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.

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.

Thesis (PhD(Agric))--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Despite adherence to fungicide spray schedules and label recommendations, table and wine grape producers invariably suffer crop losses when environmental conditions are conducive to fruit and foliar pathogens. Registered fungicides are effective and poor control is often attributed to: 1) improper spray timing, 2) reduced sensitivity to fungicides in the pathogen populations, and 3) poor spray deposition. Spray timing, management of fungicide resistance and the epidemiology of Botrytis cinerea have been thoroughly researched under South African conditions on grape crops. However, limited research regarding spray deposition exists in South Africa, probably due to a lack of proper spray deposition assessment protocols. To determine minimum spray deposition quantity and quality levels needed for effective B. cinerea control, bunches and leaves of table (Waltham Cross) and wine grapes (Chenin blanc) were sprayed at various stages using different volumes with a precision spray gun. A deposition assessment protocol using fluorometry, photomicrography and digital image analyses was improved. Deposition values correlated favourably with Botrytis infection. Increasing spray volume increased spray deposition; however, at a certain point, deposition quality remained constant and B. cinerea infections did not decrease significantly with increasing spray volume, indicating the importance of both spray deposition quantity and quality. Fluorescent pigment area that effected 75% control of B. cinerea infection (FPC75 values) was calculated for leaves, pedicels and receptacles at different growth stages. The FPC75 values obtained in this study can be used as benchmarks to evaluate future spray application. In order to study the optimisation of spray deposition with existing application technology (air blast and air shear sprayers) in commercial vineyards, spray deposition quantity and quality values were assessed from leaves and structural bunch parts of wine (Chenin blanc) and table grapes (Waltham Cross) and compared with FPC75 values. Spray trials were conducted at different growth stages at current best-practice recommendations, and with a range of spray volumes but with spray mixture concentration amended accordingly (i.e. fixed dosage per hectare). Spray trails indicated that deposition levels following current best-practice spray application were sub-optimal to control B. cinerea infections on bunches and leaves. Deposition values between air blast and air shear sprayers were generally similar. The air blast sprayer resulted in higher deposition levels with diluted spraying and increased spray volume; however, when dosage per hectare was kept constant, no significant differences were calculated between spray volumes (250-1000 L/ha), indicating that this sprayer can as effectively but more efficiently be used at lower spray volume. The air shear were not as efficient at higher spray volumes (>500 L/ha), but was superior at low volume concentrate application (≈250 L/ha at 4× concentration). This study clearly demonstrated the efficacy and cost-saving potential in optimising spray application with respect to application technology. / AFRIKAANSE OPSOMMING: Wingerdprodusente kan oesverliese ondervind indien omgewingstoestande bevorderlik is vir swampatogene. Siektes word onvoldoende beheer ten spyte van die nakoming van korrekte swamdoder aanbevelings. Geregistreerde swamdoders is effektief, mits die vatbare plantdele voldoende spuitbedekking ontvang. Onvoldoende siekte beheer kan gewoonlik toegeskryf word aan: 1) verkeerde spuit tydsberekening, 2) vermindere sensitiwiteit in patogeen-populasies teen swamdoders, en 3) swak spuitbedekking. Spuit tydsberekening, die bestuur van weerstand teen swamdoders en die epidemiologie van Botrytis cinerea is deeglik onder Suid-Afrikaanse toestande nagevors. Nietemin is daar beperkte navorsing oor spuitbedekking, waarskynlik weens 'n gebrek aan behoorlike spuitbedekking assesseringsprotokol. Om te bepaal hoeveel spuitbedekking (% area bedek deur fluoresserende pigment) nodig is om 75% van B. cinerea infeksies (FPC75 waardes) op vatbare wingerddele te beheer, is druiwetrosse en blare van tafel- en wyndruiwe (Waltham Cross en Chenin blanc, onderskeidelik) op verskillende groei stadiums en spuitvolumes in die laboratorium gespuit. ‘n Assesseringsprotokol van spuitbedekking op vatbare druifdele en blare is ontwikkel deur gebruik te maak van fluorometrie, fotomikrografie en digitale beeldanalise. Spuitbedekking het goed met Botrytis infeksies gekorreleer. Toenemende spuitvolume het bedekking laat toeneem, maar egter net tot 'n sekere punt, waar die kwantiteit van die bedekking nog toegeneem het, maar die kwaliteit van bedekking en B. cinerea infeksies nie beduidend toegeneem het nie. Dit is ‘n aanduiding van die belangrikheid van beide die kwantiteit en kwaliteit van spuitbedekking. Die FPC75 waardes wat in hierdie studie verkry is, kan as drempelwaardes om toekomstige spuittoediening te evalueer, gebruik word. Ten einde spuitbedekking met bestaande tegnologie (druk en waaierpomp spuitmasjiene) te optimiseer, is kommersiële wyn- en tafeldruiwe (Chenin blanc en Waltham Cross, onderskeidelik), volgens huidige spuit aanbevelings vir wingerde tydens verskillende groeistadiums en met ‘n reeks van verskillende spuitvolumes gespuit. Die konsentrasie van die spuitmengsel is dienooreenkomstig gewysig, i.t.v. ‘n vaste dosis per hektaar ongeag die spuitvolume. Bedekkingswaardes is met FPC75 waardes vergelyk en het aangedui dat kommersiële spuit aanbevelings aan produsente sal lei tot sub-optimale beheer van B. cinerea op beide blare en druiwetrosse. In die algemeen was bedekkingswaardes vir beide druk- en waaierpomp spuitmasjiene soortgelyk. Vir die waaierpomp teen verskillende spuitvolumes en aanbevole konsentrasie het ‘n toename in spuitvolumes tot hoër beddekingswaardes gelei, maar indien die dosis per hektaar van die spuitmengsel konstant behou is, is geen betekenisvolle verskille tussen spuitvolumes (250-1000 L/ha) voorspel nie. Hierdie dui aan dat die waaierpomp net so doeltreffend, maar meer effektief teen laer spuitvolumes gebruik kan word. Die drukpomp was nie so doeltreffend teen hoër spuitvolumes (> 500 L/ha) nie, maar was aansienlik beter by lae volume konsentraat toediening (≈ 250 L/ha op 4 × konsentrasie). Die studie toon duidelik die doeltreffendheid en moontlike kostebesparing moontlikhede deur bespuiting relatief tot bespuitingstegnologie te optimiseer. / Department of Plant Pathology, National Research Foundation, THRIP, Deciduous Fruit Producers’ Trust, Winetech, Bayer, BASF, Dow Agrosciences, DuPont, Syngenta, Nexus, Terason, UAP and Wenkem for financial assistance

Thesis (MScAgric)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Green mould is a major citrus postharvest disease caused by a wound pathogen called Penicillium digitatum. Imazalil (IMZ) is the most important postharvest fungicide that is currently registered against green mould and blue mould, given its good protective and curative control and sporulation inhibition. Imazalil can be applied in drench, dip, spray and wax coating applications. Wax coatings improve the quality of the fruit during shelf life by limiting moisture loss and providing a shiny appearance. The primary objective of this study was to investigate the application of imazalil in wax coatings in citrus packhouses to control green mould and preserve fruit quality. The first aim of this study was to evaluate the protective and curative control and sporulation inhibition of IMZ sensitive and resistant isolates of P. digitatum following single IMZ application in wax coating and IMZ application in aqueous dip as well as wax coating. For single application, Valencia orange fruit was curatively or protectively treated with a carnauba-based coating into which IMZ at 3000 μg.mL-1 was mixed. For curative treatments, fruit was treated after 24 hour's incubation. Coating was applied at 0.6, 1.2 and 1.8 L.ton-1 of fruit on a commercial coating applicator. Untreated control and fruit treated with coating only showed up to 80% infection for both sensitive and resistant isolates. Imazalil in coating proved to have better protective than curative action against the sensitive isolate, while control of the resistant isolate was poor. Imazalil residue levels increased with increasing coating load (0.85 to 1.75 μg.g-1). For double application, clementine, satsuma, navel and Valencia orange fruit were used. Curative and protective control following dip only treatment (IMZ sulphate at 500 μg.mL-1 for 45 s and 90 s) was compared with coating only treatment (IMZ at 3000 μg.mL-1 at 1.8 L.ton-1), and double application comprising dip (45 s in IMZ sulphate at 500 μg.mL-1) followed by 2000 μg.mL-1 IMZ in coating at 0.6, 1.2 and 1.8 L.ton-1. Double application resulted in improved residue loading (0.85 to 2.06 μg.g-1) compared to dip only treatments (≈ 0.49 μg.g-1). Coating only resulted in the highest residue levels (1.82 to 7.09 μg.g-1), often exceeding the maximum residue limit of 5 μg.g-1. In all treatments, poor curative and protective control of the resistant isolate (<46% and <55%, respectively) and generally no sporulation inhibition was observed. For the sensitive isolate, dip only treatments resulted in better curative control (≈ 77%) than protective control (≈ 38%). Double application showed increased protective control with increasing wax load (≈ 69%) as well as good curative control (≈ 83%). Wax coating only treatment resulted in poor curative control (≈ 26%), but good protective control (≈ 80%). Sporulation inhibition of the sensitive isolate was generally improved in treatments that included IMZ application in the wax coating. This study successfully showed the additive benefits of double application of IMZ in the wax coating following an aqueous dip application. However, despite improved control of the sensitive isolate, the resistant isolate could not be controlled. The second objective of the study was to evaluate green mould control and quality preservation following IMZ application using different coating and brush types. The inoculation and treatment procedure was similar to the single application of wax coating described above but only the sensitive isolate of P. digitatum was used. Carnauba- or polyethylene-based wax coatings, including IMZ at 3000 μg.mL-1, was applied at a dosage of 0.6, 1.2 and 1.8 L.ton-1 using commercial horsehair or synthetic brushes. Fruit was either incubated for 4 days prior to wound infection ratings or stored at -0.5oC for 26 days and 7 days shelf life. Imazalil residues on fruit increased with increasing coating loads on navel oranges (1.31 to 3.32 μg.g-1) and Valencia oranges (3.22 to 6.00 μg.g-1). Better protective (≈ 58%) than curative control (≈ 14%) was observed, with horsehair brushes resulting in lower sporulation levels (≈ 59%) than synthetic brushes (≈ 64%). Fruit weight loss and firmness loss ratios relative to uncoated control fruit were significantly higher in fruit treated with polyethylene coating compared with carnauba coating treated fruit. However, polyethylene coating resulted in shinier fruit before and after storage, whereas carnauba coating resulted in lower shine ratios. Gaseous (CO2) exchange ratios remained similar for both coatings, but higher polyethylene coating loads (1.8 L.ton-1) resulted in off-tastes similar to the uncoated control fruit and higher than the ratings for carnauba. Scanning electron micrographs revealed an amorphous crystallised natural wax layer with uncovered stomatal pores on the surface of uncoated fruit. The thickness of the applied coating layer increased with increasing coating load. Coating layers following application with synthetic brushes at 1.2 L.ton-1 appeared to be thicker than when applied using horsehair brushes. This study indicated the effects of brush type on the distribution and polishing of the wax coating on the surface of the fruit and also the importance of cautious application of coating as under or over application may lead to poor results in terms of bio-efficacy, residue loading and fruit quality. / AFRIKAANSE OPSOMMING: Groenskimmel is 'n belangrike sitrus na-oessiekte, veroorsaak deur 'n wondpatogeen, genaamd Penicillium digitatum. Imazalil (IMZ) is die belangrikste na-oesfungisied wat tans teen groenskimmel geregistreer is, gegewe sy goeie beskermende en genesende beheer en sporulasie-inhibisie. Imazalil kan in bad-, doop-, spuit- en wakslaagbehandelings toegedien word. Wakslae behou die kwaliteit van die vrugte gedurende raklewe deur vogverlies te beperk en 'n glansvoorkoms te verskaf. Die primêre doel van hierdie studie was om die toediening van imazalil in wakslaagbehandelings in sitruspakhuise vir groenskimmel en vrugkwaliteit beheer te ondersoek. Die eerste doelwit van hierdie studie was om die beskermende en genesende beheer en sporulasie-inhibisie van IMZ sensitiewe en weerstandbiedende isolate van P. digitatum, gevolg deur enkel IMZ toediening in 'n wakslaagbehandeling, en IMZ toediening in 'n doop-, asook wakslaagbehandeling, te evalueer. Vir enkelltoediening is Valencia lemoenvrugte genesend of beskermend met 'n carnauba-gebaseerde wakslaag behandel, waarin IMZ teen 3000 μg.mL-1 gemeng is. Vir genesende behandelings is vrugte ná 24 uur inkubasie behandel. Wakslaagbehandeling is teen 0.6, 1.2 en 1.8 L.ton-1 vrugte op 'n semi-kommersiële paklyn toegedien. Onbehandelde kontrole en vrugte met waks behandel, het slegs tot 80% infeksie vir beide sensitiewe en weerstandbiedende isolate getoon. Imazalil in waks het beter beskermende as genesende aksie teen die sensitiewe isolaat getoon, terwyl beheer van die weerstandbiedende isolaat swak was. Imazalil residuvlakke het met toename in wakslading toegeneem (0.85 tot 1.75 μg.g-1). Vir dubbeltoediening is clementine, satsuma, navel en Valencia lemoenvrugte gebruik. Genesende en beskermende beheer van IMZ na enkel doopbehandeling (IMZ sulfaat teen 500 μg.mL-1 vir 45 s en 90 s), is met enkel waksbehandeling (IMZ teen 3000 μg.mL-1 teen 1.8 L.ton-1) vergelyk, asook dubbeltoediening bestaande uit doop (45 s in IMZ sulfaat teen 500 μg.mL-1), gevolg deur 2000 μg.mL-1 IMZ in waksbehandeling teen 0.6, 1.2 en 1.8 L.ton-1. Dubbeltoediening het verbeterde residulading (0.85 tot 2.06 μg.g-1) tot gevolg gehad, in vergelyking met enkel doopbehandelings (≈ 0.49 μg.g-1). Enkel waksbehandeling het die hoogste residuvlakke (1.82 tot 7.09 μg.g-1) tot gevolg gehad, wat dikwels die maksimum residu-limiet van 5 μg.g-1 oorskry het. In alle behandelings is swak genesende en beskermende beheer van die weerstandbiedende isolaat (<46% en <55%, onderskeidelik), en oor die algemeen, geen sporulasie-inhibisie, waargeneem. Vir die sensitiewe isolaat, het die enkel doopbehandelings beter genesende beheer (≈ 77%) in vergelyking met die beskermende beheer (≈ 38%) tot gevolg gehad. Dubbeltoediening het verhoogde beskermende beheer met toenemende wakslading (≈ 69%) getoon, asook goeie genesende beheer (≈ 83%). Enkel wakslaagbehandeling het swak genesende beheer (≈ 26%) tot gevolg gehad, maar goeie beskermende beheer (≈ 80%). Sporulasie-inhibisie van die sensitiewe isolaat was oor die algemeen beter in behandelings wat IMZ toediening in die wakslaag ingesluit het. Hierdie studie het suksesvol die toegevoegde voordele van dubbeltoediening van IMZ in doopbehandeling en wakslaag getoon. Ten spyte van die verbeterde beheer van die sensitiewe isolaat, kon die weerstandbiedende isolaat egter nie beheer word nie. Die tweede doelwit was om groenskimmelbeheer en behoud van kwaliteit te evalueer, volgende op IMZ toediening deur gebruik van verskillende waks- en borseltipes. Inokulasie- en behandelingsprosedure was soortgelyk aan die enkeltoediening van wakslaagbehandeling soos bo beskryf, maar slegs die sensitiewe isolaat van P. digitatum is gebruik. Carnauba- of poli-etileen-gebaseerde wakslae, insluitende IMZ teen 3000 μg.mL-1, is teen 'n dosis van 0.6, 1.2 en 1.8 L.ton-1 toegedien, deur die gebruik van kommersiële perdehaar of sintetiese borsels. Vrugte is óf vir 4 dae vóór wond-infeksie evaluering geïnkubeer, óf teen -0.5oC vir 26 dae en 7 dae raklewe gestoor. Imazalil residuvlakke op vrugte het met toenemende waksladings op navellemoene (1.31 tot 3.32 μg.g-1) en Valencia lemoene (3.22 tot 6.00 μg.g-1) toegeneem. Beter beskermende (≈ 58%) as genesende beheer (≈ 14%) is waargeneem, met perdehaar borsels wat laer sporulasievlakke (≈ 59%) tot gevolg gehad het, in vergelyking met sintetiese borsels (≈ 64%). Vruggewig- en fermheidsverlies verhoudings, relatief tot onbedekte kontrole vrugte, was betekenisvol hoër in vrugte wat met 'n poli-etileenwaks behandel is, in vergelyking met carnauba-waks behandelde vrugte. Poli-etileen waksbehandeling het egter blinker vrugte vóór en ná opberging tot gevolg gehad, terwyl canauba waksbehandeling laer blink verhoudings tot gevolg gehad het. Gas (CO2) uitruilverhoudings het dieselfde vir beide waksbehandelings gebly, maar hoër poli-etileen waksladings (1.8 L.ton-1) het tot afsmake soortgelyk aan die onbedekte kontrole vrugte gelei, en hoër as die evaluerings vir carnauba. Skandeer-elektronmikrograwe het 'n amorfe gekristalliseerde natuurlike wakslaag met onbedekte stomata op die oppervlak van onbedekte vrugte getoon. Die dikte van die toegediende waks het met toename in wakslading toegeneem. Wakslae na toediening met sintetiese borsels teen 1.2 L.ton-1 het dikker vertoon in vergelyking met perdehaar borsels. Hierdie studie het die effekte van borseltipe op die verspreiding en polering van die wakslaag op die oppervlak van vrugte getoon, en versigtige toediening van wakslae onderskryf, aangesien ónder- of óórtoediening swak resultate, in terme van bio-effektiwiteit, residu-lading en vrugkwaliteit, tot gevolg kan hê. / National Research Foundation, THRIP, Citrus Academy & Citrus Research International for bursary and research funding.

Thesis (MSc)--Stellenbosch University, 2010. / ENGLISH ABSTRACT: Late blight caused by Phytophthora infestans on potato and tomato causes major economic losses worldwide. Until the 1980s, P. infestans populations outside its centre of origin (either central Mexico or the Andean region) only consisted of one mating type (A1), which prevented the pathogen from reproducing sexually. Pathogen populations outside the centre of origin most likely only consisted of a few genotypes prior to the 1980’s. Pan globally, these genotypes probably first consisted of genotype/s that had mitochondrial DNA (mtDNA) haplotype Ia, which was subsequently replaced by a mtDNA haplotype Ib genotype known as the US-1 lineage. This relative simple population structure of the pathogen changed almost worldwide in the late 1970s and early 1980s, when a second set of migrations took place from the centre of origin. These populations contained both A1 and A2 mating type isolates that consisted of several different genotypes, which were more virulent than the pre-1970s genotypes and resulted in the displacement of these genotypes almost worldwide. Some of the new genotypes were also resistant to metalaxyl, the fungicide that was most effective in controlling late blight. In Sub-Saharan Africa (SSA), the characteristics of P. infestans populations are not well documented in most countries except South Africa, Kenya and Uganda. Previous studies in SSA showed that populations were dominated by the US-1 lineage and its variants. The exceptions were reports of the presence of a few mtDNA haplotype Ia isolates in Rwanda and Ethiopia. The current study aimed to determine the population structure of P. infestans in eight selected SSA countries (Burundi, Kenya, Rwanda, Tanzania, Uganda, Malawi, Mozambique and South Africa), mainly on potato and on a limited scale on tomato and petunia, using ‘old’ markers (mating type determination, glucose-6-phosphate isomerase [Gpi] genotyping, mtDNA haplotyping, DNA fingerprinting with probe RG-57 and metalaxyl sensitivity). Populations were further also genotyped using seven recently published Simple Sequence Repeats (SSRs) markers. This information would help to define the population structure of P. infestans in SSA for the first time on a regional basis, and will also determine whether new migrations have taken place since the last characterization studies took place in 2001. A survey in the eight SSA countries yielded a total of 281 P. infestans isolates, mainly obtained from potato fields (Tanzania, Kenya, Uganda, Rwanda, Burundi, Malawi and South Africa), but also from tomato (Malawi, Mozambique and South Africa) and Petunia ´ hybrida (South Africa) that were characterized. Characterization of subsets of the isolates with the ‘old’ markers (176 isolates for mating type, 281 isolates for mtDNA, 70 isolates for [Gpi] and 49 isolates with restriction fragment length polymorphism analysis with probe RG-57), showed that most of the isolates belonged to the US-1 genotype or its variants (US-1.10 and US-1.11). The exception were isolates that belonged to genotype KE-1 (A1 mating type, mtDNA haplotype Ia, Gpi 90/100 and unique RG-57 genotype) that was identified in two fields in Kenya. Genotype KE-1, based on the ‘old’ marker data, is related to genotypes (RW-1 and RW-2) previously identified in Rwanda, and several Ecuadorean and European genotypes. Metalaxyl sensitivity testing of 64 isolates showed that metalaxyl resistant potato isolates were present in all the countries except Malawi, whereas all the tomato isolates were sensitive. Genotyping of 176 isolates with seven recently published simple sequence repeat (SSR) markers revealed a high number (79) of multi-locus genotypes (MLGs) in SSA. However, when locus D13, which was difficult to score, was excluded only 35 MLGs were identified. When locus D13 was excluded from analyses of molecular variance (AMOVA), (i) there was no significant genetic differentiation between populations from central-east Africa (Burundi, Kenya, Rwanda, Tanzania and Uganda), south-east Africa (Malawi and Mozambique) and South Africa, (ii) the KE-1 population was genetically differentiated (Fst = 0.33; P = 0.001) from the US-1 and US-1.10 populations and (iii) genetic differentiation between populations from potato and tomato was low (Fst = 0.07; P = 0.004). The study has expanded the worldwide genotypic database of P. infestans for SSA. Previously, no populations were characterized from Burundi, Malawi and Mozambique. The characterization work showed that migrations seem unlikely to have taken place in SSA, or if these did occur, it was on a very limited scale. The more severe epidemics in some SSA countries could be due to the presence of metalaxyl resistance. Furthermore, the occurrence of mutations or mitotic recombination might have resulted in more aggressive and/or better adapted genotypes, for example the US-1.10 lineage that was only detected in the Western Cape Province of South Africa. The significance of the discovery of the KE-1 genotype in Kenya needs further investigation since it might (i) be an asexual descendent of genotypes (RW-1 and RW-2) that were previously reported in Rwanda in the 1980s, (ii) previously have gone undetected due to the small surveys that were conducted in SSA, (iii) be a new migrant from countries other than SSA or (iv) have been introduced in the very first introductions into Kenya prior to the 1970s. The SSR results from the survey will allow comparison of the SSA late blight populations with other populations worldwide through the EucaBlight database in future studies. / AFRIKAANSE OPSOMMING: Laatroes, veroorsaak deur Phytophthora infestans op aartappel en tamatie, veroorsaak groot ekonomiese verliese wêreldwyd. Phytophthora infestans populasies buite hul kern van oorsprong (óf sentraal Meksiko óf die Andes area), het tot die 1980’s slegs uit een paringstipe (A1) bestaan, wat verhoed het dat die patogeen geslagtelik vermeerder. Patogeenpopulasies buite die kern van oorsprong, het heel moontlik vóór die 1980’s slegs uit ‘n paar genotipes bestaan. Wêreldwyd, het hierdie genotipes moontlik aanvanklik uit genotipe(s) bestaan wat mitokondriale DNS (mtDNS) haplotipe Ia bevat het, wat later met ‘n mtDNS haplotipe Ib genotipe, bekend as die US-1 genotipe, vervang is. Hierdie relatiewe eenvoudige populasiestruktuur van die patogeen, het omtrent wêreldwyd in die láát 1970’s en vroeë 1980’s verander, toe ‘n tweede stel migrasies vanaf die patogeen se kern van oorsprong plaasgevind het. Hierdie populasies het beide A1 en A2 paringstipe isolate ingesluit, wat uit verskeie verskillende genotipes bestaan het, wat meer virulent as die vóór-1970’s genotipes was, en wat die verskuiwing van hierdie genotipes omtrent wêrelwyd tot gevolg gehad het. Sommige van die nuwe genotipes was ook weerstandbiedend teen metalaksiel, die fungisied wat mees effektief in die beheer van laatroes was. Die kenmerke van P. infestans populasies is nie goed in die meeste lande in Sub- Sahara Afrika (SSA) gedokumenteer nie, behalwe vir Suid-Afrika, Kenia en Uganda. Vorige studies in SSA het aangedui dat populasies deur die US-1 genotipe en sy variante gedomineer word. Die uitsonderings was aantekeninge oor die teenwoordigheid van ‘n paar mtDNS haplotipe Ia isolate in Rwanda en Etiopië. Die huidige studie was daarop gemik om die populasiestruktuur van P. infestans in agt geselekteerde SSA lande (Burundi, Kenia, Rwanda, Tanzanië, Uganda, Malawi, Mosambiek en Suid-Afrika), hoofsaaklik op aartappel en op ‘n beperkte skaal op tamatie en petunia, vas te stel, deur die gebruik van ‘ou’ merkers (paringstipe-bepaling, glukose-6-fosfaat isomerase [Gpi] genotipering, mtDNS haplotipering, DNS fingerafdrukke met RG-57 en metalaksielsensitiwiteit). Die genotipe van populasies is verder ook bepaal deur gebruik te maak van sewe onlangs-gepubliseerde “Simple Sequence Repeats (SSRs)” merkers. Hierdie inligting sal help om die populasiestruktuur van P. infestans in SSA vir die eerste keer op ‘n streeksbasis vas te stel, en sal ook bepaal of nuwe migrasies sedert die laaste karakteriseringstudies wat in 2001 uitgevoer is, plaasgevind het. ‘n Opname in die agt SSA lande, het ‘n totaal van 281 P. infestans isolate opgelewer, hoofsaaklik vanaf aartappellande (Tanzanië, Kenia, Uganda, Rwanda, Burundi, Malawi en Suid-Afrika), maar ook vanaf tamatie (Malawi, Mosambiek en Suid- Afrika) en Petunia ´ hybrida (Suid-Afrika) wat gekarakteriseer is. Karakterisering van geselekteerde isolate met die ‘ou’ merkers (176 isolate vir paringstipe, 281 isolate vir mtDNS, 70 isolate vir Gpi en 49 isolate met restriksiefragment-lengte-polimorfismeanalise met RG-57), het aangetoon dat die meeste van die isolate aan die US-1 genotipe of sy variante (US-1.10 en US-1.11) behoort het. Die uitsondering was isolate wat tot die genotipe KE-1 behoort het (A1 paringstipe, mtDNS haplotipe Ia, Gpi 90/100 en unieke RG-57 genotipe) wat in twee velde in Kenia geïdentifiseer is. Genotipe KE-1, gebaseer op die ‘ou’ merkerdata, is aan genotipes (RW-1 en RW-2) verwant, wat voorheen in Rwanda, en verskeie Ekwadoreaanse en Europese lande geïdentifiseer is. Metalaksielsensitiwiteitstoetsing van 64 isolate het aangetoon dat metalaksiel-weerstandbiedende aartappel-isolate in al die lande teenwoordig was, behalwe vir Malawi, terwyl al die tamatie-isolate sensitief was. Genotipering van 176 isolate met sewe onlangs gepubliseerde “Simple Sequence Repeat” (SSR) merkers, het ‘n hoë aantal (79) multilokus genotipes (MLGs) in SSA aangedui. Met die uitsluiting van lokus D13, wat moeilik was om te evalueer, is slegs 35 MLGs egter geïdentifiseer. Met die uitsluiting van lokus D13 uit die analise van molekulêre variansie (AMOVA), was (i) daar geen betekenisvolle genetiese differensiasie tussen populasies van sentraal-oos Afrika (Burundi, Kenia, Rwanda, Tanzanië en Uganda), suid-oos Afrika (Malawi en Mosambiek) en Suid-Afrika nie, (ii) die KE-1 populasie geneties (Fst = 0.33; P = 0.001) van die US-1 en US-1.10 populasies gedifferensieerd en (iii) genetiese differensiasie tussen populasies vanaf aartappel en tamatie laag (Fst = 0.07; P = 0.004). Die studie het die wêreldwye genotipe-databasis van P. infestans vir SSA uitgebrei. Voorheen is geen populasies vanuit Burundi, Malawi en Mosambiek gekarakteriseer nie. Die karakteriseringswerk het aangetoon dat die waarskynlikheid klein is dat migrasies in SSA plaasgevind het, of indien dit wel plaasgevind het, dit op ‘n baie beperkte skaal plaasgevind. Die meer ernstige epidemies in sommige SSA lande kan die gevolg wees van die teenwoordigheid van metalaksiel-weerstand. Die voorkoms van mutasies of mitotiese rekombinasie kon verder meer aggressiewe en/of beter aangepaste genotipes tot gevolg gehad het, byvoorbeeld die US-1.10 genotipe wat slegs in die Westelike Kaapprovinsie van Suid-Afrika waargeneem is. Die betekenis van die ontdekking van die KE-1 genotipe in Kenia benodig verdere ondersoek aangesien dit (i) ‘n ongeslagtelike afstammeling van genotipes (RW-1 en RW-2) mag wees wat voorheen in die 1980’s in Rwanda aangeteken is, (ii) voorheen nie waargeneem is nie weens die klein opnames wat in SSA uitgevoer is, (iii) ‘n nuwe genotipe van lande buite die SSA kan wees of (iv) ingebring is tydens die heel eerste inkoms in Kenia vóór die 1970’s. Die SSR resultate van die opname sal vergelykings tussen die SSA laatroespopulasies en ander populasies wêreldwyd toelaat, deur gebruik te maak van die EucaBlight databasis in toekomstige studies.

Thesis (MScAgric )--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Pine pitch canker, caused by Fusarium circinatum, is a major constraint to the production of high quality timber in the Western Cape Province of South Africa. Under nursery conditions the pathogen causes a serious root and collar rot disease, and in plantations it causes stem cankers and branch die-back. In this thesis, sources of inoculum within a forestry nursery were investigated and novel approaches developed to eradicate the pathogen. Plant material was evaluated for plantation establishment in the Western Cape Province, and epidemiological studies conducted to better understand the infection, disease development, spore release and dissemination of F. circinatum in pine plantations. Fusarium circinatum was first reported in a forestry nursery in the Mpumalanga Province of South Africa in 1990, and now causes significant economic losses to most forestry nurseries in the country. The irrigation system, planting tray inserts, seeds and air at Karatara nursery were screened as potential sources of F. circinatum. Both the irrigation water and planting tray inserts were found to serve as sources of inoculum. An oxidation reduction potential (ORP)-based system, using hydrogen peroxide, was tested to sanitize irrigation water and planting tray inserts. An ORP level of 400 mV for 6 hours was effective in killing F. circinatum spores in irrigation water without being phytotoxic to pine seedlings. An ORP value of >500 mV cleaned planting tray inserts of all inoculum. Pinus radiata is the principal pine species grown in the Western Cape Province, but this species is also highly susceptible to F. circinatum. There are numerous different varieties or families of P. radiata available that can be planted, and it is known that tolerance levels between these varieties and families vary. Pinus radiata families, as well as other Pinus spp. that could potentially be planted in the Western Cape Province in future, were evaluated for tolerance to the pitch canker fungus. The P. radiata families were moderately to highly susceptible to F. circinatum. Pinus taeda was the Pinus spp. most tolerant, while P. elliottii also had good tolerance to the pathogen. None of the P. radiata families screened showed tolerance comparable to that of either P. elliottii or P. taeda. Pine pitch canker was first reported from the Tokai plantation in the Western Cape Province in 2005. In order to develop a disease management strategy for the disease in pine plantations, we investigated the effect of pruning wounds as potential entry sites for the pathogen, and how fast the disease spread under South African climatic conditions. The amount and time of sporulation was also investigated. Pitch canker increased slowly over a 3-year period, and pruning wounds appeared not to serve as primary entry points for F. circinatum. Spore quantification over a 12-month period using quantitative real-time PCR showed that inoculum was produced throughout the year, but that significantly more spores were released during the cold, rainy winter months. The role of insects in pathogen transmission and spread should be investigated in the Tokai plantation. / AFRIKAANSE OPSOMMING: Fusarium circinatum veroorsaak “pine pitch canker”. Hierdie siekte is een van die grootste beperkinge op die Suid Afrikaanse bosbou industrie. In kwekerye veroorsaak die patogeen wortelvrot en wortelkraagvrot. In plantasies veroorsaak F. circinatum kankers op die hoofstam van dennebome en die terugsterwing van sytakke. In hierdie tesis was bronne van inokulum in ‘n bosbou kwekery ondersoek. Nuwe tegnieke was gebruik om die bronne uit te wis. Plantmateriaal wat in die Wes-Kaap geplant kan word, was geëvalueer vir toleransie teen F. circinatum. Epidemiologiesie studies was uitgevoer om die infeksieproses, siekte-ontwikkeling, spoorvrystelling, en verspreiding van F. circinatum binne denneplantasies beter te verstaan. Die eerste verslag van F. circinatum in ‘n bosboukwekery was in 1990 in die Mpumalanga Provinsie. Verliese word tans veroorsaak deur die patogeen in die meeste bosbou kwekerye in Suid Afrika. Die besproeiingstelsel, planthouers en saad was getoets as potensiële bronne van inokulum. Daar was gevind dat beide die besproeiingstelsel en die planthouers dien as bronne van F. circinatum inokulum. ‘n Oksidasie reduksie potensiaal (ORP)-gebaseerde sisteem waar waterstofperoksied gebruik was, was getoets of dit die besproeingwater en planthouers kan ontsmet. ‘n ORP-vlak van 400 mV vir ‘n blootstellingstydperk van 6 ure is effektief om al die F. circinatum spore dood te maak, sonder dat dit fitotoksies is teenoor die dennesaailinge. ‘n ORP-vlak van >500 mV het die F. circinatum in die planthouers uitgewis. Pinus radiata is die belangrikste dennespesie wat geplant word in die Wes-Kaap Provinsie, maar hierdie spesie is hoogs vatbaar vir F. circinatum. Verskillende variteite van P. radiata met verskillende tolleransie vlakke bestaan. Pinus radiata families en ander spesies wat in die toekoms potensieel geplant kan word in die Wes-Kaap was geevalueer vir toleransie teen F. circinatum. Die P. radiata families was matig to hoogs vatbaar vir F. circinatum. Pinus taeda het die hoogste toleransie vlak gehad en P. elliottii was matig tolererant. Nie een van die P. radiata families wat getoets was het toleransie vlakke gehad wat vergelykbaar was met die toleransie vlakke van P. taeda of P. elliottii nie. In 2005 was F. circinatum vir die eerste keer geraporteer in die Tokai plantasie op volwasse dennebome in die Wes-Kaap Provinsie. Die rol van snoeiwonde in die infeksieproses van F. circinatum was bestudeer asook hoe vinning die siekte ontwikkel en versprei. Die hoeveelheid en tyd van sporulasie was ook bestudeer. “Pitch canker” het stadig toegeneem oor die 3-jaar moniteringsperiode en dit blyk asof snoeiwonde nie die primêre ingangspunt vir die patogeen is nie. Kwantifisering van die sporulasie van F. circinatum deur middel van qPCR het aangedui dat spore reg deur die jaar geproduseer word. Meer spore word geproduseer in die koue nat wintersmaande. Die rol van insekte in die infeksieproses en verspreiding van F. circinatum in die Tokai plantasie moet ondersoek word.

Thesis (MScAgric)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Grey mould, caused by Botrytis cinerea, is the most important foliar disease of rooibos seedlings. Although the disease is primarily controlled with applications of fungicides, the improvement of cultural methods of disease management should lessen this dependence on chemical control. Such improvements would, however, not be possible without knowledge of the inoculum sources and dispersal of the pathogen. The aim of this study was to investigate the inoculum ecology of B. cinerea in rooibos nurseries in order to identify primary sources of inoculum and to improve the environmentally friendly management of the disease. The study was conducted in four nurseries over two production seasons (March to July 2003 and 2004). Levels of airborne inoculum of B. cinerea were monitored on a monthly basis inside and around the nurseries with spore traps. Samples of plant material and organic debris were taken in the corresponding areas to determine the incidence of plant material infected by the pathogen and the incidences of grey mould in the nurseries were recorded. Low numbers of B. cinerea colonies were observed on the spore traps. Similar levels of airborne inoculum were observed inside and around the nurseries. The incidence of plant material yielding B. cinerea was higher outside the nurseries than inside, indicating the importance of such materials as potential sources of inoculum. Since patterns of airborne inoculum observed in this study confirmed reports of the local dispersal of B. cinerea, the removal of possible hosts outside the nurseries could aid in the management of grey mould in rooibos nurseries. Resistance to dicarboximide fungicides is a genetically stable trait in B. cinerea, and therefore has the potential to be used as a phenotypic marker. This marker can be used to gain knowledge on the dispersal of B. cinerea inoculum inside and outside rooibos nurseries. Isolates of B. cinerea collected from the air and from plant material in and around four rooibos nurseries were assessed for resistance to iprodione at 1 and 3 μg/ml a.i. Some of the isolates showed resistance to iprodione at 1 μg/ml a.i. However, none of the isolates showed resistance at 3 μg/ml a.i. iprodione. The initial incidence of dicarboximide-resistance at the nurseries was slightly higher than expected. As the season progressed, the incidence of iprodione-resistant isolates decreased towards May, after which an increase was observed towards July. A relatively high percentage of isolates collected outside the nurseries was found to be dicarboximide-resistant. Two of the nurseries had a significant higher incidence of resistant isolates on plant material collected inside, than on plant material collected outside the nursery. However, when looking at resistance levels of airborne isolates, no significant differences were found in the incidence of resistant isolates sampled inside and outside the four nurseries. The data indicated the importance of organic debris and seed-borne infections in the survival and dispersal of dicarboximide-resistant isolates of the pathogen. With the current emphasis on organic agriculture the knowledge gained in this study presents valuable possibilities of improving the cultural management of grey mould in rooibos nurseries. / AFRIKAANSE OPSOMMING: Vaalvrot, veroorsaak deur Botrytis cinerea, is die belangrikste bo-grondse siekte van rooibossaailinge. Alhoewel die beheer van die siekte hoofsaaklik op die gebruik van fungisiede berus, behoort die verbetering van verbouingspraktyke hierdie afhanklikheid van chemiese beheer te verminder. Sulke verbeteringe sal egter slegs moontlik wees indien voldoende kennis van die inokulumbronne en verspreiding van die patogeen beskikbaar is. Die doel van hierdie ondersoek was om die inokulum ekologie van B. cinerea in rooibos kwekerye te ondersoek sodat primêre inokulumbronne opgespoor en omgewingsvriendelike siektebestuurspraktyke verbeter kan word. Die ondersoek is in vier kwekerye oor twee produksie seisoene (Maart tot Julie 2003 en 2004) uitgevoer. Vlakke van luggedraagde inokulum van B. cinerea is op ’n maandelikse basis met behulp van spoorvangers binne en buite die kwekerye gemonitor. Monsters van plantmateriaal en organiese materiaal is in ooreenstemmende areas geneem om die voorkoms van B. cinerea geïnfekteerde plantmateriaal vas te stel en die voorkoms van vaalvrot in die kwekerye is aangeteken. Min B. cinerea kolonies is op die spoorvangers waargeneem. Soortgelyke vlakke van luggedraagde inokulum is binne en buite die kwekerye waargeneem. Die hoër voorkoms van B. cinerea geïnfekteerde plantmateriaal buite die kwekerye as binne, dui op die belang van sulke materiaal as potensiële inokulumbronne. Aangesien die patrone van luggedraagde inokulum, soos waargeneem in hierdie ondersoek, ander berigte van B. cinerea se beperkte verspreidingsvermoë bevestig, kan die verwydering van moontlike alternatiewe gashere buite die kwekerye die bestuur van die siekte binne die kwekerye verbeter. Weerstand teen dikarboksimied fungisiede is ’n geneties-stabiele kenmerk in B. cinerea en het daarom potensiaal om as ’n fenotipiese merker gebruik te word. Hierdie merker kan gebruik word om kennis aangaande die verspreiding van B. cinerea in en om rooibos kwekerye in te samel. Botrytis cinerea isolate in lug en op plantmateriaal in en om vier rooibos kwekerye is gedurende 2003 en 2004 versamel. Die isolate is vir weerstandbiedendheid teen iprodioon by konsentrasies van 1 en 3 μg/ml aktiewe bestandeel (a.b.) getoets. Isolate met weerstand teen 1 μg/ml a.b. iprodioon is waargeneem, maar nie teen 3 μg/ml nie. Die aanvanklike voorkoms van dikarboksimiedweerstand by die kwekerye was hoër as verwag. Hierdie vlak het egter gedaal met die verloop van die seisoen tot in Mei, waarna ’n toename tot in Julie waargeneem is. Die persentasie dikarboksimied-weerstandbiedende isolate buite die kwekerye was relatief hoog. In twee van die kwekerye was die voorkoms van weerstandbiedende isolate op plantmateriaal in die kwekerye betekenisvol hoër as op plantmateriaal buite die kwekerye. Daar was egter geen betekenisvolle verskille in die voorkoms van luggedraagde weerstandbiedende isolate nie, ongeag van die kwekery of posisie. Die data dui op die belang van organiese materiaal en saadgedraagde infeksies in die oorlewing en verspreiding van dikarboksimied-weerstandbiedende isolate van die patogeen. Met die huidige klem op organiese landbou bied die inligting wat in hierdie ondersoek versamel is moontlike praktyke wat geïmplementeer kan word om die beheer van vaalvrot in kwekerye met behulp van verbouingspraktyke te verbeter.