Thesis (MScAgric)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Various studies revealed that Botrytis cinerea, the causal pathogen of Botrytis bunch rot, is mostly associated with pedicels, rachises, laterals and berry bases, and not with berry skins as previously understood. Provided that sufficient coverage of inner bunch parts was achieved, laboratory studies have shown that fungicides can effectively reduce the amount of B. cinerea at the various positions in bunches, and prevent infection and symptom expression at all growth stages. The same efficacy was, however, not achieved with the same fungicides when using conventional spraying methods in vineyards. Poor disease control on fruit and leaves in vineyards is attributed to inappropriate timing of fungicide applications and/or insufficient coverage of susceptible tissue. Previously, spray coverage evaluations in South Africa were based on the use of water-sensitive cards. A variety of other methods have been used to assess spray coverage in vineyards, but none of these methods could assess spray deposits on a very small, three-dimensional area of interest such as the susceptible grape bunch parts. The methods were furthermore dependent on human objectivity, which lacks quantitative measuring and speed of measurement. Suitable technology to determine spray coverage on susceptible bunch parts is, therefore, not available. The aim of this study was to develop a protocol to visualise and quantify spray deposits in grape bunches, specifically on the inner bunch parts and to use the protocol to determine the effect of different levels of spray cover on artificially inoculated B. cinerea grape bunches, in order to facilitate future determination of minimum effective coverage levels for effective B. cinerea control. A spray coverage assessment protocol using fluorometry, photomicrography and digital image analyses was developed to measure spray coverage on susceptible grape bunch parts. Among several fluorescent pigments tested, a yellow fluorescent pigment (SARDI Fluorescent Pigment) from Australia was selected on the basis of its small particle size (2.45 - 4.90 μm). Bunches were sprayed at pea size and bunch closure with different volumes of a mixture of fenhexamid and the yellow fluorescent pigment. Sprayed parts from bunches were illuminated under black light (UV-A light in the 365 nm region) and visualised under a stereo microscope at 20 x magnification. Photos of the berry skin, pedicel and rachis were taken with a digital camera (Nikon DMX 1200). Image analysis of photos was done with Image- Pro Discovery version 4.5 for Windows (Media Cybernetics) software. The total area of deposited pigment in selected areas of interest (AOI) was calculated. The percentage area covered was subsequently calculated for each AOI. Good correlation was evident between the parameters, sum of objects and percentage area covered. Bunch parts at pea size generally had higher coverage values than at bunch closure. Spray applications earlier in the season would therefore result in higher and more effective spray coverage of the susceptible bunch parts. Similar deposition trends were observed on the inner bunch parts (pedicel and rachis). These were, however, significantly different from berry skins, which had significantly higher levels of spray deposits than the inner bunch parts. The variance component analysis indicated that the highest variance was observed for berries and bunches, and substantially less for image readings. For the same accuracy, means for percentage coverage values of at least 10 bunches per treatment (1 part per bunch and 3 readings per part) will be sufficient. In order to determine the biological efficacy of different levels of spray coverage on B. cinerea incidence on grape bunches, bunches were sprayed at pea size and bunch closure with different volumes of a mixture of fenhexamid and a yellow fluorescent pigment and the percentage fluorescent pigment coverage on pedicels was determine. Bunches were subsequently dusted with dry airborne conidia of B. cinerea in a settling tower and incubated for 24 h at high relative humidity (98%). Infection was determined by estimating the amount of B. cinerea infections occurring on sprayed bunch parts with isolations on to paraquat and Kerssies mediums. Linear regressions for the part x stage combinations of percentage B. cinerea incidence on different bunch parts were fitted on mean coverage levels. An increase in spray cover caused linear reductions in levels of B. cinerea on susceptible bunch parts. Higher B. cinerea incidences were recorded at pea size. Furthermore, higher B. cinerea incidences were found on paraquat medium for both stages, than on Kerrsies medium. The information gathered from this study will be used to facilitate future determination of minimum effective coverage levels for effective B. cinerea control in grape bunches. In these validation experiments, the results clearly showed that the protocol can be used to determine the effect of different levels of spray coverage on B. cinerea incidence and that an increase in spray coverage will decrease B. cinerea incidence. The information gathered from this study will be used to facilitate future determination of minimum effective coverage levels for effective B. cinerea control in grape bunches and subsequently be used as benchmarks to evaluate spray application in vineyards. / AFRIKAANSE OPSOMMING: Vaalvrot by wingerde word veroorsaak deur Botrytis cinerea. Verskeie studies het getoon/gewys dat die oorsaaklike patogeen meestal geassosieer word met die pedisel, ragis, laterale en die korrelbasis, en nie met die korrelskil soos voorheen beweer nie. Laboratorium studies het getoon dat swamdoders wel effektief is om B. cinerea by alle trosdele te verminder en simptoomontwikkeling te voorkom tydens alle groeistadia, mits die binne-trosdele voldoende spuit bedekking ontvang het. Dieselfde effektiwiteit is egter nie gevind in wingerde met konvensionele spuittegnieke nie. Onvoldoende siektebeheer van vrugte en blare van wingerde kan toegeskryf word aan verkeerde spuit skedulering en/of swak spuitbedekking van vatbare gasheerweefsel. Evaluering van spuitbedekking is voorheen in Suid Afrika deur middel van water-sensitiewe papier gedoen. Verskeie ander metodes is al gebruik om spuitbedekking te evalueer in wingerde, maar nie een van hierdie metodes kan gebruik word om spuitbedekking op ’n baie klein, drie-dimensionele oppervlak, soos die vatbare trosdele, te evalueer nie. Verder was die tegnieke afhanklik van menslike objektiwiteit, en gevolglik ontbreek kwantitatiewe meting en metingspoed. Daar is dus nie geskikte tegnologie vir die evaluering van spuitbedekking op vatbare trosdele nie. Die doel van hierdie studie was die ontwikkeling van ‘n protokol vir die visualisering en kwantifisering van spuitbedekking op spesifiek die binne-tros dele en om die protokol dan te gebruik om die effek van verskillende vlakke van spuitbedekking op B. cinereageinokuleerde druiwetrosse te bepaal, Protokol vir evaluasie van spuitbedekking op vatbare druifdele is ontwikkel deur gebruik te maak van fluorometrie, fotomikrografie en digitale beeldanalise. Van die verskillende fluoresensie pigmente wat getoets is, is ‘n geel flouresensie pigment (SARDI Flourescent Pigment) van Australië gekies op grond van sy klein partikelgrootte (2.45 - 4.90 μm). Druiwetrosse is gespuit tydens ertjie- en trostoemaakstadia met verskillende volumes van ’n mengsel van fenheksamied en die geel fluorosensie pigment. Die gespuite druifdele is dan verlig onder swartlig buise (UV-A lig in die 365 nm spektrum) en gevisualiseer deur ’n stereo mikroskoop by 20x vergroting. Foto’s van die korrelskil, pedisel en ragis is met ‘n digitale kamera (Nikon DMX 1200) geneem. Beeldanalise is gedoen met ImagePro Discovery weergawe 4.5 vir Windows (Media Cybernetics) sagteware. Die totale area neerslag van die pigment is in geselekteerde areas bereken. Die presentasie area bedek is bereken vir elkeen van hierdie areas. Goeie korrelasie is gevind tussen die parameters aantal fluoresserende partikels en die persentasie bedekte area. Trosdele tydens ertjie-stadium het in die algemeen hoër waardes gehad as by trostoemaak. Dit blyk dus dat spuittoediening vroeg in die seisoen meer effektief sal wees vir die bedekking van vatbare trosdele. Soortgelyke bedekkings patrone is gevind by die binne trosdele (pedisel en ragis). Dit het egter betekenisvol verskil van die korrelskil, wat betekenisvol meer spuitbedekking as die binne trosdele gehad het. ’n Variasie komponent analise het getoon dat die meeste variasie gevind is tussen korrels en trosse, en heelwat minder vir die beeld analise lesings. Om dieselfde akkuraatheid te behou, is ten minste 10 trosse per behandeling (1 deel per tros en 3 lesings per deel) nodig. Vir die bepaling van biologiese effektiwiteit van verskillende vlakke van spuitbedekking op B. cinerea voorkoms op druiwe, is druiwe gespuit tydens ertjie- en trostoemaak-stadia met verskillende volumes van ’n mengsel van fenheksamied en die geel fluorosensie pigment. Die persentasie fluoresensie pigment is bepaal op die pedisels. Trosse is vervolgens geinokuleer met droë luggedraagde konidia van B. cinerea in ’n inokulasietoring en geïnkubeer vir 24 h by hoë relatiewe humiditeit (98%). Die voorkoms van B. cinerea infeksie op gespuite tros dele is bepaal deur middel van isolasies op paraquat en Kerssies medium. Liniêre regressies vir trosdeel x stadium kombinasies van persentasie B. cinerea voorkoms op verskillende trosdele is gepas vir gemiddelde bedekkings waardes. ’n Verhoging in spuit bedekking het ‘n liniêre vermindering van B. cinerea voorkoms op vatbare trosdele veroorsaak. Verder is hoër vlakke van B. cinerea op paraquat medium as op Kerssies medium vir beide die groeistadia gevind. Die kennis wat verkry is uit hierdie studie sal gebruik word om minimum effektiewe spuitbedekkingsvlakke vir die beheer van B. cinerea op druiwetrosse te bepaal.

Thesis (MScAgric)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Fruit and Leaf Spot Disease (FLSD) of citrus, caused by Phaeoramularia angolensis, is found only in 18 countries in Africa, the Comores Islands in the Indian Ocean and Yemen in the Arabian peninsula. The major citrus export countries in Africa are Morocco, South Africa, Swaziland, and Zimbabwe. Zimbabwe is the only country affected by FLSD. FLSD is a disease of major phytosanitary and economic importance and its devastating effect on citrus is highlighted by the fact that the damage is cosmetic, which renders the fruit unmarketable. Total crop losses are not uncommon in Kenya. The aims of the present study, therefore, was was to determine the occurrence of P. angolensis in Zimbabwe and neighbouring Mozambique, to compare these isolates with the Cercospora Fresen. isolates from Swaziland and South Africa, to determine the epidemiology of the pathogen and to implement an effective control strategy to prevent the spread of FLSD. Leaf samples with citrus canker-like lesions collected in the early 1990’s in Zimbabwe were found to be infected by the fungus, Phaeoramularia angolensis. Surveys were undertaken to determine the spread and intensity of FLSD in Zimbabwe and Mozambique. In Zimbabwe, P. angolensis was limited to an area above the 19° south latitude, predominantly the moist areas and not the low-lying drier parts of the country. In Mozambique, no P. angolensis symptoms were found. Observations during the survey indicated that no proper management systems were implemented by Zimbabwean growers. A cercosporoid fungus causing a new Fruit and Leaf Spot Disease on Citrus in South Africa was identified. From morphological and rDNA sequence data (ITS 1, 5.8S and ITS 2), it was concluded that the new disease was caused by Cercospora penzigii, belonging to the Cercospora apii species complex. The genera Pseudophaeoramularia and Phaeoramularia are regarded as synonyms of Pseudocercospora, and subsequently a new combination was proposed in Pseudocercospora as P. angolensis. Cercospora gigantea was shown to not represent a species of Cercospora, while Mycosphaerella citri was found to be morphologically variable, suggesting that it could represent more than one taxon. A control strategy for the control of FLSD was evaluated in the study. The data showed that P. angolensis in Zimbabwe can be managed successfully by the removal of all old and neglected orchards, and on timely fungicide applications. Trifloxystrobin + mancozeb + mineral spray oil (20 g + 200 g + 500 ml/100 l water) applied in November, January and March was the most effective treatment. Three applications of benomyl + mancozeb + mineral spray oil (25 g + 200 g + 500 ml/100 l water) applied during the same period, was the second most effective treatment, and two applications (November and January) of trifloxystrobin + mineral spray oil (20g + 500 ml/100 l water) and difenoconazole (40 g) per 100 l/water applied twice in November and January, the third most effective treatment. The spore trap and weather data showed that P. angolensis needs high moisture and temperatures in excess of 25°C for disease development. It is concluded that P. angolensis in Zimbabwe can be managed successfully by implementing a holistic approach, which should be supported by the authorities, organised agriculture and all technical personnel involved in citrus production. / AFRIKAANSE OPSOMMING: Blaar- en vrugvleksiekte (BVVS) op sitrus, veroorsaak deur Phaeoramularia angolensis, kom in 18 lande in Afrika voor asook die Comores Eilande in die Indiese Oseaan en Yemen op die Arabiese skiereiland. Marokko, Suid Afrika, Swaziland en Zimbabwe is die belangrikste uitvoerders van sitrus in Afrika. Van dié lande het slegs Zimbabwe blaar en vrugvleksiekte op sitrus. Hierdie siekte is van fitosanitêre en ekonomiese waarde en die nadelige effek van die siekte, wat slegs kosmetiese van aard is, is venietigend aangesien vrugte onbemarkbaar is. Totale opbrengsverliese is nie ongewoon in lande soos Kenya nie. Die doelwitte van die studie was dus om die voorkoms van P. angolensis in Zimbabwe te bepaal, om die Cercospora Fresen. isolate vanaf Swaziland en Suid-Afrika met mekaar te vergelyk, om die epidemiologie van die siekte vas te stel en om ‘n effektiewe beheermaatreël teen die siekte te ondersoek. Blaarmonsters met kankeragtige letsels wat in die vroeë 1990’s in Zimbabwe gevind is, het getoon dat die blare geinfekteer is met die swam, Phaeoramularia angolensis. Ondersoeke is geloots om die verspreiding en intensiteit van BVVS in Zimbabwe en Mosambiek te bepaal. In Zimbabwe was gevind dat P. angolensis beperk was tot gebiede bo die 19° Suid breedtegraad, wat die hoër vogtiger gebiede insluit eerder as die droeër, laagliggende gebiede. Geen P. angolensis simptome kon in Mosambiek gevind word nie. Tydens die opnames was dit duidelik dat geen geskikte beheerstrategieë toegepas word deur Zimbabwe se produsente nie. ‘n Nuwe cercosporoid swam, wat blaar en vrugvleksiekte op sitrus is in Suid Afrika veroorsaak is geidentifiseer. Morfologiese en rDNA volgorde (ITS 1, 5.8S en ITS 2) data het getoon dat die siekte veroorsaak word deur Cercospora penzigii wat tot die Cercospora apii spesie kompleks behoort. Die genus Pseudophaeoramularia kan as sinoniem van Pseudocercospora beskou word en ‘n nuwe kombinasie word voorgestel in Pseudocercospora as P. angolensis. Cercospora gigantea het getoon dat dit nie ‘n spesie van Cercospora kon verteenwoordig nie terwyl Mycosphaerella citri varieërend voorkom en meer as een takson kan verteenwoordig. ‘n Beheerstrategie vir die beheer van BVVS is ondersoek. Die data wys dat P. angolensis in Zimbabwe doeltreffend beheer kan word deur die uitroeiing van ou en verwaarloosde bome, en deur goed beplande fungisied bespuiting. Trifloxystrobin + mancozeb + minerale spuitolie (20 g + 200 g + 500 ml/100 l water), wat in November, Januarie en Maart toegedien is, was die mees effektiefste behandeling. Drie bespuitings van benomyl + mancozeb + minerale spuitolie (25 g + 200 g + 500 ml/100 l water) wat oor dieselfde tydperk toegedien is, was die naas beste behandeling. Trifloxystrobin (20 g) + minerale spuitolie (500 ml) per 100 l/water en difenoconazole (40 g) per 100 l/water, beide as twee bespuitings toegedien in November en Januarie, het die derde beste resultaat opgelewer. Die spoorlokval en klimatologiese data het getoon dat P. angolensis vogtige toestande en temperature hoër as 25°C benodig vir siekteontwikkeling. Die afleiding uit die studie is dat P. angolensis suksesvol beheer kan word indien ‘n holistiese benadering gevolg word en alle rolspelers naamlik die owerheid, georganiseerde landbou en tegniese personeel die proses ondersteun.

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 (Plant Pathology))--Stellenbosch University, 2008. / In recent years, several studies have conclusively shown that numerous pathogens, including several species in the Botryosphaeriaceae, Phomopsis, Phaeoacremonium, as well as Phaeomoniella chlamydospora and Eutypa lata, contribute to premature decline and dieback of grapevines. These pathogens have the ability to infect grapevines through pruning wounds, which leads to a wide range of symptoms developing that includes stunted growth, cankers and several types of wood necrosis. Pruning wounds stay susceptible for 2 to 16 weeks after pruning and sustained levels of pruning wound protection is therefore required. The aims of this study were to (i) evaluate the ability of several biological agents to protect pruning wounds, (ii) characterise unknown Trichoderma strains and identify their modes of action and (iii) determine the optimal time of season for biological agent application. Several biological agents were initially evaluated in a laboratory for their antagonism against trunk disease pathogens. The best performing control agents were tested in a field trial conducted on Merlot and Chenin blanc vines in the Stellenbosch region. Spurs were pruned to three buds and the fresh pruning wounds were treated with benomyl as a control treatment, Trichoderma-based commercial products, Vinevax® and Eco77®, Bacillus subtilis, and Trichoderma isolates, USPP-T1 and -T2. Seven days after treatment the pruning wounds were spray inoculated with spore suspensions of four Botryosphaeriaceae spp. (Neofusicoccum australe, N. parvum, Diplodia seriata and Lasiodiplodia theobromae), Eutypa lata, Phaeomoniella chlamydospora and Phomopsis viticola. After a period of 8 months the treatments were evaluated by isolations onto potato dextrose agar. Trichodermabased products and isolates in most cases showed equal or better efficacy than benomyl, especially USPP-T1 and -T2. Moreover, these isolates demonstrated a very good ability to colonise the wound tissue. The two uncharacterised Trichoderma isolates (USPP-T1 and USPP-T2), which were shown to be highly antagonistic toward the grapevine trunk disease pathogens, were identified by means of DNA comparison, and their ability to inhibit the mycelium growth of the trunk disease pathogens by means of volatile and non-volatile metabolite production studied. The two gene areas that were used include the internal transcribed spacers (ITS 1 and 2) and the 5.8S ribosomal RNA gene and the translation elongation factor 1 (EF). The ITS and EF sequences were aligned to published Trichoderma sequences and the percentage similarity determined and the two Trichoderma isolates were identified as Trichoderma atroviride. The volatile production of T. atroviride isolates was determined by placing an inverted Petri dish with Trichoderma on top of a dish with a pathogen isolate and then sealed with parafilm. Trichoderma isolates were grown for 2 days on PDA where after they were inverted over PDA plates containing mycelial plugs. The inhibition ranged from 23.6% for L. theobromae to 72.4% for P. viticola. Inhibition by non-volatile products was less than for the volatile inhibition. Inhibition ranged from 7.5% for N. parvum to 20.6% for L. theobromae. In the non-volatile inhibition USPP-T1 caused significantly more mycelial inhibition than USPP-T2. The timing of pruning wound treatment and subsequent penetration and colonisation of the wound site was also determined. One-year-old canes of the Shiraz and Chenin blanc cultivars were grown in a hydroponic system, pruned and spray treated with a spore suspension of Trichoderma atroviride (USPP-T1) as well as a fluorescent pigment. On intervals 1, 3, 5 and 7 days after treatment, the distal nodes were removed and dissected longitudinally. From the one half, isolations were made at various distances from the pruning surface, while the other half was observed under ultra-violet light to determine the depth of fluorescent pigment penetration. Shortly after spray-inoculation of a fresh pruning wound, Trichoderma was isolated only from the wound surface and shallow depths into the wound (2 to 5 mm). One week after inoculation, Trichoderma was isolated at 10 mm depths, and after 2 weeks, at 15 mm depths. Fluorescent pigment particles were observed to a mean depth of 6 mm, which suggests that initial isolation of Trichoderma at these depths was resultant of the physical deposition of conidia deeper into the pruning wound tissue, whereas the isolation of Trichoderma from deeper depths might be attributed to colonisation of grapevine tissue. In a vineyard trial, fluorescent pigment was spray-applied to pruning wounds of Shiraz and Chenin blanc grapevines during July and September at intervals 0, 1, 3, 7 and 14 days after pruning. One week after treatment, the distal nodes were removed and dissected longitudinally. Each half was observed under UV light and the pigment penetration measured. For Chenin blanc and Shiraz, July pruning wounds showed significant deeper penetration of the pigment than pruning wounds treated in September. Moreover, pruning wounds made in September showed pigment particles in longitudinal sections up to 1 day after pruning, whereas wounds made in July showed pigment particles up to 3 days in the xylem vessels. These findings suggest that the best time for application of a biological control agent should be within the first 24 hours after pruning.

Thesis (MScAgric (Plant Pathology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: A survey was undertaken on apple and pear trees in the Western Cape Province to determine the aetiology of trunk diseases with reference to trunk diseases occurring on grapevine. Grapevine trunk diseases cause the gradual decline and dieback of vines resulting in a decrease in the vine’s capability to carry and ripen fruit. In recent years, viticulture has been expanding into several of the well established pome fruit growing areas. The presence of trunk pathogens in pome fruit orchards may affect the health of the pome fruit trees as well as cause a threat to young vineyards planted in close proximity to these potential sources of viable inoculum. Several genera containing species known to be involved in trunk disease on pome fruit and grapevine were found, including Diplodia, Neofusicoccum, Eutypa, Phaeoacremonium and Phomopsis. Diplodia seriata and D. pyricolum, were isolated along with N. australe and N. vitifusiforme. Four Phaeoacremonium species, P. aleophilum, P. iranianum, P. mortoniae and P. viticola, two Phomopsis species linked to clades identified in former studies as Phomopsis sp. 1 and Phomopsis sp. 7, and Eutypa lata were found. In addition, Paraconiothyrium brasiliense and Pa. variabile, and an unidentified Pyrenochaetalike species were found. Of these the Phaeoacremonium species have not been found on pear wood and it is a first report of P. aleophilum occurring on apple. This is also a first report of the Phomopsis species and Eutypa lata found occurring on pome trees in South Africa Two new coelomycetous fungi were also found including a Diplodia species, Diplodia pyricolum sp. nov., and a new genus, Pyrenochaetoides gen. nov. with the type species, Pyrenochaetoides mali sp. nov., were described from necrotic pear and apple wood. The combined ITS and EF1-α phylogeny supported the new Diplodia species, which is closely related to D. mutila and D. africana. The new species is characterised by conidia that become pigmented and 1-septate within the pycnidium, and that are intermediate in size between the latter two Diplodia species. Phylogenetic inference of the SSU of the unknown coelomycete provided bootstrap support (100%) for a monophyletic clade unrelated to known genera, and basal to Phoma and its relatives. Morphologically the new genus is characterised by pycnidial with elongated necks that lack setae, cylindrical conidiophores that are seldomly branched at the base, and Phoma-like conidia. The phylogenetic results combined with its dissimilarity from genera allied to Phoma, lead to the conclusion that this species represents a new genus. A pathogenicity trial was undertaken to examine the role of these species on apple, pear and grapevine shoots. N. australe caused the longest lesions on grapevine shoots, while Pyrenochaetoides mali, Pa. variabile, D. seriata and P. mortoniae caused lesions that were significantly longer than the control inoculations. On pears, D. pyricolum and N. australe caused the longest lesions, followed by D. seriata and E. lata. On apples, the longest lesions were caused by N. australe and P. iranianum. D. seriata, D. pyricolum, E. lata, N. vitifusiforme, Pa. brasiliense, P. aleophilum and P. mortoniae also caused lesions on apple that were significantly longer than the control. The study demonstrated that close cultivation of grapevine to apple and pear orchards may have inherent risks in terms of the free availability of viable inoculum of trunk disease pathogens. / No Afrikaans abstract available.

Thesis (MScAgric)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: The aim of this study was to determine the effect of a grass herbicide, a broadleaf herbicide with some grass control capabilities, method of tillage (tine and no-tillage) at planting of wheat as well as different medic cultivars on the regeneration, dry matter (OM) production and quality of a medic pasture. The trial was conducted at Langgewens experimental farm in the Swartland wheat producing area. Nine medic cultivars of three different species were evaluated after being sprayed with either haloxyfop-R-methyl (HAL) ester or imazamox (IMI) and subjected to either a tine tillage or a no tillage treatment at planting of wheat. Soil samples were taken during January 2000 to determine the size of the medic and weed seedbank as well as the degree of dormancy in the medic seeds, while OM samples were taken throughout the growing season to determine the OM production of the different medic cultivars and weed species. OM samples taken during October 1998 on the same pasture, were used to determine the crude protein (CP) and neutral detergent fibre (NOF) content of the pasture. The samples were subjected to in vitro digestion and the digestibility of pasture CP (OCP), NOF(ONOF) and DM (DOM)were determined. Results showed that seedling establishment differed between cultivars used, herbicide treatments applied as well as the crop stage in the rotation. The cultivars produced more seedlings where IMI was applied compared to HAL as well as where the area consisted of two year pasture compared to one year pasture (1998) and one year wheat (1999). After a year of pasture and a year of wheat, cultivars Sephi and Paraggio produced the most seedlings, while Caliph and Orion produced the least. Caliph however, showed a very high degree of seed dormancy while Orion's low seedling establishment was due to its sensitivity to the IMI herbicide used. Little difference was found between the nine cultivars early in the season (July - August) with regard to cumulative OM production, except for Orion, whose growth was severely damaged by the IMI treatment. At the end of the growing season (October), the cultivar Caliph's cumulative OM production (2010.1 kg/ha) was significantly higher than all the other cultivars, except for Parabinga (1053. 4 kg/ha). Oifferent pasture samples, of which the botanical composition was known, was analysed for CP, NOF, OOM, OCP and ONOF. There was no significant difference in pasture composition during 1998 but variation in the pasture composition did however cause the IMI treatment, compared to the HAL treatment, to have a lower ONOFand OOMcontent. A modelling procedure was used to predict the pasture quality parameters (CP, NOF, OOM,OCP and ONOF) from the pasture composition (medic hay, medic pods, grassy and broadleaf weeds). This prediction of CP, NOF, OOM, ONOF and OCP from the pasture components had a relative low accuracy (49 -74.1 %) and a further refinement of this model for possible use on farms in order to improve grazing management and animal production is advised. In conclusion it could be said that broadleaf weed control caused a definite increase in medic seed and OMproduction, but Orion should not be used with an IMI herbicide. All the cultivars, except for Orion, produced enough seedlings up to the second year to ensure sustainability of the medic pasture. All the cultivars, except for Orion, produced a sufficient amount of OM early in the growing season. Caliph however, produced by far the most OM later in the growing season. A reduction of broadleaf weeds and medic pods will increase the digestibility of NOFand OMand therefore increase the quality of the pasture. Pods however are an important part of summer forage and the aim should therefore rather be to reduce the number of broadleaf weeds in the pasture. / AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om die effek van 'n gras en breëblaar onkruiddoder (wat sekere grasse beheer), metode van bewerking tydens die saai van koring asook nege verskillende medic kultivars op die regenerasie, droë materiaal produksie en kwaliteit van medic weidings te bepaal. Die proef is gedoen op Langgewens proefplaas wat geleë is in die Swartland koring produserende gebied. Nege medic kultivars is geëvalueer nadat die weiding met of haloxyfop-R-metiel ester (HAL) of imazamox (IMI) onkruiddoders gespuit is en onderwerp is aan of 'n vlak tand of geen bewerking tydens die saai van koring. Grondmonsters is geneem in Januarie 2000 om die grootte van die medic en onkruid saadbank asook om die graad van dormansie in die verskillende medic kultivars se sade te bepaal. Droë materiaal monsters is gedurende die 2000 groeiseisoen geneem om die droë materiaal produksie van die verskillende medic kultivars asook onkruid spesies te bepaal. Droë materiaal monsters is gedurende Oktober 1998 geneem en gebruik om die ruproteïn (CP) en neutraaloplosbare vesel (NDF) inhoud van die weiding te bepaal. Die monsters is in vitro verteer en die verteerbaarheid van CP (OCP), NDF (ONOF) en droë materiaal (DOM) is bepaal. Resultate wys dat saailing vestiging verskil tussen die verskillende kultivars wat gebruik is, verskillende onkruiddoder behandelings asook die stadium van die weidings/koring. Die kultivars het meer geproduseer waar die weiding met IMI behandel is in vergelyking met waar HAL toegedien is, asook waar koring nog nie gesaai is nie. Na 'n jaar van weiding en 'n jaar van koring, het die kultivars Sephi en Paraggio die meeste saailinge, en Caliph en Orion die minste saailinge gehad. Caliph het egter 'n hoë graad van dormansie in sy saad getoon, terwyl die swak vestiging van Orion die gevolg is van die kultivar se hoë sensitiwiteit teenoor IMI. Min verskil is gevind tussen die nege kultivars, vroeg in die groei seisoen (Julie - Augustus), wat kumulatiewe droë materiaal produksie betref, behalwe vir Orion wat erg beskadig is deur die IMI behandeling. Aan die einde van die groeiseisoen (Oktober 2000) was die kumulatiewe droë materiaal produksie van die kultivar Caliph (2010.1 kg/ha) betekenisvol hoër as al die ander kultivars behalwe vir Parabinga (1053.4 kg/ha). Weidingsmonsters, waarvan die botaniese samestelling bekend was, is ontleed vir CP, NDF, DDM, DCP en DNDF. Daar is geen betekenisvolle verskille gevind in die botaniese samestelling van die weidingmonsters geneem in 1998 nie,maar die variasie in botaniese samestelling het veroorsaak dat IMI in vergelyking met HAL 'n laer DNDF and DDM inhoud het. 'n Model is opgestel wat die weidingskomponente (medic hooi, medic peule, gras en breëblaar onkruide) gebruik om die kwaliteits parameters (CP, NDF, DDM, DCP en DNDF) van die weiding te skat. Hierdie skatting van CP, NDF, DDM, DCP en DNDF deur van die weidingskomponente gebruik te maak het 'n relatiewe lae akuraatheid gehad (49 -74.1 %) en verdere verfyning van hierdie model vir moontlike gebruik op plase, ten einde weidings bestuur en diere produksie te verbeter, word voorgestel. Die gevolgtrekking kan gemaak word dat breëblaar onkruidbeheer 'n definitiewe verbetering in die medic saailing en droë materiaal produksie van die medies te weeg gebring het, maar die kultivar Orion behoort nie saam met 'n IMI gebruik te word nie. AI die getoetste kultivars, behalwe Orion, het voldoende saad oorlewing vertoon tot en met die tweede jaar van die rotasie om lewensvatbaarheid van die sisteem te verseker en alle kultivars, behalwe Orion, het voldoende droë materiaal produseer vroeg in die groeiseisoen. Caliph het egter laat in die groeiseisoen by verre die meeste droë materiaal geproduseer. 'n Vermindering in die hoeveelheid breëblaar onkruide en peule in die weiding sal tot 'n verhoging in die verteerbaarheid van NDF en DM lei en dus 'n verhoging in die kwaliteit van die weiding tot gevolg hê. Peule is egter 'n belangrike bron van voedsel aan weidende diere gedurende droë somermaande en die verbetering van weidings moet eerder gedoen word deur te poog om breëblaar onkruide te beheer.

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(Agric)--Stellenbosch University, 1973. / ENGLISH ABSTRACT: no abstract available / AFRIKAANSE OPSOMMING: geen opsomming

Thesis (MScAgric)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Grapevine trunk diseases are responsible for reduced wine and table grape production world-wide. Trunk disease infections are caused by xylem-inhabiting pathogens which include species of Botryosphaeriaceae, Diatrypaceae, Hymenochaetales and Diaporthales, as well as Phaeomoniella chlamydospora and Phaeoacremonium spp. Winter pruning wounds are regarded as the main infection-sites for trunk disease pathogens. However, the role of sucker wounds as portals of trunk disease infections has been minimally investigated. Knowledge of the potential role of grapevine trunk pathogen infections that occur through sucker wounds is important for better wound protection strategies. The aim of this study was to determine the role of grapevine sucker wounds as portals of entry for trunk disease pathogens and to assess the use of Trichoderma spp. for sucker wound protection. The susceptibility of sucker wounds to different trunk disease pathogens was assessed from natural as well as artificial infections. In addition the duration of sucker wound susceptibility in the field was also ascertained. Sucker wounds were sampled from three wine and two table grape vineyards during 2011 and 2012 in the Western Cape province of South Africa. Thereafter, fungal isolations were made from 161 sucker wounds and the cultures were identified based on cultural and morphological characteristics as well as the internal transcribed spacer regions and 5.8S ribosomal RNA gene. Sixty-two percent of the wounds were naturally infected by at least one of the trunk pathogens. Phomopsis (Po.) viticola (46%; 18%), Diplodia (D.) seriata (30%; 9%) and Phaeomoniella (Ph.) chlamydospora (27%; 5%) were the most predominant trunk disease pathogens isolated from sucker wounds of field wine and table grape cultivars, respectively. Lower incidences of Phaeoacremonium aleophilum (18%), Eutypella sp. (3%), Cryptovalsa ampelina (2%), Diplodia sp. (1%) and Neofusicoccum australe (1%) were obtained, however, only from wine grapes. Sucker wounds on 1-year-old potted grapevine plants of Chardonnay cultivar were inoculated with spore suspensions of Eutypa lata, N. parvum, Pa. aleophilum, Ph. chlamydospora and Po. viticola in the glasshouse. After 4 months all the inoculated pathogens could be re-isolated at the following incidences: N. parvum (85%), Ph. chlamydospora (75%), Po. viticola (65%), Pa. aleophilum (55%) and E. lata (45%). Sucker wound susceptibility was further ascertained under field conditions on 12-year-old Cabernet Sauvignon vines by artificial inoculation of the same pathogen species. After 5 months three pathogens could be re-isolated at the following incidences: Po. viticola (65%), N. parvum (32.5%) and Ph. chlamydospora (7.5%). The duration of susceptibility of field sucker wounds to Ph. chlamydospora was assessed for a period of 4 weeks. The wounds remained susceptible for 4 weeks with a decline in susceptibility after one week. This study showed that sucker wounds are susceptible to the major trunk disease pathogens and thus could play an important role in grapevine trunk disease epidemiology. In the second part of this thesis a possible management strategy to prevent infections of sucker wounds was investigated. The use of Trichoderma (T.) harzianum against two trunk pathogens on sucker wounds was tested in the field. Additionally the sensitivity of T. harzianum and T. atroviride was tested in vitro against 16 fungicides that are used to control powdery mildew, downy mildew, Botrytis rot and Phomopsis cane and leaf spot. In October 2012, sucker wounds were made on 1-year-old wood of Cabernet Sauvignon and spray-treated with Eco-77® immediately after desuckering, and then inoculated with spore suspensions of either Ph. chlamydospora or Po. viticola after 24 hours. After 5 months, isolations were made from the sucker wounds to evaluate the efficacy of the Trichoderma treatment. Trichoderma harzianum reduced the incidence of Ph. chlamydospora by 66.65%. Although the incidence of Po. viticola was reduced by 15.37%, it was not significantly different from the control treatment. The inhibition of mycelial growth and conidial germination of T. harzianum and T. atroviride were screened against 16 fungicides. The fungicides were applied at 0, 0.25, 0.5, 1 and 2 times the recommended dosages. Systemic fungicides boscalid, metrafenone and trifloxystrobin, as well as contact fungicides quinoxyfen and meptyldinocap were least toxic to Trichoderma spp. isolates. For the conidial germination assay, boscalid, trifloxystrobin, penconazole and metrafenone (systemic) plus quinoxyfen and folpet (contact) were compatible with Trichoderma spp. These fungicides were regarded as being compatible with Trichoderma spp. isolates because they gave mean percentage inhibitions of less than 50% at all the tested dosages. Spiroxamine and pyrimethanil gave the highest mean percentage inhibitions for both mycelial inhibition and conidial germination. The findings of this study showed that T. harzianum can protect sucker wounds against Ph. chlamydospora in the field. Furthermore, some fungicides applied for the control of powdery mildew and Phomopsis cane and leaf spot can be alternatively or simultaneously applied with T. harzianum and T. atroviride, however, this will have to be verified with field trials. / AFRIKAANSE OPSOMMING: Wingerd stamsiektes is wêreldwyd verantwoordelik vir verminderde wyn- en tafeldruif produksie. Stamsiektes word veroorsaak deur patogene wat in die xileem voorkom, insluitend verskeie spesies in die Botryosphaeriaceae, Diatrypaceae, Hymenochaetales en Diaporthales, asook Phaeomoniella chlamydospora en Phaeoacremonium spp. Winter snoeiwonde word beskou as die hoof bron van infeksies vir stamsiekte patogene. Die rol van suierwonde as poorte van infeksie vir stamsiektes is nog nie goed bestudeer nie. Kennis van die potensiële rol van wingerd stamsiekte patogeen infeksies wat deur suierwonde plaasvind is belangrik vir die formulasie van beter wondbeskerming strategieë. Die mikpunt van hierdie studie was om die rol van suierwonde as ingangsportale vir wingerd stamsiekte patogene te bepaal en om die gebruik van Trichoderma spp. vir suierwond beskerming te evalueer. Die vatbaarheid van suierwonde vir verskillende stamsiekte patogene is geëvalueer vanuit natuurlike, sowel as kunsmatige infeksies. Die duur van suierwond vatbaarheid in die veld is ook bepaal. Suierwonde is versamel vanuit drie wyn- en twee tafeldruif wingerde gedurende 2011 en 2012 in die Wes Kaap provinsie van Suid Afrika. Hierna is swam isolasies gemaak vanuit 161 suierwonde en die kulture is geïdentifiseer volgens kultuur en morfologiese kenmerke, sowel as die interne transkribeerde spasieerders en 5.8S ribosomale RNA geen. Twee-en-sestig persent van die wonde was geïnfekteer deur ten minste een van die stamsiekte patogene. Phomopsis (Po.) viticola (46%; 18%), Diplodia (D.) seriata (30%; 9%) en Phaeomoniella (Ph.) chlamydospora (27%; 5%) was die mees algemene stamsiekte patogene wat, respektiewelik, vanuit die wyn- en tafeldruif kultivars verky is. Laer hoeveelhede Phaeoacremonium aleophilum (18%), Eutypella sp. (3%), Cryptovalsa ampelina (2%), Diplodia sp. (1%) en Neofusicoccum australe (1%) is verkry, en slegs vanaf wyndruiwe. Suierwonde op 1-jaar oue Chardonnay wingerdplante in potte is in die glashuis geïnokuleer met spoorsuspensies van Eutypa lata, N. parvum, Pa. aleophilum, Ph. chlamydospora en Po. viticola. Na 4 maande kon al die geïnokuleerde patogene her-isoleer word teen die volgende hoeveelhede: N. parvum (85%), Ph. chlamydospora (75%), Po. viticola (65%), Pa. aleophilum (55%) en E. lata (45%). Suierwond vatbaarheid is verder geëvalueer onder veld kondisies op 12-jaar oue Cabernet Sauvignon plante deur kunsmatige inokulasie van die selfde patogeen spesies. Na 5 maande kon drie patogene her-isoleer word teen die volgende hoeveelhede: Po. viticola (65%), N. parvum (32.5%) en Ph. chlamydospora (7.5%). Die duur van vatbaarheid van suierwonde teen Ph. chlamydospora in die veld is geevalueer oor ‘n periode van 4 weke. Die wonde het vatbaar gebly vir 4 weke met ‘n afname in vatbaarheid na ‘n week. Hierdie studie demonstreer dat suierwonde vatbaar is vir die hoof wingerd stamsiektes en dus ‘n belangrike rol in die epidemiologie van wingerd stamsiektes kan speel. In die tweede deel van hierdie tesis is ‘n moontlike bestuurs-strategie ondersoek om infeksie van suierwonde te verhoed. Die gebruik van Trichoderma (T.) harzianum teen twee stampatogene op suierwonde is getoets in die veld. Verder is die in vitro sensitiwiteit van T. harzianum en T. atroviride getoets teen 16 fungisiedes wat gebruik word in die beheer van poeieragtige meeldou, donsskimmel, Botrytis vrot en Phomopsis streepvlek. Gedurende Oktober 2012 is suierwonde gemaak op 1-jaar oue hout van Cabernet Sauvignon en onmiddelik behandel met Eco-77® na suiering. Wonde is dan geïnokuleer met spoorsuspensies van óf Ph. chlamydospora óf Po. viticola na 24 uur. Na 5 maande is isolasies gemaak vanaf suierwonde om die doeltreffendheid van van die Trichoderma behandeling te evalueer. Trichoderma harzianum het die voorkoms van Ph. chlamydospora met 66.65% verminder. Alhoewel die voorkoms van Po. viticola verminder is met 15.37%, was dit nie ‘n beduidende verskil in vergelyking met die kontrole behandeling nie. Die inhibisie van miselium groei en konidia ontkieming van T. harzianum en T. atroviride is getoets teen 16 fungisiedes. Die fungisiedes is aangewend teen 0, 0.25, 0.5, 1 en 2 keer die aanbevole dosisse. Sistemiese fungisiedes boscalid, metrafenone en trifloxystrobin, sowel as kontak fungisiedes quinoxyfen en meptyldinocap was die minste toksies teen Trichoderma spp. Gedurende die konidia ontkiemingstoets was boscalid, trifloxystrobin, penconazole en metrafenone (sistemies) en quinoxyfen en folpet (kontak) versoenbaar met Trichoderma spp. Die fungisiedes is beskou as bruikbaar met Trichoderma spp. isolate omdat hulle gemiddelde persentasie inhibisies van minder as 50% teen al die getoetste dosisse gelewer het. Spiroxamine en pyrimethanil het die hoogste gemiddelde persentasie inhibisie gelewer vir beide die miselium inhibisie en konidia ontkieming. Die bevindings van hierdie studie het gewys dat T. harzianum suierwonde kan beskerm teen Ph. chlamydospora in die veld. Verder sou sommige fungisiedes wat aangewend word vir die bestuur van poeieragtige meeldou en streepvlek moontlik alternatiewelik of gelyktydig met T. harzianum en T. atroviride aangewend word, alhowel dit met veldproewe bevestig moet word.

Thesis (PhDAgric (Plant Pathology))--Stellenbosch University, 2010. / ENGLISH ABSTRACT: In the Western Cape onion industry in South Africa, Fusarium oxysporum Schlechtend.:Fr. f.sp. cepae (H.N. Hans.) W.C. Snyder & H.N. Hans. (Focep) has been identified as the leading cause of harvest and storage losses. This pathogen is of world-wide importance and causes Fusarium basal rot of onions (Allium cepa), affecting all onion growth stages. No information is available on the evolution, genetic diversity, molecular detection and inoculum sources of the South African Focep population. Similar to what is the case for South Africa, limited information is available on Focep in other regions of the world. World-wide, four vegetative compatibility groups (VCGs) and two single-member VCGs (SMVs) have been identified among two Japanese and 19 Colorado (USA) isolates. This polyphyletic origin of Focep suggested by VCG analyses was confirmed through molecular analyses of isolates from a few countries. Only the mating type (MAT)1-1 idiomorph has been reported for Focep isolates from Welsh onion (Allium fistulosum). The development of sustainable management strategies of Focep is dependent on knowledge of (i) the genetic diversity and evolution of Focep, (ii) whether high throughput molecular methods can be developed for identifying the most virulent and widespread Focep genotypes and (iii) the role of seedlings and seeds as primary inoculum sources, and the Focep genotypes associated with these growth stages. Therefore, the three main aims of the current study were to investigate the aforementioned three aspects. In the first aim of the study, the genetic diversity and evolution of Focep was investigated using a collection of 79 F. oxysporum isolates from South Africa (27 Focep and 33 non-pathogenic isolates) and Colorado (19 Focep isolates). VCG analyses revealed the presence of six VCGs, four among the Colorado Focep isolates (VCGs 0421, 0422, 0423 and 0424) and two among the South African bulb-associated isolates (VCGs 0425 and 0426). VCG 0421 and VCG 0425 were the two main VCGs in Colorado and South Africa, respectively. Four SMVs and one heterokaryon selfincompatible (HSI) isolate were also identified. The polyphyletic nature of Focep in South Africa and Colorado was shown through a combined translation elongation factor 1α (EF-1α) and mitochondrial small-subunit (mtSSU) phylogeny. The phylogeny divided the Focep isolates into two main clades, of which one contained the two main VCGs (0421 and 0425), SMVs and non-pathogenic isolates. The second, ancestral clade contained the HSI isolate, VCGs 0422, 0423 and 0424, and non-pathogenic isolates. Unlike the clade containing the two main VCGs, which were highly virulent toward onion bulbs, the ancestral clade contained isolates that were mostly moderately virulent. The incongruence of the EF-1α and mtSSU datasets with an intergenic spacer (IGS) region data set, and the presence of both MAT idiomorphs within the same isolate for some isolates, suggested possible exchange of genetic material between isolates. The second aim of the study was to develop molecular methods for identifying the two main Focep VCGs (0425 and 0421), using DNA fingerprinting methods and sequence-characterized amplified region (SCAR) markers. These techniques were first developed using the F. oxysporum isolates from the first aim, and were then used to investigate the prevalence of VCG 0425 among 88 uncharacterized F. oxysporum isolates from onion bulbs in South Africa. Two random amplified polymorphic DNA primers provided two diagnostic amplicons for VCG 0425, but attempts to develop SCAR markers from these amplicons were unsuccessful. In contrast, an interretrotransposon amplified polymorphism (IRAP) fingerprinting method enabled the developed of a multiplex IR-SCAR polymerase chain reaction method that detected the VCG 0421, 0425 and SMV 4 isolates as a group. Fingerprinting and SCAR marker testing of the 88 uncharacterized F. oxysporum isolates from South Africa (65 Focep and 23 non-pathogenic) confirmed that VCG 0425 is the main VCG in South Africa associated with mature onion bulbs, since 63 of the Focep isolates had the molecular characteristics of VCG 0425. The third aim of the study was to determine whether seed and seedling transplants are inoculum sources of Focep, and whether the same genotype (VCG 0425) that dominated on mature bulbs could be detected from these sources. Focep isolates were obtained from seven of the 13 investigated onion seed lots, as well as from onion seedling transplants that were collected from all five onion nurseries in the Western Cape. Focep seedling infection more than doubled from the 6-week growth stage to the 14-week growth stage. Seed infections by Focep were low, but the seedborne nature of Focep was confirmed by showing that a green fluorescent protein labelled Focep transformant could be transmitted from infected soil to onion seed via the onion bulbs and seedstalks. It is thus clear that commercial seed and seedlings are inoculum sources of Focep. However, the Focep genotypes on seed and seedlings are different from those in mature bulbs and were not dominated by VCG 0425. Furthermore, most (≤ 60%) of the seed and seedling isolates were moderately virulent, as compared to the mostly highly virulent isolates from mature bulbs. / AFRIKAANSE OPSOMMING: In die Wes-Kaapse uiebedryf in Suid-Afrika is Fusarium oxysporum Schlechtend.:Fr. f.sp. cepae (H.N. Hans.) W.C. Snyder & H.N. Hans. (Focep) geïdentifiseer as die vernaamste oorsaak van oes- en opbergingsverliese. Hierdie patogeen is van wêreldwye belang; dit veroorsaak Fusarium-bolvrot van uie (Allium cepa) en affekteer alle plantgroeistadia. In Suid-Afrika is daar geen inligting beskikbaar oor die evolusie, genetiese diversiteit, molekulêre opsporing en inokulumbronne van die Focep-populasie nie. Soortgelyk aan wat die geval in Suid-Afrika is, is daar beperkte inligting beskikbaar oor Focep in ander wêrelddele. Wêreldwyd is daar vier vegetatiewe versoenbaarheidsgroepe (VVGe) en twee enkellid VVGe (ELVe) geïdentifiseer onder twee Japannese en 19 Colorado (VSA) isolate. Hierdie veelvuldige oorsprong van Focep wat deur VVG-analise voorgestel was, is deur die molekulêre analises van isolate uit ’n paar ander lande bevestig. Slegs die paringstipe (PT)1-1 idiomorf is vir Focep-isolate uit Walliese-tipe uie (ook bekend as ‘lenteuie’ in Suid Africa) (Allium fistulosum) berig. Die ontwikkeling van volhoubare bestuurstrategieë vir Focep steun op kennis van (i) die genetiese diversiteit en evolusie van Focep, (ii) of hoë-deurset molekulêre metodes ontwikkel kan word vir die identifisering van die mees virulente en wydverspreide Focep-genotipes en (iii) die rol van saailinge en saad as primêre inokulumbronne, en die Focep-genotipes wat met hierdie groeistadia geassosieer word. Daarom was die hoof doelstellings van hierdie studie om die bogenoemde drie aspekte te bestudeer. Om die eerste doel van die studie te bereik is die genetiese diversiteit en evolusie van Focep bestudeer deur gebruik te maak van ‘n versameling van 79 F. oxysporum-isolate uit Suid-Afrika (27 Focep en 33 nie-patogeniese isolate) en uit Colorado (19 Focep-isolate). VVG-analises het die teenwoordigheid van ses VVGe aangetoon – vier onder die Colorado Focep-isolate (VVGe 0421, 0422, 0423 en 0424) en twee onder die Suid-Afrikaanse bol-geassosieerde isolate (VVGe 0425 en 0426). VVG 0421 en VVG 0425 was die twee hoof VVGe in onderskeidelik Colorado en Suid-Afrika. Vier ELVe en een meerkernige self-onversoenbare (MSO) isolaat is ook geïdentifiseer. Die veelvuldige oorsprong van Focep in Suid-Afrika en Colorado is ook aangetoon deur ‘n gekombineerde translasie verlengings faktor 1α (VF-1α) en mitokondriale klein-subeenheid (mtKSE) filogenie. Dié filogenie het die Focepisolate in twee groepe verdeel, waarvan die een groep die twee hoof VVGe (0421 en 0425), ELVe en nie-patogeniese isolate bevat het. Die tweede, basal groepering het die MSO-isolaat, VVGe 0422, 0423 en 0424, en nie-patogeniese isolate bevat. In teenstelling met die eersgenoemde groepering wat hoogs virulente isolate van uiebolle bevat het, het die basale groepering isolate bevat wat meestal matig virulent was. Die inkongruensie van die VF-1α en mtKSE-datastelle met ‘n intergeen-gespasieerde (IGS) area datastel – asook die teenwoordigheid van beide PT-idiomorwe binne dieselfde isolaat by sommige isolate – het op ’n moontlike uitruiling van genetiese materiaal tussen isolate gedui. Die tweede doel van die studie was om molekulêre metodes te ontwikkel vir die identifisering van die twee hoof Focep VVGe (0425 en 0421) deur gebruik te maak van DNA-vingerafdrukke en nukleotied-gekarakteriseerde geamplifiseerde area (NKAA) merkers. Hierdie tegnieke is ontwikkel deur van die F. oxysporum-isolate van die eerste doelstelling gebruik te maak en is daarna gebruik om die frekwensie van VVG 0425 onder 88 ongekarakteriseerde F. oxysporum-isolate van uiebolle in Suid-Afrika te ondersoek. Twee gerandomiseerde geamplifiseerde polimorfiese DNS (RAPD) merkers het twee diagnostiese nukleotiedbasis-areas vir VVG 0425 gelewer, maar pogings om NKAA-merkers uit hierdie geamplifiseerde nukleotiedbasis-areas te onwikkel was onsuksesvol. In teenstelling hiermee het ‘n inter-retrotransposon geamplifiseerde polimorfisme (IRAP) vingerafdrukmetode die ontwikkeling van ‘n multipleks IR-NKAA polimerase kettingreaksiemetode moontlik gemaak wat die VVG 0421-, VVG 0425- en ELV 4-isolate as ’n groep aangedui het. Vingerafdruktoetsing en NKAA-merkertoetsing van die 88 ongekaraktariseerde F. oxysporum isolate van Suid-Afrika (65 Focep en 23 nie-patogenies) het bevestig dat VVG 0425 die hoof VVG in Suid-Afrika is wat met volwasse bolle geassosieer word, aangesien 63 van die Focep-isolate die molekulêre eienskappe van VVG 0425 gehad het. Die derde doel van die studie was om vas te stel of saad en saailinge inokulumbronne van Focep is, en of dieselfde genotipe (VVG 0425) wat op volwasse bolle dominant is, waargeneem kon word op hierdie bronne. Focep-isolate is verkry van sewe van die 13 uiesaadlotte asook van uiesaailinge wat in al vyf uiesaailingkwekerye in die Wes-Kaap versamel is. Focep-saailinginfeksie was meer as dubbel in die 14-week groeistadium as wat dit in die 6-week stadium was. Saadinfeksies deur Focep was laag, maar die saadgedraagde aard van Focep is bevestig deur aan te toon dat ’n Focep-transformant wat met ‘n groen fluoreserende proteïen geëtiketeer is, van geïnfekteerde grond na uiesaad oorgedra kon word via die uiebolle en -saadstele. Dit is dus duidelik dat kommersiële saad en saailinge as inokulumbronne van Focep dien. Die Focep-genotipes op saad en saailinge verskil egter van dié in volwasse bolle en is nie deur VVG 0425 gedomineer nie. Verder was die meeste (≤ 60%) saad- en saailingisolate matig virulent, in teenstelling met die meestal hoogs virulente isolate uit volwasse bolle.