tot bio-antagoniste wat natuurlik voorkom beperk nie. Deur die proses van genetiese transformasie kan ander swam- of gisspesies verbeter word om hul eie chitinases of ander antimikrobiese substanse meer effektief te produseer, wat aanleiding sal gee tot kragtige bio-antagoniste. Verskeie tipes chitinases is al in die produksie van swambestande plante ingespan en uitgebreide navorsing is gedoen op die toepassing van 'n reeks chitinases, afkomstig van 'n verskeidenheid van mikroërganismes, as biologiese beheeragente. In teenstelling is baie min bekend oor die antifungiese aktiwiteite van die Saccharomyces cerevisiae chitinase ensiem, wat deur die CTS1-2 geen ge-enkodeer word. Die CTS1-2-geen is in hierdie studie gebruik vir ooruitdrukking in beide gis- en plantuitdrukkingsisteme om die chitinase se vermoë om swamgroei te inhibeer, te ondersoek. Die eerste oorkoepelende oogmerk van hierdie studie het hoë-vlak uitdrukking en optimalisering van sekresie van die CTS1-2-geen in S. cerevisiae behels, met die toekomstige doelwit om 'n rekombinante gis met verbeterde antifungiese eienskappe en met moontlike toepassings as 'n bio-antagonis teen plantpatogeniese swamme te ontwikkel. Die hipotese was dat hoë-vlak uitdrukking en voldoende sekresie voorvereistes vir 'n bio-antagonisras is. Omdié rede is twee sterk promotors en termineerders by hierdie studie ingesluit en is die sekresie van die chitinase-geen geëvalueer deur drie verskillende sekresieseine te toets. Die sekresieseine sluit in: die wilde-tipe CTS1-2 sekresiesein, die S. cerevisiae paringsferomoon a-faktor (MFa1) sekresiesein, en die Trichoderma reesei p-xilanase (XYN2) sekresiesein. Die fosfogliseraat kinase 1 (PGK1) en alkohol dehidrogenase 2 (ADH2) promotors en termineerders is gebruik om hoë-vlak uitdrukking te dryf. Die resultate wat vanaf die analises van die rekombinante giste verkry is, het getoon dat die PGK1 promotor-termineerder konstrukte hoë-vlak CTS1-2-uitdrukkende en chitinase-produserende S. cerevisiae PRY488 rasse opgelewer het. Die vermoë van die verskillende sekresieseine om die ooruitgedrukte chitinase voldoende uit te skei, is geanaliseer, en daar is gevind dat die heteroloë sekresieseine aansienlik meer proteïene na die ekstrasellulêre omgewing geloods het. Dit was dus duidelik dat die MFa1 en XYN2 sekresieseine beter as die wilde-tipe sekresiesein presteer het. Die antifungiese aktiwiteit van die rekombinante chitinases wat deur hierdie konstrukte geproduseer is, is ook in in vitrotoetse teen Botryits cinerea getoets. Die teenwoordigheid van die ensieme het gelei tot 'n aansienlike afname in hife-ontwikkeling, veroorsaak deur ekstreme strukturele skade aan die hifepunte, die hifeselwande, asook die vermoë van die swam om voortplanting- en oorlewingstrukture te vorm. Die ADH2 promotor-termineerderkonstrukte het CTS1-2 transkripte vertoon, maar geen chitinase-aktiwiteite kon in hierdie konstrukte waargeneem word nie. Die redes hiervoor is tot op hede onbekend. Die tweede oogmerk van hierdie studie was om die potensiaal van die gischitinase om swaminfeksie in planta teë te werk, te ondersoek. Die CTS1-2-geen is konstitutief ooruitgedruk in tabakplante, waarin die chitinase na beide die intra- en ekstrasellulêre omgewing geteiken is. Resultate het getoon dat die geregenereerde transgeniese tabaklyne die transgeen stabiel geïntegreer het, transgeenuitdrukking vertoon en dat 'n biologies aktiewe chitinase-ensiem geproduseer is. 'n F1-generasie is aan strawwe toetse onderwerp om weerstand teen B. cinerea te ondersoek. Beide die in vitro en in planta toetse het bevestig dat die gischitinase die plant se verdraagsaamheid teenoor swaminfeksie verhoog het; sommige lyne het siekteweerstand van tussen 65 en 70% getoon. Die plante wat 'n ekstrasellulêre chitinase produseer, word steeds geëvalueer. Interessante resultate word verwag aangaande die effek van die chitinase op die plant se oppervlak met betrekking tot siekteweerstand teen swampatogene. Ten slotte, die gekombineerde stel resultate wat vanaf beide die gis- en plantuitdrukkingstudies verkry is, het die sterk antifungiese effek van gischitinases bevestig. Die gis CTS1-2 kan instrumenteel wees in die ontwikkeling van 'n nuwe generasie gisrasse met verbeterde antifungiese eienskappe. Die ensiem kan ook 'n belangrike rol in genetiese transformasietegnologieë, wat op verbeterde siekteweerstand gemik is, speel.

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.

Dissertation (PhD(Agric)) -- University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Net blotch of barley, caused by Pyrenophora teres, is one of the most important diseases of this cereal in the south Western Cape Province of South Africa. This fungus exists as two different types (forms), namely a nettype and a spot-type that are distinguished by differential symptom expression on barley leaves. Based on this specific plant pathological difference a series of studies of agricultural importance were executed to investigate the effects of sexual recombination between these two types. In addition, studies were done to determine the difference between local net- and spot-type populations with regards to population structure and fungicide sensitivity. This dissertation therefore, consists of a collection of separate publications and as a result a certain degree of redundancy has been unavoidable. Recombination is one of the most important evolutionary forces involved with sexual reproduction. In plant-fungal agricultural ecosystems this may result in pathogenic fungal populations adapting more rapidly to control programs such as fungicide applications. The first section of the review in part 1 of this dissertation covers different aspects of sexual reproduction in ascomycetes, specifically focussing on mating-type genes, vegetative incompatibility and recombination. The major part of the review is then dedicated to various plant pathological aspects of P.teres, specifically addressing the differences between the two types, and in various cases highlighting the significance of sexual recombination within and between the net- and spot-type. Using morphological criteria for identification purposes there have been many conflicting reports concerning the identity of leaf spot isolates in the Western Cape Province of South Africa. In part 2, the correct identity was eventually achieved employing mating studies and molecular markers .: This was accomplished after single ascospores were obtained from pseudothecia after in vitro mating had occurred between a verified P. teres net-blotch isolate from Denmark and a representative Pyrenophora leaf spot isolate from South Africa. Using amplified fragment length polymorphism (AFLP) and RAPD markers, recombination was demonstrated in the progeny that had DNA banding patterns different from the two parental isolates. Pathogenicity trials also confirmed that recombination had taken place during mating. Inoculations were conducted on the differential cultivars susceptible to the net-blotch and leaf spot forms. The two parents induced typical net-blotch or leaf spot symptoms whereas the progeny mostly induced a jagged spot symptom on each cultivar. Fungicide sensitivity tests using the ergosterol biosynthesis inhibitors showed that, due to recombination, some progeny could have increased resistance to these fungicides. Due to mating and subsequent recombination between a net blotch isolate of P. teres and a representative leaf spot isolate, it was concluded that the latter was P. teres f. maculata. Fifteen of the net-spot hybrid progeny (F1) produced from the mating study in Part 2 were screened in Part 3 to assess their viability and genetic stability. Hybrid progeny (F1) inoculated onto barley seedlings consisting of the cultivars Stirling (differentially susceptible to net-type isolates), B87/14 and Clipper (both differentially susceptible to spot-type isolates) produced intermediate symptoms on all cultivars. Axenic cultures (F1-1) isolated from foliar lesions, followed by repeated inoculation and isolation (F1-2) onto a healthy set of seedlings produced similar intermediate symptoms. RAPDs conducted with two 1Q-mer primers on all isolates of F1-1and F1-2progeny revealed profiles similar to those obtained for F1 isolates. RAPD molecular data, therefore, indicated that hybrid progeny of this net x spot mating were genetically stable after having been subjected to two repetitive inoculation and reisolation cycles. Phylogenetic analysis of DNA sequences of the internal transcribed spacers (ITS1 and ITS2) flanking the 5.8S nuclear ribosomal RNA gene and the 5' end partial histone-3 gene confirmed the genetic stability of the hybrid progeny. These results also indicated that the hybrid progeny produced consistent symptoms throughout the series of experiments, and maintained their virulence to the differential cultivars screened. Both types of P. teres are prevalent in the south Western Cape Province of South Africa, found on susceptible cultivars often grown within close proximity of each other. In Part 4, a net- and spot-type population were characterised in terms of their population structure using RAPD markers. Samples were collected from infected barley leaves from two separate quadrants in each field, the two quadrants positioned in corners of the fields, diagonal to one another. A total of 65 loci were produced of which 54 were polymorphic. Total gene diversities determined for all loci resulted in mean indices of 0.063 and 0.082 being obtained respectively for the net- and spottype populations. A coefficient of genetic differentiation (Gs) of 0.0149 was obtained between sites within populations while a coefficient (GT) of 0.63 was obtained between the two populations. Genotypic variation revealed 13 distinct multilocus genotypes (haplotypes) in the net-type population while there were 12 in the spot-type population. UPGMA cluster analysis done on the two populations together with six progeny from the mating between a netand spot-type isolate resulted in three main clusters being produced, one for each population and one for the progeny. One isolate collected from the nettype population also contained a unique spot-type RAPD fragment. This suggested that sexual recombination may be taking place between isolates of the net- and spot-type under field conditions. Fungicide application is the most important method used in the control of net blotch in South Africa. In Part 5 the fungicide sensitivities (ICsD values) of 89 monoconidial isolates (46 net-type and 43 spot-type) of P. teres to sterol demethylation inhibiting fungicides were determined, based on the inhibitory effect on radial mycelial growth. The fungicides evaluated were triadimenol, bromuconazole, flusilazole, propiconazole and tebuconazole. Both net- and spot-type isolates revealed strong resistance to triadimenol while flusilazole was shown to be the strongest inhibitor of fungal growth. Spot-type isolates showed a higher resistance than net-type isolates to all five fungicides screened. The ICsD values indicated significant differences between four of the fungicides (triadimenol, tebuconazole, flusilazole and propiconazole). The ICsD values between propiconazole and bromuconazole were not significant. This study suggested that spot-type isolates showed a higher degree of resistance to commercially used fungicides than net-type isolates. The overall conclusion of this study is that the spot-type of P. teres is the pathogen associated with leaf spots of barley in the south western Cape province of South Africa and not P. japonica as earlier reported. Together with the net-type, both types exist as genetically variable populations in this barley production region. Mating between the two types results in sexual progeny that are genetically stable. This implies that barley fields adjacent to one another in which either net- or spot-type susceptible cultivars are being cultivated may lead to sexual progeny being produced. This in turn may lead to an increased rate at which fungal populations may become resistant to commercially used fungicides. It is furthermore suggested that an alternative fungicide seed treatment is used instead of triadimenol due to high resistance of P. teres to this fungicide. / AFRIKAANSE OPSOMMING: Netvlek op gars is een van die belangrikste siektes van hierdie graansoort in die suidelike deel van die Westelike Kaapprovinsie. Dié siekte word veroorsaak deur die swam Pyrenophora teres. Hierdie swam kom voor as twee verskillende tipes, naamlik 'n net-tipe en 'n kol-tipe wat onderskei word op grand van die voorkoms van hulle simptome op garsblare. Hierdie planpatologiese verskil in ag genome, is 'n reeks studies van landboukundige waarde uitgevoer om die effek van geslagtelike rekombinasie tussen die twee tipes te ondersoek. Daarbenewens is ook studies uitgevoer om om die verskil te bepaal tussen plaaslike net- en koltipe populasies ten opsigte van populasiestruktuur en fungisiedsensitiwiteit. Hierdie verhandeling bestaan dus uit 'n versameling afsonderlike publikasies en as gevolg daarvan is daar onvermydelik'n mate van oorvleueling. Rekombinasie is een van die belangrikste evolusionêre kragte betrokke by geslagtelike voortplanting. In plant-swam landboukundige ekostelsels kan dit veroorsaak dat patogene swampopulasies vinniger aanpas by beheerpragramme soos fungisiedtoediening. Die eerste gedeelte in deel 1 van hierdie verhandeling dek die verskillende aspekte van geslagtelike voortplanting van ascomycetes, met spesifieke verwysing na paringstipe gene, vegetatiewe onverenigbaarheid en rekombinasie. Die grootste gedeelte van die oorsig word gewyaan verskeie plantpatologiese aspekte van P. teres,en wys veralop die verskille tussen die twee tipes. In verskeie gevalle word die betekenis van geslagsrekombinasie binne en tussen die net- en koltipe uitgelig. Deur morfologiese kenmerke vir identifikasiedoeleindes te gebruik, is daar baie teenstrydige verslae rakende die identifikasie van blaarvlekisolate in die Westlike Kaapprovinsie van Suid-Afrika. In deel 2 is die korrekte identifikasie eventueel verkry deur gebruik te maak van paringstudies en molekulêre merkers. Dit is bereik nadat enkel ascospore verkry is uit pseudothecia gevorm na in vitro paring plaasgevind het tussen 'n bevestigde P. teres netvlek isolaat uit Denemarke en 'n verteenwoordigende Pyrenophora blaarvlekisolaat van Suid- Afrika. Deur gebruik te maak van versterkte fragmentlengte polimorfisme [AFLP] en RAPD merkers, is rekombinasie gedemonstreer in die nasate wat DNA bandpatrone gehad het wat verskil het van dié van die "ouer" isolate. Patogenisiteitstoetse het ook bevestig dat rekombinasie tydens paring plaasgevind het. Inokulasies is uitgevoer op die verskillende cultivars wat vatbaar is vir die netvlek en blaarvlek vorme. Die twee ouers het tipiese netvlek of blaarvlek simptome veroorsaak, terwyl die nasate hoekige vlekke veroorsaak het op elke cultivar. Toetse vir fungisiedsensitiwiteit deur gebruik van die ergosterol biosintese inhibeerders het gewys dat a.g.v. rekombinasie sekere nasate verhoogde weerstand teen hierdie fungisiedes het. As gevolg van paring en daaropvolgende rekombinasie tussen 'n netvlek isolaat van P. teres en 'n verteenwoordigende blaarvlek isolaat is afgelei dat laasgenoemde P. teres f. maculata is. Vyftien van die netvlek hibried nakomelinge (F1) verkry van die paringstudie in deel 2 is ondersoek in deel 3 om hul lewensvatbaarheid en genetiese stabiliteit te bepaal. Hibried nasate (F1) geïnokuleer op garssaailinge bestaande uit die volgende cultivars: Stirling (soms vatbaar vir net-tipe isolate) , B87/14 en Clipper (albei soms vatbaar vir kol-tipe isolate) het intermediêre simptome op al die cultivars veroorsaak. Akseniese kulture (F1-1) geïsoleer uit blaarletsels gevolg deur herhaalde inokulasie en isolasie (F1-2) op 'n gesonde stel saailinge het dieselfde intermediêre simptome veroorsaak. RAPDs uitgevoer met twee 10-mer inleiers op al die isolate van F1-1 en F1-2 nasate het profiele opgelewer soortgelyk aan dié wat vir F1 isolate verkry is. RAPD molekulêre data het dus gewys dat die hibried nasate van hierdie net x kol paring geneties stabiel was nadat dit onderwerp is aan twee inokulasie en reïsolasie siklusse. Genetiese stabiliteit van die hibried nageslag is bevestig deur filogenetiese analise van die DNA volgorde van die interne getranskribeerde spasieerders (ITS1 en ITS2) reg langs die 5.8S nukluêre ribosomale RNA geen en die 5' end gedeeltelike histoon-3 geen. Hierdie resultate het ook gewys dat die hibried nasate konstante simptome getoon het tydens die hele reeks eksperimente en hulle virulensie behou het vir die kultivars wat getoets is. Beide tipes van P. teres kom algemeen voor in die suidelike deel van die Westelike Kaapprovinsie en word gevind op vatbare cultivars wat dikwels naby mekaar groei. In deel 4 is 'n net- en kol-tipe populasie gekarakteriseer in terme van hulle populasiestruktuur deur gebruik van RAPD merkers. Monsters is versamel van geïnfekteerde garsblare van twee aparte kwadrante in elke saailand. Die twee kwadrante is geplaas in die hoeke van die saailand, diagonaal tot mekaar. 'n Totaal van 65 lokusse is gevorm, waarvan 54 polimorfies was. Die algehele genetiese verskeidenheid bepaal vir alle lokusse, het gelei tot gemiddelde indekse van 0.063 en 0.082 soos gevind vir die net- en kol-tipe populasies. 'n Koëffisiënt van genetiese differensiasie (Gs ) van 0.0149 is gevind tussen gebiede tussen populasies, terwyl 'n koëffisiënt (GT) van 0.63 gevind is tussen die twee populasies. Genotipiese variasie het 13 duidelike multilokus genotipes (haplotipes) getoon in die net-tipe populasie, terwyl daar twaalf was in die kol-tipe populasie. UPGMA groeperingsanalises wat gedoen is op die twee populasies tesame met ses nasate van die paring van 'n net- en koltipe isolaat het tot gevolg gehad dat drie hoof groepe gevorm is, een vir elke populasie en een vir die nasate. Een isolaat wat versamel is, van die net-tipe populasie het 'n unieke kol-tipe RAPD fragment bevat. Dit wys daarop dat geslagtelike rekombinasie in veldomstandighede mag voorkom tussen isolate van die net- en kol-tipe. Fungisiedtoediening is die belangrikste metode wat gebruik word om netvlek in Suid-Afrika te beheer. In deel 5 is die fungisiedsensitiwteit (Ieso waardes) van 89 enkelkonidiale isolate (46 net-tipe en 43 kol-tipe) van P. teres teen sterol demetielasie inhiberende fungisiedes bepaal, op die basis van die onderdrukkende effek op die radiale groei van die miselium. Die volgende fungisiedes is geëvalueer: triadimenol, bromuconazole, flusilazole, propiconazole en tebuconazole. Beide net- en kol-tipe isolate het 'n sterk weerstand teen triadimenol openbaar, terwyl flusilazole gevind is as die sterkste onderdrukker van swamgroei. Kol-tipe isolate het 'n hoër weerstand as die net-tipe isolate teen al vyf fungisiedes wat getoets is, gehad. Die lesowaardes het aangedui dat daar beduidende verskille tussen vier van die fungisiedes IS (triadimenol, tebuconazole, flusilazole en propiconazole). Die leso waardes tussen propiconazole en bromuconazole was nie beduidend nie. Die gevolgtrekking van hierdie studie is dus dat die kol-tipe isolate 'n hoër graad van weerstand teen kommersiëel gebruikte fungisiedes as die net-tipe isolate gehad het. Die algehele gevolgtrekking van hierdie studie is dat die kol-tipe van P. teres, die patogeen is wat geassosieer word met blaarvlekke op gars in die suidwestelike Kaapprovinsie van Suid-Afrika, en nie P. japonica soos voorheen gerapporteer nie. Tesame met die net-tipe, kom altwee tipes voor as geneties veranderlike populasies in hierdie gars verbouingstreek. Paring tussen die twee tipes lei tot geslagtelike nasate wat geneties stabiel is. Dit impliseer dat aangrensende garsvelde waarop net- óf kol-tipe vatbare kultivars verbou word, mag lei tot die produksie van geslagtelike nasate. Dit kan weer lei tot 'n verhoogde tempo waarteen swampopulasies weerstandbiedend teenoor kommersiële fungisiedes raak. Daar word verder ook voorgestel dat alternatiewe fungisied saadbehandelings gebruik word in plaas van triadimenol as gevolg van verhoogde weerstand van P. teres teenoor laasgenoemde.