Dissertationen zum Thema „Fungal diseases of plants“

Thesis (Master of Conservation Science)--Cape Peninsula University of Technology, 2018.
Introduction pathways of fungal pathogens in South Africa are far less quantified in the literature than those for plants, animals and human infectious diseases. Phytopathogens continue to be introduced to South Africa via several pathways at an unprecedented rate. A number of these species pose a significant threat to South African ecosystems and biodiversity. Despite this, fungal pathogens could also be beneficial when they are used as bio-control agents to control alien invasive plant species. Nevertheless, recent studies revealed pathogens are most likely to be studied after they have caused a detrimental impact on the environment. Invasive fungal pathogens, such as Phytophthora cinnamomi (Oomycota) do not only pose a threat to native species of the family Proteaceae but could also potentially be bio-control agents for emerging alien plant invaders. In this thesis, firstly, I review current knowledge of phytopathogenic fungi introduction pathways in South Africa; secondly, I aim to understand the importance of fungi in limiting plant invasions using Banksia as a case study in the Cape Floristic Region. In chapter two I investigate introduction pathways and dispersal vectors that facilitate the spread of fungal pathogens. I compiled comprehensive list of fungal pathogens in South Africa, and evaluated the dispersal vectors and introduction pathways for each species. I found fifty five casual species, three naturalised species, six invasive species and thirty six pathogens for which invasion status was not classified due to insufficient data. Agriculture is responsible for the introduction of most fungal pathogens in South Africa. Wind was identified to be the prominent dispersal vector facilitating the spread of pathogens. I conclude that knowing introduction pathways of pathogens and their dispersal vectors will assist in developing quarantine protocols that could improve bio-security. Lastly, I provide recommendations for the national invasive microbe species list. In chapter three the study investigates the variability in mortality rate of Banksia species in the Cape Floristic. Species abundance was calculated across known Banksia populations in the Cape Floristic Region to determine survival and mortality rates. Soil and leave samples were taken from Banksia plants to evaluate potential microbial pests that were present. Also, acetone leaf extracts of twelve Banksia species were screened for antimicrobial activity against P. cinnamomi (Oomycota). Lastly, a post-border risk assessment was conducted for 14 Banksia species− present in South Africa − using the Australian Weed Risk Assessment protocol, to evaluate potentially invasive species. The results indicated that survival and mortality rate varied across species; I found the two invasive species, B. integrifolia and B. ericifolia to have the highest survival rate. Phytophthora cinnamomi was the most prominent isolated fungal pathogen sampled from Banksia species roots. The detection of antifungal activities in the minimum inhibitory concentration (MIC) bioassay provided evidence that some Banksia species (B. ericifolia, B. integrifolia, B. hookeriana and B. formosa) have antimicrobial chemical constituents that could possibly inhibit infection and colonisation by P. cinnamomi. The weed risk assessments conducted on Banksia species showed five species pose a high risk of invasion while seven species required further evaluation. I conclude that P. cinnamomi could potentially regulate invasive Banksia species such as B. speciosa with minimal antimicrobial activity against the pathogen. I recommend an in-situ and ex-situ inoculation trials of Banksia species against P. cinnamomi to be conducted to evaluate pathogenicity, under different watering regimes since the pathogens proliferation is favoured by soils that are high in moisture. I present the main conclusions from this thesis in chapter four and provide recommendations for management and invasive species legislation.

A survey of dipterocarp forest in four sites revealed that the incidence of stem canker was relatively low but high localized incidences were recorded. No consistent association was obtained between the presence of mechanical damage and cankers. Cankers occurred more frequently on dipterocarps and less frequently on euphorbs. Field studies and experimental manipulations were used to compare sapling resistance and responsiveness to wounding and stem breakage in relatively nutrient-rich, alluvial forest and relatively nutrient-poor, sandstone ridge forest. Species found on sandstone ridges showed greater resistance to damage (e.g., greater stem flexibility, narrower crowns) than those on alluvial soils. Species common on alluvial soils tended to be more responsive to damage (e.g., faster wound closure rates, more likely to re-sprout). Results from manipulation experiments conducted on pot-grown seedlings were consistent with results from the field studies, where conditions of greater nutrient availability, saplings closed wounds at faster rates, had less flexible stems, more narrow crowns, and lower levels of foliar total phenolics. Species showed differential rezones to resource availability which, in part, may relate to contrasting strategies for investment in passive defence (i.e., resins and phenolics) over investment in growth. Through their narrower crowns, greater whole stem flexibility, and lesser stem taper, tree species characteristic of sandstone ridges had greater resistance to mechanical damage from debris falling from above than congeneric species characteristic of alluvial soils. Tree species characteristic of alluvial soils were more responsive to damage than congeners on sandstone ridges, by producing earlier and longer sprouts following stem snapping and more rapid rates of wound closure following wounding.

The fungus Botrytis cinerea is an opportunistic pathogen on a wide variety of crops, causing disease known as grey mould through infections via wounds or dead plant parts. Synthetic fungicides for controlling this disease are fast becoming ineffective due to the development of resistance. This, coupled with consumers world wide becomng increasingly conscious of potential environment and health problems associated with the build up of toxic chemicals, (particularly in food products), have resulted in pressure to reduce the use of chemical pesticide volumes as well as its residues. An emerging alternative to random synthesis is the study and exploitation of naturally occurring products with fungicidal properties. There have been reports on the uses of synthetic fungicides for the control of plant pathogenic fungi. When utilized in two-way mixtures, such fungicides may maintain or enhance the level of control of a pathogen at reduced rates for both components utilized in combinations, or alone at normal rates. For this study it was hypothesize that the addition of plant extracts may enhance the antifungal efficacy of the synthetic strobilurin fungicide, kresoxim-methyl against Botrytis cinerea.

The purpose of this thesis was to study interactions between systemic fungal diseases and perennial plants. Using the systemic rust Puccinia minussensis on the host plant Lactuca sibirica, and the rust Puccinia pulsatillae on the host plant Pulsatilla pratensis, this thesis focused on: (i) the effects of systemic diseases on their hosts (ii) host and pathogen responses to abiotic factors, (iii) the importance of life history strategies for understanding host-pathogen interactions, and (iv) the evolutionary consequences of living in close associations. Results of greenhouse experiments showed that Lactuca sibirica had a high plasticity in growth, since it produced significantly more shoots in favourable than in unfavourable growth conditions. Both the disease levels and the number of healthy shoots (i.e. escape) were significantly higher under favourable conditions. Disease spread within the rhizome was found to be incomplete, and the risk of aecidial- infection decreased with distance from the parent. Furthermore, one isolate of the fungus had highest success and reduced the host plant biomass and shoot production more on the clone it was collected on compared to four other clones . In the field, disease levels were found to fluctuate more at localities subjected to disturbance, the host and pathogen abundances were found to be in phase and the pathogen showed no delayed response to increasing host densities. The rust Puccinia pulsatillae on Pulsatilla pratensis showed no fluctuations between years, low infection rates, and disease levels were higher in ungrazed compared to grazed sites. There was no escape from the disease in this system. A comparison of characteristics of different systemic fungi and hosts with different growth patterns indicated that the life history strategies of both host plants and pathogens need to be studied if the long-term consequences of host-pathogen interactions are to be predicted.

Diss. (sammanfattning) Umeå : Umeå universitet, 1993, härtill 5 uppsatser.

digitalisering@umu

Eutypa dieback of grapevines, caused by Eutypa lata, is a major cause of reduced longevity in vineyards worldwide. The fungus grows in the woody tissue of infected vines, producing translocatable toxins that cause foliar symptoms of the disease. By the time foliar symptoms are evident the pathogen may have become well established in the vine. One aim of this study was to develop DNA markers to allow rapid reliable identification of E. lata and to detect the pathogen in infected wood. The second aim was to analyse secondary metabolite production by E. lata in order to gain information on the compounds responsible for the foliar symptoms of the disease and to identify metabolites which could be used as markers to detect the early stages of the disease prior to the expression of foliar symptoms. In addition, genetic variation of the pathogen was assessed using RFLP and RAPD analysis. Two techniques were used to develop DNA markers; first, SCAR markers derived from RAPD fragments were developed and, second, an E. lata genomic DNA library was constructed, from which DNA fragments specific to E. lata were identified. These markers were used in either PCR- or Southern hybridisation-based assays to detect the pathogen in infected wood. PCR-based detection of the pathogen in infected wood was prone to inhibition by phenolic compounds, however, Southern hybridisation techniques were capable of detecting E. lata in wood. Genetic variation among 38 isolates of E. lata was assessed using six randomly selected clones from the genomic DNA library. A subset of 11 isolates was subjected to RAPD analysis using 10 random primers. Considerable genetic diversity, in terms of RFLP and RAPD profiles, was observed among isolates. There was no apparent correlation between grouping of isolates following neighbour joining analysis and either host species or geographic origin of isolates. The RAPD and RFLP profiles of two isolates differed significantly from the majority of the other isolates. These isolates, which were morphologically similar to all other isolates, were subsequently found not to be E. lata. Secondary metabolite production of 11 isolates was analysed by HPLC following growth on a range of media. A wider range of secondary metabolites was detected in E. lata than has previously been reported. Two of the secondary metabolites, eutypine and an unidentified compound with a retention time of 19.6 min, were produced by eight of nine isolates of E. lata. Neither of the non-E. lata isolates produced these compounds. It was concluded that the remaining isolate of E. lata may have lost the ability to produce these compounds following storage. Whilst a wider range of isolates needs to be screened before a candidate marker can be selected, these results suggest that certain compounds are present in the majority of E. lata isolates and, hence, may prove suitable markers for the detection of the pathogen prior to the expression of foliar symptoms. The molecular probes developed in this study will allow the rapid and reliable identification and detection of E. lata in grapevine cane or wood. These probes also have the potential to be used as a research tool to gather information on the epidemiology of the disease and to assess the efficacy of potential control agents against E. lata. Suitable control measures could then be applied to vines which have been shown by the use of chemical markers to have latent infection. Used in combination, therefore, the DNA and biochemical markers could facilitate improved management of eutypa dieback.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture and Wine, 2003.

Polyclonal antisera produced against spores, soluble protein and the whole mycelium fractions of Didymella lycopersici reacted with the homologous and heterologous antigens. The most sensitive antiserum was that raised against the whole mycelium, the soluble protein and the spore, in decreasing order of sensitivity. Using the antiserum raised against the whole mycelium it was possible to detect D. lycopersici on diseased plants and infested seeds. Cross reactivity was observed between the antisera produced to D. lycopersici and D. applanata, D. bryoniae and other tomato fungal pathogens including Fusarium spp. and B. cinerea. ELISA was most sensitive and reliable compared to double immunodiffusion, and latex tests. No reactions were obtained using the latex agglutination procedure and no antiserum detected spores in double diffusion tests. Protein profiles on SDS-PAGE revealed that the total number of protein bands decreased with increased age of cultures of D. lycopersici incubated in liquid media. Western blots probed with the antiserum raised against the whole mycelium showed that protein bands from extracts of both D. lycopersici and D. applanata were antigenic.
Land and Food Systems, Faculty of
Graduate

Studies presented in this dissertation highlight the importance of fungal pathogens in forestry nurseries in South Africa. Both Acacia meamsii seedlings and Eucalyptus hybrid cuttings are shown to be affected by important nursery pathogens. Chapter one presents an evaluation of the potential importance of pathogens to Eucalyptus hedge plants maintained in hydroponics. Hydroponics is a new technology being used in South African forest nurseries, which allows for the rapid establishment of Eucalyptus hedge plants. However, no information is available on pathogens affecting Eucalyptus in hydroponics. By applying information on pathogens of other hydroponic crops, several potentially important pathogens were identified and these reside in the genera Phytophthora, Pythium and Fusarium. Possible disease symptoms in Eucalyptus caused by these pathogens include wilting, stem cankers and root rots. Implementation of appropriate control measures that include cultural, biological and chemical practices could prevent and/or reduce disease impact in hydroponics. Chapter two presents the results of a survey of the roots of Eucalyptus hedge plants grown in an ebb and flow hydroponic system. An interesting result of the survey was the discovery of Cylindrocladium pauciramosum in the hydroponic system. This is the first report of the pathogen in a hydroponic system. Other important pathogens in the genera Phytophthora and Pythium were also isolated. Two Pythium species, namely P. dissotocum and P. helicoids, found in the roots and nutrient solution are new to Eucalyptus. Several Fusarium species were also isolated of which two, namely F. nygamai and F. lateritium, are also new to Eucalyptus. Chapter three of this dissertation presents the results of a survey of Eucalyptus cuttings conducted at four forestry nurseries in KwaZulu-Natal, South Africa. Several well-known Eucalyptus nursery pathogens were isolated. Cylindrocladium pauciramosum was identified as the dominant pathogen on Eucalyptus cuttings. This was confirmed based on morphological characteristics and DNA sequence comparisons. Pathogenicity tests conducted using a spore suspension of C. pauciramosum indicated that this pathogen is capable of infecting most commercial Eucalyptus clones used in South Africa. Chapter four considers a serious disease of Acacia mearnsii seedlings caused by an unidentified species of Cylindrocladium. Cylindrocladium pauciramosum was isolated from A. mearnsii seedlings showing girdling and stem canker symptoms. The pathogen was identified based on morphological characteristics and DNA sequence comparisons. Pathogenicity tests with Acacia seedlings confirmed the susceptibility of this tree to C. pauciramosum infection. This dissertation clearly indicates that Cylindrocladium pauciramosum is an important nursery pathogen in South African forestry nurseries. This pathogen has already been shown to be limiting during production of planting stock. I hope to have highlighted the importance of C. pauciramosum and other nursery pathogens in forestry nurseries in South Africa. This study will also hopefully provide information to forestry nursery managers and help them improve production.
Dissertation (MSc)--University of Pretoria, 2004.
Microbiology and Plant Pathology
Unrestricted

Thesis (MScAgric)--Stellenbosch University, 1998.
ENGLISH ABSTRACT: Eyespot is an important disease of spring wheat (Triticum aestivum L.). Four species of Ramulispora are associated with this disease, of which Tapesia yallundae and T. acuformis. are common. This thesis investigates the broader subjects of genetic variability, reproductive dynamics and fungicide resistance in Tapesia yallundae. Each of the chapters treats specific but related topics. T. yallundae, which is the only species thus far reported from South Africa, has been associated with yield losses of up to 50%. To enable the implementation of more accurate and effective control measures, understanding the dynamics of reproduction and the genetics of the pathogen is of utmost importance. Of the many plant disease control measures such as cultural practices, sanitation, biological control, etc., fungicide application is the most commonly resorted to measure in eyespot control. This thesis investigates the broader subjects of genetic variability, reproductive dynamics and fungicide resistance of Tapesia yallzll7dae. Fungicide application, however, is not without problems. The pathogen can build up resistance to fungicides. The most commonly used fungicides in eyespot control include the benzimidazole carbendazim, triazoles such as flusilazole, tebuconazole, propiconazole, bromuconazole, flutriafol, fenbuconazole, triademinol, and the imidazole, prochloraz. Cases of resistance to the groups listed above have been reported. Frequent monitoring for resistance is thus crucial to prevent wastage of fungicide and unnecessary impregnantation of the environment with potentially ineffective chemicals. In chapter 2 of this thesis 300 isolates of T. yallundae from 15 fields were evaluated for resistance against carbendazim, flusilazole, tebuconazole, propiconazole, bromuconazole, flutriafol and fenbuconazole. These results indicated that to some triazoles, such as fenbuconazole, a high level of resistance was already present in field populations. In a sexually reproducing fungus such as T. yallundae, knowledge pertaining to its ability to pass resistance factors to offspring is equally important. Mating studies were, therefore, also conducted with parental strains that showed signs of triazole resistance. Three generations were subsequently tested for resistance to five triazoles, namely flusilazole, tebuconazole, propiconazole, bromuconazole and flutriafol. Results of this study showed variable sensitivity in progeny, which indicated quantitative inheritance of resistance to triazoles. Although the sexual stage has not yet been observed in the field in South Africa, this knowledge lays the foundation for the long-term understanding of the population dynamics of the fungus. The ability of a heterothallic ascomycete population to reproduce sexually is dependent on the availability of its two mating types, MATI-I and MATI-2, their distribution, and female fertility amongst other factors. In the UK. the teleomorph is commonly observed in the field, which is in contrast to the situation in South Africa, where it has only been induced in the laboratory. A comparative study between the South African and the UK. populations was therefore undertaken. Isolates representative of the two populations were mated with tester strains as both sperm recipients and as sperm donors. This allowed the percentage of hermaphrodites to be determined. No difference in terms of female fertility was observed between the South African and the UK. populations, with both populations showing low effective population numbers. These data suggested, therefore, that the teleomorph would also occur more frequently in South Africa if the climate was more indusive to its development. The overall results of this study indicated that eyes pot could still be controlled by means of fungicide application in South Africa. Although a shift in sensitivity was observed towards fenbuconazole and flusilazole, no resistance was detected towards carbendazim. The latter might be due to the absen<.:eof the sexual stage in the field, coupled by the monocyclic nature of the pathogen and sensible fungicide regimes. The absence of T. acujormis makes the disease situation less complicated in terms of fungicide application and management. Continuous surveys will have to be conducted, however, to monitor this situation in future.
AFRIKAANSE OPSOMMING: Hierdie studie ondersoek die genetiese variasie, reproduksie dinamika en fungisied weerstand in Tapesia yallundae. Elke hoofstuk handel oor spesifieke maar verwante onderwerpe. Oogvlek is 'n belangrike siekte van lentekoring (Triticum aestivum L.). Vier spesies van Ramulispora word geassosieer met die siekte, waarvan Tapesia yallundae en T. acuformis mees algemeen voorkom. T. yallundae, wat tans die enigste spesie is wat in Suid-Afrika aangeteken is, het al verliese van tot 50% veroorsaak. Om meer akkurate en effektiewe beheermaatreels te implementeer, is dit noodsaaklik om die oorlewingsdinamika van die patogeen te verstaan. Van al die siektebeheermaatreels soos kulturele praktyke, sanitasie, biologiese beheer ens., bly fungisiedbehandeling die mees algemene maatreel vir die beheer van oogvlek. Fungisiedtoediening het egter ook verskeie probleme. Die patogeen kan weerstand opbou teen die fungisied. Die mees algemene fungisiedes wat vir oogvlekbeheer aangewend word sluit onder meer die benzimidasool karbendazim in, triasole soos flusilasool, tebukonasool, propikonasool, bromukonasool, flutriafol, fenbukonasool, triadimenol, en die imidasool, prochloraz. Weerstand is egter reeds teen hierdie middels bekend. Gedurige monitering vir weerstand is dus krities om die vermorsing van fungisied en besoedeling van die omgewing met oneffektiewe middels te beperk. In hoofstuk 2 van hierdie manuskrip word 300 isolate van T. yallundae van 15 lande geevalueer vir weerstand teenoor karbendazim, flusilasool, tebukonasool, propikonasool, bromukonasool, flutriafol en fenbukonasool. Resultate dui daarop dat teen sommige van hierdie triasole, soos bv. fenbukonasool, daar reeds 'n hoe vlak van weerstand teenwoordig was in veldpopulasies. In 'n seksueel reproduserende fungus soos T. yalluJ1dae, is dit noodsaaklik om te bepaal wat sy vermoe is om weerstandbiedenheid aan die nageslag oor te dra. Om die rede is paringstudies ook op ouers wat tekens van weerstand teenoor triasole getoon het uitgevoer. Drie generasies was gevolglik getoets vir weerstand teenoor vyf triasole, naamlik flusilasool, tebuconasool, propikonasool, brumukonasool en flutriafol. Resultate van die studie het 'n variasie in sensitiwiteit van die nageslag getoon, wat op 'n kwantitatiewe oorerwing van weerstand teen £riasole dui. Alhoewel die teleomorf nog nie in lande in Suid-Afrika opgemerk is nie, Ie hierdie kennis die fondament vir die langtermyn vertolking van die populasie dinamika van hierdie fungus. Die vermoe van 'n heterotalliese askomiseet populasie om seksueel voort te plant is afhanklik van die beskikbaarheid van sy twee paringstipes, MATI-I en MATl-2, hul verpreiding, vroulike vrugbaarheid en ander faktore. Alhoewel die teleomorf algemeen in lande in die Verenigde Koninkryk opgemerk word, is dit in kontras met die situasie in Suid-Afrika, waar hierdie stadium nog slegs in die laboratorium gelnduseer kon word. 'n Studie is dus onderneem om die Suid-Afrikaanse en V.K. populasies met mekaar te vergelyk. Isolate van die twee populasies is dus gepaar met paringsisolate as beide sperm ontvangers en sperm donors. Hierdie prosedure het dit moontlik gemaak om die persentasie hermafrodiete te bepaal. Geen verskille in vroulike fertiliteit is tussen die Suid-Afrikaanse en V.K. populasies bespeur nie, en beide populasies het ook 'n lae effektiewe populasie getal getoon. Hierdie data het dus voorgestel dat die teleomorf ook meer algemeen in Suid-Afrika sou voorkom as die klimaat meer geskik was vir teleomorf vormmg. Die resultate van hierdie studie het tot die slotsom gelei dat oogvlek steeds deur fungisiedbehandeling in Suid-Afrika beheer kan word. Alhoewel daar 'n merkbare verskuiwing in sensitiwiteit teenoor fenbukonasool en flusilasool was, was geen weerstand teenoor karbendazim waargeneem nie. Laasgenoemde kan dalk toegeskryf word aan die afwesigheid van die teleomorf in die veld, gekombineer met die monosikliese natuur van die patogeen en gebruik van alternerende fungisiedes. Die afwesigheid van T. acuformis maak die plaaslike siektetoestand minder gekompliseerd in terme van fungisied aanwending en bestuur. Voortdurende opnames sal egter uitgevoer moet word om hierdie situasie ook in die toekoms te monitor.

Dissertation (PhD (Agric))--University of Stellenbosch, 2002.
ENGLISH ABSTRACT: Fungi belonging to the genus Botryosphaeria are heterotrophic micromycetes that can be pathogens on woody plants. They cause serious, and in some cases devastating losses to crops through leaf necrosis, stem cankers and plant death. The Proteaceae cut-flower industry in South Africa accounts for 70% of the national cut-flower enterprise. Botryosphaeria diseases are a major impediment to production and trade of Proteaceae and there is an urgent need to investigate the etiology, epidemiology and control of these diseases. Losses of one of the most important proteas, P. magnifica, amount to 50% or more, locally. The main aims of this study were therefore to establish the etiology and aspects of epidemiology of Botryosphaeria stem cankers on P. magnifica and other Proteaceae, and to investigate methods of disease control. Although there is a vast body of information pertaining to this fungus, which was reviewed in Chapter 1, there is relatively little information available on Botryosphaeria on Proteaceae. The taxonomy of Botryosphaeria requires thorough review, and molecular techniques need to be employed to resolve species identities. In Chapter 2, it was found that Phyllachora proteae, a leaf pathogen of proteas, produced a Fusicoccum anamorph, which is described as F. proteae. A sphaeropsis-like synanamorph was associated with F. proteae and a new combination for P. proteae is proposed in Botryosphaeria, as B. proteae. The taxonomy of Botryosphaeria is in disarray at both the generic and the specific level. In Chapter 3 the taxonomic history of Botryosphaeria is reviewed, and the genus circumscribed and distinguished from other morphologically similar genera. Although several anamorph genera have been linked to Botryosphaeria, based on morphological observations and phylogenetic analysis of lTS rDNA sequence data, two anamorph genera are now recognised, those with pigmented conidia (Diplodia), and those with hyaline conidia (Fusicoccum). Botryosphaeria proteae should thus be excluded from Botryosphaeria. Several pathogenic Botryosphaeria spp. have an endophytic phase within their hosts. They are therefore imported unwittingly into other countries where they may pose a risk to agriculture and indigenous vegetation. The current global distribution of Botryosphaeria spp. associated with Proteaceae is clarified and a key to these taxa associated with Proteaceae is provided in Chapter 4. Five Botryosphaeria spp. are associated with cut-flower Proteaceae worldwide viz. B. lute a, B. obtusa, B. protearum, B. proteae and B. rib is. B. protearum is described as a new species. A thorough understanding of disease epidemiology is essential to effect a reduction of losses. In Chapter 5, I show that on P. magnifica, lesions caused by Botryosphaeria protearum, which lead to the formation of stem cankers, are initiated in the mid-rib vein or margin of leaves. Koch's postulates were satisfied and it was found that the number of lesions that developed from artificial inoculations correlated with starch levels present in leaves at the time of inoculation. In Chapter 6 it is shown that B. protearum exists as an endophyte in leaves of P. magnifica in naturally occurring as well as cultivated plants. In natural stands of proteas stem cankers are rare, but in cultivated plantations the incidence is high. Nutritional analyses indicate that higher levels of nitrogen occur in leaves of cultivated plants in spring, which could enhance disease development. High levels of sodium in the leaves of wild plants may restrict disease development. The severe economic losses caused by B. protearum make the search for improved methods of disease control essential. Fungicide applications form an important component of an integrated approach to disease management. In Chapter 7, in vitro tests demonstrate that tebuconazole, benomyl, prochloraz me, iprodione and fenarimol reduce the mycelial growth of B. protearum effectively. In the field there was a 25-85% reduction in the occurrence of stem cankers by applying fungicides or sanitation pruning. The best control was achieved by using benomyl, bitertanol, fenarimol, iprodione, prochloraz manganese chloride alternated with mancozeb and tebuconazole prophylactically. If sanitation pruning is combined with regular applications of fungicides, disease can be combated.
AFRIKAANSE OPSOMMING: Mikrofungi wat tot die genus Botryosphaeria behoort, is heterotrofiese organismes, wat patogenies op houtagtige plante kan wees. Hulle veroorsaak ernstige, en in sommige gevalle, verwoestende verliese, deur blaarnekrose, stamkankers en plantafsterwing. Die Proteaceae snyblom-industrie in Suid-Afrika maak 70% van die nasionale snyblomindustrie uit. Botryosphaeria siektes is 'n belangrike struikelblok in die produksie en handeldryf van Proteaceae, en daar is 'n ernstige behoefte om die etiologie, epidemiologie en beheer van siektes te ondersoek. Verliese van een van die belangrikste proteas, P. magnifica, beloop plaaslik 50% of meer. Die hoof doelstellings van hierdie studie was dus om die etiologie en epidemiologie van Botryosphaeria stamkankers op P. magnifica en ander Proteaceae vas te stel en metodes van siektebeheer te ondersoek. Hoewel daar 'n wye hoeveelheid inligting rakende die swam bestaan, wat in Hoofstuk I hersien is, is daar relatief min inligting oor Botryosphaeria op Proteaceae beskikbaar. Die taksonomie van Botryosphaeria benodig deeglike hersiening, en molekulêre tegnieke word benodig om spesie-identiteite op te klaar. In Hoofstuk 2 is gevind dat Phyllachora proteae, 'n blaarpatogeen van proteas, 'n Fusicoccum anamorf produseer, wat as F. proteae beskryf word. 'n Sphaeropsis-agtige synanamorf is met F. proteae geassosieer en 'n nuwe kombinasie vir P. proteae is as B. proteae in Botryosphaeria voorgestel. Die taksonomie van Botryosphaeria is, beide op die genus- as die spesievlak, in wanorde. In Hoofstuk 3 word die taksonomiese geskiedenis van Botryosphaeria hersien, en die genus word omskryf en van ander morfologies soortgelyke genera onderskei. Hoewel verskeie anamorf genera al met Botryosphaeria op grond van morfologiese waarnemings en filogenetiese analise van ITS rDNA volgorde data verbind is, word twee anamorf genera nou herken, dié met gepigmenteerde konidia (Diplodia), en dié met deurskynende konidia (Fusicoccum). Botryosphaeria proteae moet dus van Botryosphaeria uitgesluit word. Verskeie patogeniese Botryosphaeria spp. het 'n endofitiese fase in hul lewenssiklus. Hulle word dus onwetend in ander lande ingevoer waar hulle 'n gevaar vir landbou en inheemse plantegroei kan inhou. Die huidige wêreldverspreiding van Botryosphaeria spp. wat met Proteaceae geassosieer word is opgeklaar, en in Hoofstuk 4 word 'n sleutel tot die taksa wat met Proteaceae geassosieer word verskaf. Vyf Botryosphaeria spp. word met snyblom Proteaceae wêreldwyd geassosieer, naamlik B. lutea, B. protearum, B. proteae, B. ribis en B. obtusa. B. protearum word as 'n nuwe spesie beskryf. 'n Deeglike kennis van siekte-epidemiologie is noodsaaklik ten einde verliese te verminder. In Hoofstuk 5 dui ek aan dat letsels wat lei tot stamkankers, veroorsaak deur Botryosphaeria protearum op P. magnifica, in die hoofnerf of rant van blare ontstaan. Koch se postulate is uitgevoer en daar is vasgestel dat die aantal letsels wat vanuit kunsmatige inokulasies ontwikkel het korreleer met die styselvlakke teenwoordig in die blare ten tye van die inokulasie. In Hoofstuk 6 word getoon dat B. protearum as 'n endofiet in die blare van P. magnifica. In natuurlike standplase van proteas is stamkankers skaars, maar in verboude plantasies is die voorkoms hoog. Voedingsanalises dui aan dat hoër vlakke van stikstof in die blare van verboude plante in die lente voorkom, wat siekte-ontwikkeling moontlik kan bevorder. Hoë vlakke van natrium in die blare van natuurlike plante mag siekteontwikkeling beperk. Die ernstige ekonomiese verliese wat deur B. protearum veroorsaak word, maak die soektog na verbeterde metodes van siektebeheer noodsaaklik. Fungisiedtoedienings maak 'n belangrike deel uit van 'n geïntegreerde benadering tot siektebeheer. In Hoofstuk 7 dui in vitro toetse aan dat tebuconazole, benomyl, prochloraz me, iprodione en fenarimol die miseliumgroei van B. protearum effektief verminder. 'n Vermindering van 25-85% is aangetoon in die voorkoms van stamkankers in die veld, deur die toediening van fungisiedes en sanitasiesnoei. Die beste beheer is verkry deur die voorkomende toediening van benomyl, bitertanol, fenarimol, iprodione en prochloraz manganese chloride, afgewissel met mancozeb en tebuconazole, op plante in die veld. Indien sanitasiesnoei met gereelde toedienings van fungisiedes gekombineer word, kan die siekte bekamp word.

Thesis (MScAgric)--University of Stellenbosch, 2000.
ENGLISH ABSTRACT: Petri grapevme decline, also known as black goo, slow die-back and Phaeoacremonium grapevine decline, causes significant losses of young vines worldwide. Species of Phaeoacremonium, Phaeomoniella chlamydospora and related genera are associated with this grapevine disease. This study investigates the Phaeoacremonium-complex and Phaeomoniella chlamydospora, focussing on the species isolated from grapevines. Fungicide sensitivity of Pa. chlamydospora and the possibility of employing molecular techniques for the detection of Pa. chlamydospora in grapevines were also investigated. In an overview of the literature on Petri grapevine decline the disease history and the relatedness of Petri grapevine decline to esca is discussed. Petri grapvine decline occurs in propagation material or young vines. Infected material can appear asymptomatic and therefore the possibilities of molecular techniques for identification were also investigated in the literature. In South Africa Pa. chlamydospora is the dominant organism causing Petri grapevine decline and therefore different fungicides were evaluated to control this fungus. Six isolates of Pa. chlamydospora, from Stellenbosch, Wellington, Somerset West and Malmesbury of Western Cape province, South Africa, were screened against twelve fungicides testing their effect on mycelial inhibition in vitro. These fungicides included benomyl, chlorothalonil, fenarimol, fosetyl-Al, iprodione, kresoxim-methyl, mancozeb, metalaxyl, prochloraz manganese chloride, quintozene, tebuconazole and thiram. Results provided the base-line sensitivity of South African isolates of Pa. chlamydospora. Benomyl, fenarimol, kresoxim-methyl, prochloraz manganese chloride and tebuconazole were the most effective (with EC50 values ranging from 0.01 to 0.05 ug/ml) for inhibiting mycelial growth of Pa. chlamydospora in vitro. This in vitro test gave a good indication of which fungicides could be selected for further studies in glasshouses and nurseries. The molecular phylogeny of Phaeoacremonium and Phaeomoniella isolates from grapevines of South Africa, or isolates obtained from the Centraalbureau voor Schimmelcultures (CBS) in the Netherland, were investigated. Sequence data were created from the rONA region and partial B-tubulin gene of 33 of these isolates using the PCR technique. This sequence data were analysed with PAUP* version 4.Ob2a. An analysis of the sequence data confirmed the genus Phaeomoniella to be distinct from Phaeoacremonium (Pm.) based on DNA phylogeny. Although morphologically similar, the species status of Pm. aleophi/um and Pm. angustius was confirmed with DNA phylogeny and cultural characteristics. Pm. aleophilum has an optimum growth rate at 30°C and the ability to grow at 35°C, where as Pm. angustius has an optimum growth rate at 25°C and cannot grow at 35°C_ Pm. viticola was shown to be synonymous with Pm. angustius, and a new species, Pm. mortoniae, was newly described from grapevine occurring in California. Futhermore, Pm. aleophilum was newly reported from South Africa and grapevine isolates thought to be Pm. inflatipes were all re-identified as Pm. aleophilum. These findings therefore also shed some doubt on the possible role of Pm. inflatipes in Petri grapevine decline. It was confirmed that Pa. chlamydospora, Pm. aleophilum and Pm. angustius are the species involved in Petri grapevine decline. Pm. mortoniae was isolated from grapevines, but its pathogenicity should still be confirmed and the role of Pm. injlatipes in Petri grapevine decline remains unclear. Pa. chlamydospora has been routinely isolated from symptomless propagation and nursery material. Because the disease can take years to develop, it is crucial that healthy propagation material is used at planting. Pa. chlamydospora is a slowgrowing fungus, and positive identification from symptomless grapevine tissue can take up to 4 wks. The possibility of employing molecular techniques for the detection of Pa. chlamydospora in apparently healthy grapevines was investigated. Speciesspecific primers (PCLI and PCL2) based on the regions ITSI and ITS2 were designed for Pa. chlamydospora. These primers were highly sensitive and amplification was achieved from genomic DNA of Pa. chlamydospora from as low as 16 pg. Phaeoacremonium spp., related genera and common fungal taxa from grapevines were tested with these primers, but positive amplification was achieved for Pa. chlamydospora only. The presence of Pa. chlamydospora in symptomless grapevine tissue culture plants was confirmed by PCR within 24 hours. These primers therefore allow rapid and accurate identification of Pa. c~lamydospora. Testing on a larger scale with nursery material should be conducted to determine the feasibility of using these species-specific primers in the grapevine industry.
AFRIKAANSE OPSOMMING: Petri-terugsterwing van jong wingerde, ook algemeen bekend as "black goo" en Phaeoacremonium-terugsterwing, veroorsaak wêreldwyd groot geldelike verliese in die wingerdbedryf. Spesies van Phaeoacremonium, Phaeomoniella chlamydospora en verwante genera word met hierdie wingerdsiekte geassosieer. In die tesis word In oorsig gegee van die geskiedenis van hierdie siekte, die verwantskap tussen Petriterugsterwing en esca, en moontlike maniere van siektebestuur. Swamme wat by die siektekompleks betrokke is, kan in simptoomlose plantweefsel voorkom en daarom is die moontlikhede van die gebruik van molekulêre tegnieke vir swamidentifikasie in oënskou geneem. In Suid-Afrika is Pa. chlamydospora die dominante swam wat met Petriterugsterwing geassosieerword, gevolglik is verskillende fungisiedes vir die chemiese beheer van Pa. chlamydospora geëvalueer. Ses isolate van Pa. chlamydospora, versamel vanaf verskillende areas in die Wes-Kaap provinsie, is in dié studie gebruik. Benomyl, chlorothalonil, fenarimol, fosetyl-Al, iprodione, kresoxim-methyl, mancozeb, metalaxyl, prochloraz manganese chloride, quintozene, tebuconazole en thiram se effek op miselium inhibisie van Pa. chlamydospora is in vitro geëvalueer. Benomyl, fenarimol, kresoxim-methyl, prochloraz manganese chloride en tebuconazole was die mees effektiewe middels. Die effektiewe konsentrasie waarby 50% van die miselium groei geïnhibeer is (EKso),was tussen 0.01 en 0.05 ug/ml vir die mees effektiewe groep middels. Benomyl, fenarimol, kresoxim-methyl, prochloraz manganese chloride en tebuconazole het in vitro goeie potensiaal getoon, en verder toetse moet in vivo uitgevoer word. 'n Molekulêre studie is van Phaeoacremonium en Phaeomoniella isolate; verkry uit Suid-Afrikaanse wingerde, of vanaf die "Centraalbureau voor Schimmelcultures" (CBS) van Nederland; gedoen. Deur van die PKR tegniek gebruik te maak, is die basispaaropeenvolgingsdata van 33 isolate, van die ITSl, 5.8S, ITS2 rDNA area en die gedeeltelike B-tubullen geen verkry. Gekombineerde molekulêre data het die teorie ondersteun dat Phaeomoniella (Herpotrichiellaceae) gedistansieerd is van Phaeoacremonium (Magnaporthaceae). Pm. aleophilum en Pm. angustius was morfologies moeilik onderskeibaar, maar kon op grond van molekulêre data en kulturele eienskappe onderskei word. Pm. aleophilum se optimum groeitemperatuur was by 30°C en die swam besit die vermoë om by 35°C te groei. Pm. angus/ius se optimum groeitemperatuur was by 25°C, maar het nie by 35°C gegroei nie. 'n Studie van molekulêre en kulturele eienskappe het getoon dat Pm. angus/ius en Pm. viticola sinoniem is. 'n Nuwe spesie, Pm. mortoniae, wat uit wingerde van Kalifornie geïsoleer is, is beskrywe. Verder is Pm. aleophilum die eerste keer in Suid-Afrikaanse wingerde aangetref en Pm. tnflatipes isolate, wat vanuit wingerde geïsoleer is, is almal met molekulêre data gewys om Pm. aleophilum te wees. Hierdie bevindinge trek die rol van Pm. inflatipes in Petri-terugsterwing van wingerde in twyfel. Phaeomoniella chlamydospora IS m voortplantingsmateriaal en kwekerystokkies opgespoor. Omdat dit jare kan duur voordat siektesimptome ontwikkel, is dit belangrik om vroegtydig te weet of jong stokkies met Pa. chlamydospora geïnfekteer is. Pa. chlamydospora groei baie stadig en positiewe identifikasie van simptoomlose infeksies duur tot vier weke. Die toepassing van molekulêre tegnieke vir die vinnige identifikasie van Pa. chlamydospora in wingerde is dus ondersoek. Spesie-spesifieke oligonukleotiedes (PCU en PCL2) is vir Pa. chlamydospora ontwerp. Hierdie oligonukleotiedes is uiters sensitief en genomiese DNA van Pa. chlamydospora is van so laag as 16 pg geamplifiseer. Phaeoacremonium spp., verwante genera en algemene swamme vanuit wingerdmateriaal is met die oligonukleotiedes getoets, maar positiewe amplifikasie was slegs met Pa. chlamydospora moontlik. Die teenwoordigheid van Pa. chlamydospora is binne 24 uur in asimptomatiese wingerd weefselkultuurplantjies bevestig. Hierdie oligonukleotiedes identifiseer Pa. chlamydospora vinnig en akkuraat en toetsing op 'n groter skaal moet vervolgens met kwekerymateriaal onderneem word.

Thesis (MScAgric)--University of Stellenbosch
ENGLISH ABSTRACT: An understanding of the infection pathways of Botrytis cinerea in grape bunches will help to combat this devastating pathogen of grape. Many studies have been done to determine the possible infection pathways of B. cinerea. Most of these studies made use of artificial inoculations that deposit groups of conidia on the plant surface. The deposition of clusters of conidia is not a common phenomenon in nature. The aim of this study was to investigate the infection pathways of (i) naturally- as well as (ii) artificially inoculated B. cinerea conidia during all the phenological stages of three wine grape cultivars, and to compare the (iii) pathogenicity and virulence, on grape and nectarine fruit, of isolates obtained from different host plants. In the natural infection study the occurrence of Botrytis cinerea and subsequent disease expression at different positions in bunches of wine grapes (cultivars Chenin Blanc, Shiraz and Chardonnay) was determined from 1999 to 2001. Different techniques were used to detect viable inoculum at different positions (rachises, laterals, pedicels, and the peicel end, cheek and style end of berries) in bunches. Isolations were made on Kerssies' B. cinerea selective medium, or bunches were used untreated, or treated with paraquat. Paraquat was used to terminate host resistance and to promote the development of the pathogen from the tissues. The material was used untreated to detect the pathogen on the surface, or were surface-sterilized to detect mycelia (latent infection) in the tissue. In the artificial inoculation study, bunches of wine grapes (cultivars Chenin Blanc, Chardonnay and Shiraz) at pea size, bunch closure, and harvest were dusted with dry conidia of Botrytis cinerea in a settling tower and incubated for 24 h at high relative humidity (±93%). Following incubation, the bunches were divided in two groups. The one group was surface-sterilised in 70% ethanol for 5 s, the other group was left untreated. Bunches of the sterile group, and from the untreated group were used for isolation. From each bunch rachis segments, laterals, pedicels and berry skin segments (from the pedicel-end and cheek) were removed. The sections were placed in Petri dishes on Kerssies' B. cinerea selective medium and on a water agar medium supplemented with paraquat, and incubated at 22°C under diurnal light. Occupation by the pathogen was positively identified by the formation of sporulating colonies of B. cinerea on the different tissues. Lastly, in the virulence and pathogenicity experiment on grape and nectarine fruit Botrytis cinerea isolates, which were obtained from different host plants, were compared by simulating natural infection. Cold-stored fruit, considered highly susceptible to B. cinerea were therefore inoculated with single, airborne conidia of the pathogen. Different tests were conducted to assess surface penetration and lesion formation. Isolations were made from fruit skins on Kerssies' B. cinerea selective medium. Nectarine fruit were treated with paraquat, and grape berries were frozen for 1 h at -12°C. Paraquat and freezing were used to terminate host resistance and to promote the development of the pathogen from the tissues. In the natural infection studies B. cinerea occurred in a consistent pattern in bunches of the three cultivars. B. cinerea consistently developed from the tissue of the rachis, laterals, pedicel and pedicel-end, but not from the berry cheek. The rachis, lateral and pedicel contained much higher levels of B. cinerea than any position on the berry. Furthermore, the pathogen consistenly occurred at relatively high levels on the rachises throughout the season. Collectively, the data showed that in the Western Cape province, B. cinerea occured more regularly in wine grape bunches during the early part of the season, than later in the season. The data of the artificial studies confirmed the findings made with the natural infection studies. In these experiments the pathogen resided more often on the structural bunch parts than on the berries. Overall, the isolation studies revealed that conidia occurred predominantly on the rachis. The incidence of B. cinerea was furthermore constantly high in the inner bunch after each inoculation, and in bunches of different maturities. The data therefore indicated that, when available, conidia penetrated loose and tight clustered bunches in a similar way. Finally, in the virulence and pathogenicity experiments the results showed clearly that no host specialisation exists in the B. cinerea isolates used in this study. From these studies it is clear that in the Western Cape province B. cinerea occurs more readily in the inner structural parts of the bunches and more so during the earlier parts of the season. These findings should be considered when planning and implementing disease control programmes.
AFRIKAANSE OPSOMMING: INFEKSIEWEË VAN BOTRYTIS CINEREA OP GESELEKTEERDE WYNDRUIF KULTIVARS Indiepte kennis van die infeksieweë van Botrytis cinerea op druiwetrosse word benodig vir die beheer van dié vernietigende patogeen van druiwe. Vele studies is al gedoen om die moontlike infeksieweë van die swam op druiwe trosse te ondersoek. Die meeste van die studies het gebruik gemaak van kunsmatige inokulasie tegnieke waar die konidia van die swam in groepe op die korreloppervlak gedeponeer is. In die natuur is dit 'n rare verskynsel dat konidia in groepe op die korreloppervlak land. Die doel van die studie was om die infeksieweë van B. cinerea op drie wyndruif kultivars te ondersoek wat (i) natuurlik- en (ii) kunsmatig geïnokuleer is met konidia gedurende al die fenologiese stadia, en om die (iii) virulensie en patogenisisteit van isolate wat van verskillende gashere verkry is, op druiwe en nektariens te vergelyk. In die natuurlik-geïnokuleerde druiwe is die voorkoms van B. cinerea en die gevolglike siektevoorkoms op verkillende posisies in trosse van wyndruiwe (Chenin Blanc, Chardonnay, Shiraz) gedurende 1999 tot 2001 bepaal. Verskillende tegnieke is gebruik om lewensvatbare inokulum by verskillende posisies (ragis, lateraal, pedisel en pedisel-end van die korrel) in die tros waar te neem. Isolasies is op Kerssies' B. cinerea selektiewe medium gemaak, of trosse is onbehandeld gebruik, of behandel met paraquat. Paraquat is gebruik om die gasheer se natuurlike weerstand te verlaag en om die ontwikkeling van die patogeen te bevorder. Die plantmateriaal is onbehandeld gelaat om die patogeen op die oppervlak waar te neem, of die oppervlak is gesteriliseer om die latente myselium in die weefsel waar te neem. In die kunsmatige inokulasiestudies is trosse, van wyndruiwe (Chenin Blanc, Chardonnay, Shiraz), geïnokuleer met droë spore, van B. cinerea, in 'n inokulasietoring en die plantmateriaal is dan geinkubeer vir 24 h by 'n hoë relatiewe humiditeit (93%). Na die inkubasie proses is die trosse in twee groepe verdeel. Die een groep druiwe het oppervlak sterilisasie ondergaan in 70% etanol vir 5 s, en die ander groep was onbehandeld gelaat. Trosse van die onbehandelde en gesteriliseerde groep druiwe is gebruik vir isolasies. Vanuit elke tros is daar segmente van die ragis, laterale, pediselle en korrels (van die pedisel-end en wang gedeeltes) geïsoleer. Die segmente is in Petri bakkies met Kerssies' B. cinerea selektiewe medium en op water agar medium, wat paraquat bevat het, geïsoleer en geïnkubeer onder 'n 12 h dagligperiode teen 22°C. Die patogeen is positief geïdentifiseer deur sporuierende kolonies op die onderskeie weefseltipes. Laastens, in die virulensie- en patogenisiteitsproewe op druiwe en nektariens is verskillende isolate van B. cinerea, verkry vanaf verskillende gasheerplante, vergelyk deur natuurlike inokulasie toestande na te boots. Koue opgebergde vrugte, wat beskou word as hoogs vatbaar vir die infeksie van B. cinerea, is geïnokuleer met droë, enkel luggedraagde spore van die patogeen. Verskillende toetse is gedoen om die oppervlak penetrerende en letselvormende vermoëns van die onderskeie isolate te toets. Isolasies is van die skille van die vrugte gemaak en op Kerssies' B. cinerea selektiewe medium geplaas. Die nektarienvrugte is met paraquat behandel en die druifkorrels is gevries vir 1 h teen -12°C. Paraquat en bevriesing is gebruik om die gasheer se weerstand te verlaag en om die ontwikkeling van die patogeen te bevorder. In die natuurlik-geïnokuleerde studies het B. cinerea 'n konstante patroon getoon in die trosse van die drie verskillende wyndruif kultivars. B. cinerea het konstant ontwikkel uit die ragis, laterale, pedisel en pedisel-end, maar selde uit die korrelwang. Die ragis, lateral en pedisel dele het baie hoër vlakke van van die swam bevat as enige deel op die korrel. Die patogeen het ook konstant volop deur die hele seisoen op die ragis voorgekom. Gesamentlik wys die data dat, B. cinerea in wyndruiwe, in die Wes Kaap provinsie, meer geredelik vroeër in die seisoen voorkom, eerder as later. Data van die kunsmatige inokulasiestudies het die bevindinge van die natuurlike inokulasiestudies tot 'n groot mate bevestig. In dié studies het die patogeen meer geredelik die strukturele dele van die tros, eerder as op die korrels, bewoon. Oor die algemeen het die isolasieproewe gewys dat die konidia meer op die ragis voorkom as op enige ander deel. Die voorkoms van B. cinerea was ook oor die algemeen baie hoër in die strukturele dele van die tros, as op die korrel self. Die verskynsel het onder trosse van verskillende ontwikkelingsvlakke voorgekom. Die data het dus ook gewys dat konidia, wanner dit beskikbaar is, minder- sowel as meer kompakte trosse op 'n soortgelyke manier penetreer. Laastens, in die virulensie en patogenisiteitseksperimente het die resultate duidelik gewys dat daar geen gasheer spesifieke gedrag onder B. cinerea isolate is nie. In die studies het dit duidelik na vore gekom dat, B. cinerea meer geredelik in die strukturele binne dele van die wyndruif tros, in die Wes Kaap provinsie voorkom. En so ook eerder aan die begin van die seisoen, as later in die seisoen. Dié kennis moet in aanmerking geneem word by die beplanning en implementering van siektebeheerprogramme.

The fungus Stagonospora nodorum is the causal agent of leaf and glume blotch disease on wheat and is an emerging model for the study of the interaction between plants and necrotrophic fungal pathogens. Signal transduction plays a critical role during infection by allowing the pathogen to sense and appropriately respond to environmental changes. The role of signal transduction in the pathogenicity of S. nodorum was analysed by the targeted inactivation of genes encoding a G[alpha] subunit (Gna1) and a mitogen-activated protein kinase (Mak2). Strains carrying the inactivated genes were impaired in virulence and demonstrated a host of phenotypic impairments such as abolished sporulation. Therefore, it was hypothesised that Gna1 and Mak2 regulate downstream effector molecules that are critical for pathogenic development. A 2D gel-based proteomic approach was used to compare the extracellular and intracellular proteomes of the wild-type fungus and signalling mutants for differences in protein abundance. Tandem mass spectrometry (LC-MS/MS) analysis and patternmatching against the S. nodorum genome sequence led to the identification of 26 genes from 34 differentially abundant protein spots. These genes possess probable roles in protein cycling, plant cell wall degradation, stress response, nucleotide metabolism, proteolysis, quinate and secondary metabolism. A putative short-chain dehydrogenase gene (Sch1) was identified and its expression was shown to be reduced in both signalling mutants. The transcript level of Sch1 increased during the latter period of infection coinciding with pycnidiation. Sch1 was inactivated by targeted gene deletion. Mutants were able to effectively colonise the host but asexual sporulation was dramatically reduced and pycnidial ontogeny was severely disrupted. Furthermore, the sch1 mutants showed alterations in the metabolome. GC-MS analysis identified a metabolite which accumulated in the sch1 mutants. Computational and database analyses indicated that the compound possesses a cyclic carbon backbone. Based on these findings, Sch1 may be a suitable target for fungicides that inhibit asexual sporulation and the accumulated compound may be used to design novel antifungal compounds. 2D SDS-PAGE analysis identified increased abundance of another putative short-chain dehydrogenase (Sch2) and a nitroreductase in the sch1-deleted background. It was also shown that Sch2 was regulated by Gna1.

The fungus Stagonospora nodorum is the causal agent of leaf and glume blotch disease on wheat and is an emerging model for the study of the interaction between plants and necrotrophic fungal pathogens. Signal transduction plays a critical role during infection by allowing the pathogen to sense and appropriately respond to environmental changes. The role of signal transduction in the pathogenicity of S. nodorum was analysed by the targeted inactivation of genes encoding a G[alpha] subunit (Gna1) and a mitogen-activated protein kinase (Mak2). Strains carrying the inactivated genes were impaired in virulence and demonstrated a host of phenotypic impairments such as abolished sporulation. Therefore, it was hypothesised that Gna1 and Mak2 regulate downstream effector molecules that are critical for pathogenic development. A 2D gel-based proteomic approach was used to compare the extracellular and intracellular proteomes of the wild-type fungus and signalling mutants for differences in protein abundance. Tandem mass spectrometry (LC-MS/MS) analysis and patternmatching against the S. nodorum genome sequence led to the identification of 26 genes from 34 differentially abundant protein spots. These genes possess probable roles in protein cycling, plant cell wall degradation, stress response, nucleotide metabolism, proteolysis, quinate and secondary metabolism. A putative short-chain dehydrogenase gene (Sch1) was identified and its expression was shown to be reduced in both signalling mutants. The transcript level of Sch1 increased during the latter period of infection coinciding with pycnidiation. Sch1 was inactivated by targeted gene deletion. Mutants were able to effectively colonise the host but asexual sporulation was dramatically reduced and pycnidial ontogeny was severely disrupted. Furthermore, the sch1 mutants showed alterations in the metabolome. GC-MS analysis identified a metabolite which accumulated in the sch1 mutants. Computational and database analyses indicated that the compound possesses a cyclic carbon backbone. Based on these findings, Sch1 may be a suitable target for fungicides that inhibit asexual sporulation and the accumulated compound may be used to design novel antifungal compounds. 2D SDS-PAGE analysis identified increased abundance of another putative short-chain dehydrogenase (Sch2) and a nitroreductase in the sch1-deleted background. It was also shown that Sch2 was regulated by Gna1.

Dissertation (PhD (Agric))--University of Stellenbosch, 2005.
ENGLISH ABSTRACT: During the past few years a drastic reduction has been noted in the survival rate of grafted grapevines in nurseries, as well as in young vineyards in the Western Cape Province of South Africa. Circumstantial evidence suggested that Cylindrocarpon spp., which cause black foot disease of grapevine, were associated with this decline. Black foot disease of grapevine is a relatively new, and as yet poorly known disease affecting vines in various countries where grapevines are cultivated. Primary aims of this research have been (1) to conduct nursery surveys in order to determine which fungi are involved in the decline phenomenon, with special reference to the involvement of Cylindrocarpon spp., (2) to identify and characterise the organisms believed to be the causal organisms of black foot disease, and (3) the development of control and/or management strategies to prevent or eradicate Cylindrocarpon infections. Nursery grapevines were sampled at different stages from three commercial nurseries in the Wellington area of the Western Cape Province and were investigated during the 19992000 season by means of destructive sampling. The first samples were taken in September from callused cuttings prior to planting in nurseries. After planting, asymptomatic rooted cuttings were selected from nurseries after 3, 6 and 9 months. Isolation studies clearly demonstrated that different “Cylindrocarpon spp.” infected cuttings from nursery soils. These species rarely occurred in rootstock propagation material prior to planting. At the time of planting, the susceptible basal ends (especially the pith area) of most of the nursery cuttings are partly or even fully exposed. Callus roots also break during the planting process, resulting in small wounds susceptible to infection by soilborne pathogens. The isolation studies revealed that the first infections occurred in the roots, followed by infections of the rootstocks. These infections increased progressively during the course of the growing season. Substantial variation in cultural and morphological characters was observed among the Cylindrocarpon isolates obtained from the nursery survey, as well as from isolations that were made from diseased grapevines. Morphological and phylogenetic studies were conducted to identify these “Cylindrocarpon spp.” and to establish their association with black foot disease. Sequences of the partial nuclear large subunit ribosomal DNA (LSU rDNA), internal transcribed spacers 1 and 2 of the rDNA including the 5.8S rDNA gene (ITS), and partial β-tubulin gene introns and exons were used for phylogenetic inference. Phylogenetic analyses confirmed the diversity observed among the isolates and four Cylindrocarpon-like species were identified. One of these species was initially identified as Cylindrocarpon destructans. However, further research revealed C. destructans to represent a species complex. Grapevine isolates of “C. destructans” proved to be identical to the ex-type strain of Cylindrocarpon liriodendri, which also produced a teleomorph, Neonectria liriodendri in culture. A second species was newly described in this study as Cylindrocarpon macrodidymum (Neonectria macrodidyma). The two remaining Cylindrocarpon-like species were placed in a new genus, Campylocarpon. The two species were named Campylocarpon fasciculare and Campylocarpon pseudofasciculare. Pathogenicity studies confirmed that all four species were able to reduce root and shoot mass significantly. Knowledge obtained pertaining to the disease cycle of black foot disease suggest that suitable management strategies should focus on prevention of primary infection in nurseries. However, at present, no fungicides are registered for control of this disease in South African vineyards or nurseries. Thirteen fungicides were screened in vitro for mycelial inhibition of these pathogens. Prochloraz manganese chloride, benomyl, flusilazole and imazalil were the most effective fungicides tested, and were subsequently included in semi-commercial field trials. Basal ends of grafted cuttings were dipped (1 min) in various chemical and biological treatments prior to planting in open-rooted nurseries. Black foot pathogens were not isolated from grafted cuttings prior to planting in nurseries. Additional treatments involved soil amendments with Trichoderma formulations and hot water treatment (50°C for 30 min) of dormant nursery grapevines. Field trials were evaluated after a growing season of eight months. The incidence of black foot pathogens was not significantly and/or consistently reduced by the majority of chemical or biological treatments. However, these pathogens were not isolated from uprooted plants that were subjected to hot water treatment. It is therefore recommended that hot water treatment of dormant nursery plants be included in an integrated strategy for the proactive management of black foot disease in grapevine nurseries.
AFRIKAANSE OPSOMMING: Gedurende die afgelope paar jaar is ‘n drastiese afname waargeneem in die sukses van geënte wingerdplante in kwekerye, sowel as jong wingerde van die Wes-Kaap. Omstandigheidsgetuienis dui daarop dat Cylindrocarpon spp., wat die wingerdsiekte swartvoet veroorsaak, geassosieer word met hierdie agteruitgang. Swartvoet is ‘n relatiewe nuwe siekte waarvan daar baie min inligting bekend is, alhoewel dit voorkom in verskeie lande waar wingerd verbou word. Die primêre doel van navorsing was (1) om opnames in wingerdkwekerye uit voer om te bepaal watter swamme betrokke is by die verskynsel van agteruitgang, met spesiale verwysing na die betrokkenheid van Cylindrocarpon spp., (2) om die organismes te identifiseer en te karakteriseer wat daarvan verdink word dat hulle die siekte swartvoet veroorsaak, en (3) om beheer en/of bestuurspraktyke te ontwikkel om Cylindrocarpon infeksies te voorkom of uit te wis. Kwekeryplantjies in drie kommersiële kwekerye in die Wellington omgewing van die Wes-Kaap is gedurende verskillende tye gedurende die groeiseisoen gemonitor. Die opnames het plaasgevind gedurende die 19992000 seisoen deur middel van destruktiewe monsterneming. Die eerste monsters is geneem in September nadat die stokkies geënt en gekallus is en voordat dit in die kwekery geplant is. Na plant is asimptomatiese, gewortelde plante vanuit die kwekerye na 3, 6 en 9 maande uitgehaal. Isolasiestudies dui duidelik daarop dat verskillende “Cylindrocarpon spp.” plante vanuit die kwekerygrond geïnfekteer het. Hierdie spesies het selde voorgekom in onderstok-voortplantingsmateriaal voor plant. Tydens plant is die vatbare basale gedeelte, veral die pit, van die meeste geënte stokkies gedeeltelik of selfs volledig blootgestel. Kalluswortels breek ook tydens plant wat wonde laat vir infeksie deur grondgedraagde siektes. Die isolasiestudies dui ook daarop dat die eerste infeksies in die wortels plaasgevind het, gevolg deur infeksies van die onderstokke. Hierdie infeksies het toenemend voorgekom gedurende die verloop van die groeiseisoen. Substansiële variasie in kultuur- en morfologiese eienskappe is waargeneem in die Cylindrocarpon isolate wat tydens die kwekeryopnames versamel is, sowel as van isolasies wat gemaak is uit siek plante. Morfologiese en filogenetiese studies is uitgevoer om hierdie “Cylindrocarpon spp.” te identifiseer en hul betrokkenheid by die siekte swartvoet uit te klaar. Gedeeltelike DNS volgordes van die groot ribosomale subeenheid (“LSU rDNA”), interne getranskribeerde spasiëerderarea (“ITS1, “ITS2”), insluitend die 5.8S rRNS geen, en gedeeltelike β-tubilien geen introns and eksons is gebruik vir filogenetiese analise. Filogenetiese analises het die diversiteit wat waargeneem is tussen die verskillende isolate bevestig deurdat vier Cylindrocarpon-agtige spesies geïdentifiseer is. Een van hierdie spesies is aanvanklik geïdentifiseer as Cylindrocarpon destructans. Verdere navorsing het egter daarop gedui dat C. destructans ‘n spesie-kompleks verteenwoordig. “C. destructans” afkomstig van wingerd blyk identies te wees aan die ex-tipe isolaat van Cylindrocarpon liriodendri, wat ook ’n teleomorf, Neonectria liriodendri in kultuur vorm. ’n Tweede spesie is nuut beskryf in hierdie studie as Cylindrocarpon macrodidymum (Neonectria macrodidyma). Die twee oorblywende Cylindrocarpon-agtige spesies is geplaas in ‘n nuwe genus, Campylocarpon. Die twee spesies staan bekend as Campylocarpon fasciculare en Campylocarpon pseudofasciculare. Patogenisiteitstudies het bevestig dat al vier spesies die vermoë het om wortel- en lootmassa van wingerdplant drasties te verlaag. Kennis wat opgedoen is rakende die lewensiklus van swartvoet dui daarop dat bestuurspraktyke daarop moet fokus om primêre infeksies in wingerdkwekerye te voorkom. Op die oomblik is daar egter geen fungisiedes geregistreer vir die beheer van die siekte in Suid- Afrikaanse wingerde of kwekerye nie. Dertien fungisiedes is in vitro geëvalueer om te bepaal of dit miseliumgroei van hierdie swamme kan inhibeer. Prochloraz mangaan chloried, benomyl, flusilasool en imazalil was die effektiefste fungisiedes wat ondersoek is, en is gevolglik ingesluit in semi-kommersiële veldproewe. Die basale gedeelte van geënte stokkies is gedoop (1 min) in verskeie chemies en biologiese behandelings voordat dit geplant is in die kwekerye. Patogene wat geassosieer word met swartvoet is nie vanuit geënte stokkies geïsoleer voordat dit in die kwekerye geplant is nie. Addisionele behandelings het bestaan uit grondtoevoegings met Trichoderma formulasies, sowel as warmwaterbehandeling (50°C vir 30 min) van dormante kwekeryplante. Die veldproewe is geëvalueer na ‘n groeiseisoen van 8 maande. Die voorkoms van swartvoet patogene is nie betekenisvol/konstant verlaag deur die meeste chemies en biologiese behandelings nie. Hierdie patogene is egter nie vanuit plante geïsoleer wat na uithaal aan warmwaterbehandeling blootgestel is nie. Dit word dus aanbeveel dat warmwaterbehandeling van dormante kwekeryplante deel word van ‘n geïntegreerde strategie vir die pro-aktiewe beheer van swartvoet in wingerdkwekerye.

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

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

The tropical rainforest has interested ecologists for hundreds of years because of its vast species diversity. The distribution and establishment of trees is related to soil properties and rootassociated microorganisms. The coexistence of hyper-diverse plant communities in tropical rainforests has resulted in associations being formed with belowground communities, mycorrhizas (particularly arbuscular mycorrhizal (AM), ectomycorrhizas (ECM)) and root associated bacterial communities. The rapid deforestation in Southeast Asia is causing the loss of the dominant and important tree species belonging to the family Dipterocarpaceae. It is important to understand whether different host species in the same environment maintain mycorrhizal and bacterial diversity, especially mycorrhizas with a restricted host range. In this study, I examine the ecology of mycorrhizas and bacteria associated with Dipterocarpaceae and also the plant community as a whole. The aim of this project is to understand the effect of host properties (e.g. species, size), soil factors (e.g. nutrient concentrations) and spatial factors on mycorrhizal fungi and bacterial diversity and community structure. The research took place in two Centre for Tropical Forest Science (CTFS) plots in Malaysia: Pasoh Forest Reserve (in Negeri Sembilan) and Danum Valley Conservation Area (in Sabah). Molecular protocols and a modern technique, Next Generation Sequencing (NGS), were adopted to quantify mycorrhizal and bacterial loads in tropical plants. ITS1 and ITS2 regions were used for ECM, 18S rRNA were used for AM, and 16S rRNA were used for bacteria. Mycorrhizas and bacteria present in the roots of Dipterocarpaceae from 60 individual plants belonged to 25 species within 6 genera were traced and sampled in 2015. To my knowledge, this study is the first attempt to study root-associated bacteria across multiple species within a single family, Dipterocarpaceae. Dipterocarpaceae's species was found to significantly influence root bacteria. Analyses showed that mycorrhizal communities are similar on the host, unlike the null model. Dipterocarpaceae was previously believed to solely host ECM, but this study disproves this. This study shows that Dipterocarpaceae can have dual colonization, as it iv can also associate with AM fungi. One soil core of 10 cm × 10 cm × 7.5 cm were collect randomly in three subplot and further divided at 2.5 cm each slice into 75 individual 'microcubes' of 2 cm × 2 cm × 2.5 cm depths enumerates a total of 192 fine root samples. Multivariate analysis revealed that AM fungi tend to associate with non-dipterocarp (as well as unidentified families) while ECM fungi tend to associate with dipterocarps. Data was also collected on host attributes, plant size, and root density. Dipterocarpaceae size does not influence the distribution of mycorrhizal or bacterial communities. The root density reduces as depth increases. Therefore, root density does have a significant influence on mycorrhizal community structure. The diversity of ECM and AM fungal communities within cubes decreased significantly with depth (p < 0.001), whereas the mycorrhizal communities did not change across horizontal distances within cubes. To investigate whether there is a relationship between belowground communities and soil properties, soil macro and micro nutrients were examined and a multivariate analysis was performed. The results showed that communities of belowground (mycorrhizal and bacterial) species correlate with soil parameters. Spatial scale also had an effect on community assembly, independent of environmental variation. These results demonstrate that mycorrhizal fungal communities can vary substantially over very fine spatial scales, and that the distribution of roots from different species do not reflect their proximity aboveground. This study clearly demonstrates the widespread presence of mycorrhizal fungi and root associated bacteria in tropical rainforest plants.

Thesis (MScAgric)--University of Stellenbosch, 2001.
ENGLISH ABSTRACT: Several attempts have been made to reduce Botrytis cinerea grey mould in vineyards and in storage by means of biological control. However, the so called "silver bullet" approach in utilising a single antagonist, has its limitations when compared with synthetic fungicides. Often the antagonist has a limited spectrum of activity and the duration of its effectiveness is less than that provided by synthetic fungicides. Furthermore, antagonists are more likely to be effective in preventing initial infection rather than resumption of latent infection. Therefore, due to the various infection sites in grape bunches utilised by B. cinerea and the fact that the pathogen can remain latent in the grapevine tissue, it may be possible to obtain effective control of the pathogen by integrating fungicides and different biological control agents each aimed at a different site in grape bunches, protecting the bunch at the various phenological stages of growth and under different micro climatic conditions. In this study the potential of three fungal antagonists (Glioc/adium roseum, Uloc/adium atrum and Trichoderma harzianum) and one yeast (Trichosporon pullulans) to colonise different sites in grape bunches, and to reduce B. cinerea infection, was investigated in commercial vineyards. As the biological control agents were used in an integrated system, the effect of various fungicides frequently applied to local vineyards on the organisms was also investigated. Fungicide trials were conducted taking into account two possible scenarios. Firstly, the possible effect of fungicides applied to the vineyard after an application of the biological control agent or shortly before the application of the biocontrol agent. This entailed exposing the biocontrol agents to relatively low concentrations of the active ingredient of the fungicides, similar to the residue levels to which these organisms would be exposed under field conditions. Secondly, the possibility of applying the organisms and the fungicides at the same time by making use of spray tank mixtures. This meant exposing the biocontrol agents to relatively high doses of the active ingredient of the various fungicides. Mycelial growth and germination tests were performed on agar in Petri dishes to determine the effect of fungicides. It was assumed that if the fungicide effectively inhibits the antagonist at 2.5 !-lg a.Uml, the fungicide and antagonist can not be used in an integrated programme. Based on this criterium, T harzianum can not be applied to vineyards with penconazole, mancozeb/metalaxyl, pyrifenox or mancozeb. In addition T harzianum can not be applied as tank mixtures with iprodione. However, T harzianum can be used in conjunction with pyrimethanil, folpan, iprodione, fosetyl-Al and copperhydroxide, provided the chemicals and the antagonist are applied alternately. Gliocladium roseum can not be applied in a tank mixture with pyrimethanil and penconazole, but can be used on grapevine in conjunction with penconazole, pyrifenox, pyrimethanil, iprodione and fosetyl-Al. Ulocladium atrum can not be applied with pyrimethanil and iprodione. Ulocladium atrum can be applied in conjunction with penconazole, pyrifenox, pyrimethanil, iprodione, fosetyl-Al and mancozeb. The fungus can be applied in a tank mixture with penconazole and pyrifenox. The antagonists were applied as conidial suspensions to bunches at various phenological stages in commercial vineyards planted with the wine grape cultivar Chardonnay in the Stellenbosch region, or the table grape cultivar Dauphine planted in Paarl region. Bunches were collected 2 wk after application, surface-sterilised and used for determining antagonist colonisation and B. cinerea infection at specific sites in the bunches. In Chardonnay, the antagonists colonised the different sites, but colonisation during the three seasons was inconsistent and sporadic. Ulocladium atrum and G. roseum colonised floral debris to a degree in the 1996 season. However, in the 1997 season these two antagonists did not develop from floral debris. Trichoderma harzianum colonised floral debris extensively in the 1996 season. In the 1997 season colonisation by T harzianum dropped, but unlike G. roseum and U atrum, T harzianum occurred at a low level in flowers. Ulocladium atrum only colonised bunches during bloom, and was not found in bunches monitored from pea-size stage to véraison. This finding suggests that the saprophyte colonised moribund and dead flower parts occurring in bunches during full bloom to the pre-pea size stage, and is not likely to be found in living tissue. Gliocladium roseum colonised grape berries and pedicels to some degree and T harzianum colonised these grape parts extensively. Botrytis cinerea occurred inconsistently and at low frequencies in the different sites in bunches. It was therefore not possible to comment on the effectivity of the various antagonists in the three seasons during which the trials were performed. However, it was noted that, during the peasize stage in 1996, when high levels of B. cinerea were recorded, T harzianum controlled these infections in the pedicels more effectively than any other treatment.
AFRIKAANSE OPSOMMING: ONDERDRUKKING VAN BOTRYTIS CINEREA DEUR ANTAGONISTE IN LEWENDE, AFSTERWENDE EN DOOIE WINGERDWEEFSEL Die benadering om Botrytis cinerea verrotting van wingerd met behulp van 'n enkele biologiese beheeragent in plaas van met sintetiese fungisiede te beheer, het sekere beperkinge. Antagoniste het dikwels 'n beperkte spektrum van aktiwiteit, en die duur van hul effektiwiteit is minder as dié van fungisiede. Antagoniste is gewoonlik ook minder effektief in die beheer van latente infeksie. Die patogeen het verder die opsie om druiwetrosse deur verskillende infeksieweë te koloniseer. Fungisiede kan druiwetrosse beter teen infeksie deur veelvuldige infeksieweë beskerm as 'n enkele antagonis. In die lig hiervan is die beheer van die patogeen deur 'n kombinasie van fungisiede en verskillende biologiese beheeragente, wat elk gemik is om 'n ander infeksiepunt in die druiwe te beskerm, ondersoek. Drie swamagtige antagoniste (Glioc/adium roseum, Uloc/adium atrum en Trichoderma harzianum) en een gis (Trichosporon pullulans) is in die ondersoek gebruik. Voorloper ondersoeke, waar twee moontlike scenarios in ag geneem is, is met fungisiede uitgevoer. In die eerste scenario is die effek van fungisiede, aangewend op wingerd kort vóór aanwending van die biologiese beheeragent, of kort ná aanwending, ondersoek. Hierdie proef het die blootstelling van die biologiese beheeragent aan relatief lae konsentrasies van die aktiewe bestanddeel van die fungisied, vergelykbaar met residuvlakke waaraan die organismes onder veldtoestande blootgestel sou word, behels. Tweedens is die moontlikheid om antagoniste en fungisiede gelyktydig as spuitpompmengsels toe te dien, ondersoek. In hierdie proef is die biologiese beheeragente aan relatief hoë dosisse van die aktiewe bestanddeel van verskillende fungisiede blootgestel. Miseliumgroei en ontkiemingstoetse is op agar in Petribakkies uitgevoer om die effek van die fungisiede te bepaal. As kriterium is aanvaar dat indien 'n fungisied die antagonis effektief by 2.5J..lglml aktiewe bestanddeel inhibeer, die fungisied en antagonis nie in 'n geïntegreerde program gebruik kan word nie. Gebaseer op hierdie kriterium kan T harnzianum nie aangewend word in 'n wingerd wat met penconazole, mancozeb/metalaxyl, pyrifenox of mancozeb behandel is nie. Ook kan T harzianum nie in 'n spuitpompmengsel met iprodione aangewend word nie. Trichoderma harzianum kan egter saam met pyrimethanil, folpan, iprodione en fosetyl-Al gebruik word, mits dié chemikalieë en die antagonis afwisselend aangewend word. Glioc/adium roseum kan nie in 'n spuitpompmengsel met pyrimethanil en penconazole aangewend word nie, maar kan saam met penconazole, pyrifenox, pyrimethanil, iprodione en fosetyl-Al gebruik word. Uloc/adium atrum kan nie saam met pyrimethanil, iprodione en fosetyl-Al gebruik word nie. Die swam kan wel in 'n spuitpompmengselmet penconazole en pyrifenox aangewend word. In verdere proewe is die antagoniste as spoorsuspensies op trosse op verskillende groeistadia in kommersiële wingerde, wat met die wyndruitkultivar Chardonnay of die tafeldruifkultivar Dauphine aangeplant is, ondersoek. Trossies is twee weke na toediening versamel, oppervlakkig gesteriliseer en gebruik om vlakke van antagoniskolonisasie en B. cinerea infeksie op spesifieke nisse in die trosse te bepaal. In die geval van Chardonnay het die antagoniste die verskillende nisse gekoloniseer, maar die kolonisasie was sporadies en nie konstant gedurende die drie seisoene van ondersoek nie. Uloc/adium atrum en G. roseum het blomdeeltjies tot 'n beperkte mate in die 1996 seisoen gekoloniseer, maar nie in die daaropvolgende seisoen nie. Daarteenoor het T. harzianum blomdeeltjies ekstensief in die 1996 seisoen gekoloniseer, en in 'n beperkte mate in die daaropvolgende seisoen. Uloc/adium atrum kon nie trosse van ertjiekorrelgrootte tot deurslaan vestig nie. Hierdie bevinding dui daarop dat die saprofiet afsterwende en dooie blomdeeltjies, wat van volblom tot ertjiekorrelstadium in die trosse voorkom, koloniseer, maar dat dit nie in lewende weefsel voorkom nie. Daarteenoor het T. harzianum die verskillende trosdele ekstensief gekoloniseer. Botrytis cinerea het gedurende die drie seisoene wisselvallig en teen lae frekwensies in die verskillende nisse in die trosse voorgekom. Dit was gevolglik nie moontlik om 'n konkrete afleiding oor die effektiwiteit van die verskillende antagoniste as biobeheeragente van B. cinerea te maak nie. In die geval van Dauphine was die onderskeie organismes swak koloniseerders van blomdeeltjies. Trichoderma harizanum kon egter die lewende trosdele koloniseer. Kolonisasievlakke was laag en was nooit meer as 50% nie. In beide seisoene het die kolonisasievermoë van T. harzianum drasties ná trostoemaak gedaal. Daarteenoor het beide G. roseum en U atrum tydens al die ontwikkelingstadia die lewende trosdele swak gekoloniseer. Botrytis cinerea het ook uiters sporadies en teen baie lae vlakke voorgekom. Die bevindinge het getoon dat klimaatsomstandighede wat in tafeldruifwingerde in die Wes-Kaap heers, nie geskik is vir die vestiging van die biologiese beheeragente wat in die studie ondersoek is nie.

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

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.

This research has focused on the determination of natural populations in the fields, the effect of different inoculum densities on lettuce growth and a study of the association of this fungus with two nematodes (Pratylenchus penetrans Cobb and Meliodogyne hapla Chitwood). Under conditions of artificial infestation of soil the results were satisfactory, but in trials with naturally infested soil the fungus could not be detected. The effect of different inoculum densities was measured at different stages of growth, and only in those plants inoculated 2 weeks after seeding were differences significant and consistent. Some evidence of the detrimental effect of wounding the root system prior to attack by the fungus led to studies of the relationship between this fungus with either P. penetrans or M. hapla. In the first case a negative interaction seemed to exist; no significant increase of the damage caused to the lettuce was observed. In contrast, when the root-knot nematodes and P. tracheiphilum were combined there was a marked reduction of lettuce growth. The interaction was found to be additive.

Thesis (MScAgric)--University of Stellenbosch, 2002.
ENGLISH ABSTRACT: Botrytis bunch rot of grape is caused by Botrytis cinerea Pers. :Fr. Conidia of the pathogen, which is dispersed by wind, water droplets and by insects, can penetrate the intact grape berry cuticle, but disease expression occurs only under predisposing conditions. Since relatively high infection rates often occur in vineyards, predisposing factors must play a fundamental role in primary infection and subsequent disease occurrence. Insects can play a very important role in this regard by depositing inocula at wound sites during feeding and by providing fresh wounds during their oviposition and feeding activities. The aim of this study was (i) to determine the potential of the Mediterranean fruit fly to transfer B. cinerea and other bunch and fruit rot fungi in natura, (ii) to investigate the transport, deposition and subsequent disease expression on grape berries in vitro, and (iii) to investigate fruit fly activities and the nature of deposited conidia and mycelia of B. cinerea by aid of digital photography and epifluorescence microscopy, respectively. Two Sensus fruit fly traps containing the para-pheromone, Capilure, were installed in orchards and five neighboring vineyards on four farms in the Stellenbosch region. Ceratitis fruit flies were collected weekly, identified and counted to determine the fluctuations in fruit fly population. Following field collection, the fruit flies were plated on Kerssies' B. cinerea selective medium and the number of flies yielding the pathogen was recorded. Two fruit fly species, C. capitata and C. rosa, were captured during the study period. Ceratitis rosa numbers comprised only 1% of the total number of fruit flies captured. Ceratitis capitata numbers, and the percentage B. cinerea contaminated flies generally increased after harvest in the different orchards and vineyards. Following harvest, the percentage flies yielding B. cinerea was higher in vineyards compared to orchards. Furthermore, in each vineyard an increase in percentage B. cinerea contaminated fruit flies was preceded by a corresponding increase in its neighboring orchard. The levels of both Penicillium and Alternaria contaminated fruit flies stayed high throughout the investigation period, especially after harvest of the orchard cultivars. Low incidence of Aspergillus, Mucor and Rhizopus spp. were recorded on C. capitata. These findings suggest that the Mediterranean fruit fly may play an important role in the dispersal of inocula of fungi associated with postharvest decay from early-maturing stone fruit orchards to mid- and late-maturing wine grape vineyards, and in disease induction under conditions unfavourable for natural infection. Three experiments were conducted to determine the potential of fruit flies in provoking B. cinerea decay. In the first experiment, transport of conidia and disease expression were investigated on rachis segments bearing unwounded berries only. In the second experiment, the effect of wounding on disease expression was investigated. In the third experiment, the effect of inoculum type (mycelia and conidia) on transportation and disease expression was investigated on rachis segments bearing unwounded berries, and on segments with wounded berries. The table grape cultivar, Dauphine, and the wine grape cultivar, Shiraz, were used at véraison, two weeks before harvest and harvest, and the transport studies were conducted in ethanol-disinfected perspex cages. Disease expression was studied in dry (~56% RH), ethanol-disinfected perspex chambers incubated at 22°C. The isolations from berries revealed that the flies deposited, without preference, high amounts of B. cinerea at various positions on the grape berry's surface. The freezing studies showed that the deposited conidia germinated and penetrated the berry skin at various positions. However, B. cinerea developed more often at the pedicel end than on the cheek or style end, which indicated a peculiar interaction between B. cinerea, the fruit fly and host tissue at this part of the berry. This phenomenon was substantiated by the finding that B. cinerea also developed more often at the pedicel end of berries that were not frozen. Further evidence for this interaction was found on intact berries exposed to flies that carried mycelia after feeding on berries without sporulating colonies of the pathogen, but showing symptoms of slippery skin. Significantly more decay developed on wounded berries compared to the unwounded berries and more so at the wound site. In addition, female fruit flies were responsible for significantly more decay development than male fruit flies. The study thus proved that the Mediterranean fruit fly can promote B. cinerea disease development under conditions unfavorable to natural infection. The activities of the Mediterranean fruit fly, Ceratitis capitata, on grape berries were monitored by aid of digital photography. In addition, the deposition of conidia and mycelia of Botrytis cinerea at three sites (pedicel end, cheek and style end) on the grape berry, germination of the fungal structures after dry (±56% RH) and moist (±93% RH) incubation and wounds inflicted during ovipositioning were examined with an epifluorescence microscope. The observations revealed that the fruit fly's activities were generally restricted to the grape berry. They visited the grape berry cheek more often, but visitations to the pedicel end of berries increased substantially from véraison to harvest, indicating the possibility of nutrient leakages at this site. Microscopy revealed that the flies deposited conidia singular, in feeding packages and in faecal excrements on the berry surface. The conidia in feeding packages were ensheathed by salivical fluids and occurred in clusters of 10 to 50 conidia. An average of 60% of the conidia in feeding packages germinated under dry conditions (±56% RH). Conidia that passed through the intestinal tract of the fruit fly and that were deposited in faecal excrements were deformed and low in viability. These conidia did not occur in cluster format, but were proportionally spread with the faeces on the surface of the grape berry. Conidia that were deposited singular and in faecal excrements did not germinate unless incubated under moist conditions (± 93% RH). Wounds inflicted by female fruit flies during ovipositioning were most frequently observed on the cheek. This predisposition to B. cinerea infection of grape berries by the activities of fruit flies, suggested an important role for the flies in the initiation of Botrytis bunch rot epidemics in vineyards.
AFRIKAANSE OPSOMMING: DIE ROL VAN DIE MEDITERREENSE VRUGTEVLIEG, CERATITIS CAPITATA, IN BOTRYTIS CINEREA TROSVERROTTING VAN DRUIWE Botrytis-trosverrotting van druiwe word deur Botrytis cinerea Pers. :Fr. veroorsaak. Konidia van die patogeen wat deur wind, waterdruppels en insekte versprei word, kan die intakte druiweskil binnedring, maar siekte-uitdrukking vind slegs onder spesiale omstandighede plaas. Aangesien relatief hoë infeksie vlakke algemeen in wingerde voorkom, moet predisponerende faktore 'n fundamentele rol in die primêre infeksie, en die daaruit voortspruitende siektetoestand speel. Insekte kan 'n baie belangrike bydrae lewer deur inokuia tydens voeding by wonde te deponeer. Nuwe wonde kan ook tydens oviposisionering en voeding ontstaan. Die doel van hierdie studie was om (i) die potensiaal van die Mediterreense vrugtevlieg om B. cinerea en ander tros- en vrugverrottingswamme in natura oor te dra, te bepaal; om (ii) die verspreiding, deponering en daaropvolgende siekteuitdrukking op druiwekorrels in vitro te ondersoek; en om (iii) die aktiwiteite en aard van die gedeponeerde konidia en miselia met behulp van digitale fotografie sowel as epifluoressensiemikroskopie waar te neem. Twee Sensus-vrugtelokvalle met die paraferomoon, Capilure, IS In vrugteboorde en aangrensende wingerde in die Stellenbosch-omgewing aangebring. Ceratitis-vrugtevlieë is weekliks versamel, geïdentifiseer en getel om fluktuasies in die vrugtevliegpopulasie te bepaal. Na die veldversameling is die vrugtevlieë op Kerssies se B. cinerea-selektiewe medium uitgeplaat. Gedurende die studie is twee spesies vrugtevlieë, C. capitata en C. rosa, gevang. Na oesstyd het die aantal Ceratitis-vrugtevlieë en die persentasie vrugtevlieë, besmet met B. cinerea, in die verskillende boorde en wingerde toegeneem. Na oestyd was die persentasie vrugtevlieë wat B. cinerea gedra het, hoër in die wingerde as in die boorde. Elke toename in die persentasie B. cinerea-besmette vrugtevlieë in 'n wingerd is voorafgegaan deur 'n ooreenkomstige toename in die aangrensende vrugteboord. Die aantal vrugtevlieë besmet met Penicillium en Alternaria spp. het tydens die navorsingstydperk deurgaans hoog gebly, veral nadat die vrugteboord-kultivars geoes is. Die voorkoms van Aspergillus-, Mucor- en Rhizopus spp. op Ceratitis-vrugtevlieë was deurgaans laag. Hierdie bevinding wys daarop dat vrugtevlieë 'n belangrike rol speel in die verspreiding van swarninokula, wat met na-oes verrotting geassosieer word, van vroegrypwordende steenvrugteboorde na mid- en laatrypwordende wyndruifwingerde. Drie eksperimente is in vitro onderneem om vrugtevlieë se potensiaal om B. cinereaverrotting te veroorsaak te bepaal. In die eerste eksperiment is ragi met slegs ongewonde korrels gebruik om die oordrag van konidia en siekte-ontwikkeling te ondersoek. In die tweede eksperiment is die effek van verwonding op siekte-ontwikkeling ondersoek. In die derde eksperiment is die effek van inokulumtipe (miselia en konidia) op verspreiding en siekte-ontwikkeling ondersoek deur ragis-segmente met gewonde korrels sowel as ragissegmente met ongeskonde korrels te gebruik. Die tafeldruif-kultivar Dauphine en die wyndruif-kultivar Shiraz, by kleurbreuk, twee weke voor oes en by oestyd, is in die eksperimente gebruik. Die oordragstudies is in etanol-ontsmette perspex-hokke uitgevoer. Siekte-ontwikkeling is bestudeer in droeë (±56% RH), etanol-ontsmette perspex-kamers en geinkubeer by 22°C. By ondersoek is gevind dat vlieë, sonder voorkeur, groot hoeveelhede B. cinerea op verskeie dele op die druiwekorrel-oppervlak deponeer. Bevriesingstudies het aangetoon dat die gedeponeerde konidia op verskeie dele van die korrelontkiem en die skil binnedring. Botrytis cinerea het egter meer dikwels by die korrelsteelkant as by die stempelkant, of op die wang, ontwikkel. Hierdie bevinding het 'n eiesoortige interaksie tussen B. cinerea, die vrugtevlieg en gasheerweefsel by die korrelsteelkant van die korrel aangetoon. Die verskynsel is gestaaf deur die bevinding dat B. cinerea ook meer dikwels by die korrelsteelkant van die korrels wat nie gevries is nie, ontwikkel het. Verdere bewys van hierdie interaksie is gevind by ongeskonde korrels wat aan die vlieë wat miselia gedra het blootgestel is. Die siekte het beduidend meer dikwels op gewonde as ongewonde korrels en verder aansienlik meer dikwels op die wondoppervlakte ontwikkel. Dit was ook duidelik dat vroulike vrugtevlieë baie meer vir verrotting verantwoordelik was as manlike vrugtevlieë. Die studie bewys dus dat Mediterreense vrugtevlieë die ontwikkeling van B. cinerea kan bevorder in omstandighede wat ongunstig is vir natuurlike infeksie. Die aktiwiteite van die Mediterreense vrugtevlieg C. capitata op die druiwekorrels is met behulp van digitale fotografie waargeneem. Verder is die deponering van konidia en miselia van B. cinerea op die verskillende dele (korrelsteelkant, wang en stempelkant) van die korrel, ontkieming van die swamstrukture na droeë (±56% RH) en nat (±93% RH) inkubasie en wonde wat tydens oviposisionering veroorsaak is, met epifluoressensie-mikroskopie ondersoek. Die waarnemings het onthul dat die vrugtevlieg se aktiwiteite gewoonlik tot die druiwekorrel beperk is. Hulle het korrelwange meer dikwels besoek. Besoek aan die korrelsteelkant het aansienlik toegeneem van kleurbreuk tot oestyd, wat op die moontlikheid van voedingstof-lekkasie by die deel aandui. Mikroskoopstudies het aangedui dat vlieë konidia enkel, in voedingspakkies en in fekale uitskeidings op die korreloppervlakte deponeer. Die konidia in die voedingspakkies is deur speekselvloeistof omhul en het in groepe van 10 tot 50 konidia voorgekom. Gemiddeld 60% van die konidia in voedingspakkies het in droeë omstandighede (±56% RH) ontkiem. Konidia wat deur die spysverteringskanaal van die vrugtevlieg gegaan het en in die fekale ekskresie gedeponeer is, was misvorm en het lae lewensvatbaarheid gehad. Laasgenoemde konidia was nie in groepe gedeponeer nie, maar is proporsioneel met die feces op die oppervlak van die druiwekorrel versprei. Konidia wat enkel en in feces gedeponeer is, het nie ontkiem nie, tensy toestande vogtig (±56% RH) was. Wonde wat deur die vroulike vrugtevlieë tydens oviposisionering veroorsaak is, is meer dikwels op die wang van die korrelopgemerk. Hierdie predisposisie van druiwekorrels tot B. cinerea-infeksie, meegebring deur die aktiwiteit van die vrugtevlieg, dui daarop dat die rol wat die vrugtevlieg in die inisiëring van Botrytis trosverrottingepidemies in wingerde speel, van beduidende belang is.

Common barley diseases observed in North Dakota include net blotch, spot blotch, leaf and stripe rust, bacterial leaf streak, and Fusarium head blight. The first objective of this research was to determine the effect of variety and fungicide timing on disease development of barley under conventionally tilled systems. Five field trials were performed in 2016-2017 to test the effect of common varieties and fungicide applications on foliar disease of barley. Overall, varietal selection had a greater effect on the level of foliar disease observed than fungicide application. The second objective focused on the efficacy and timing of adepidyn and prothioconazole + tebuconazole on Fusarium head blight. An inoculated greenhouse experiment was performed the fall of 2017 to determine the effectiveness of fungicide timing at half-spike, full-spike, and five days after full-spike. The protectant capabilities of the fungicides were greater than their curative properties.

Thesis (MScAgric)--University of Stellenbosch, 2002.
ENGLISH ABSTRACT: The evaluation of fungicide efficacy in commercial vineyards can be influenced by the sporadic occurrence of Botrytis cinerea at various positions on vines, differences in bunch structure during bunch development and the phenomenon that symptom expression in shoots and bunches is governed by the resistance reaction of the various shoot and bunch parts. It has been postulated that, following air and water dispersal, infection by solitary conidia should playa prominent role in the epidemiology of B. cinerea on grapevine. The aim of this study was to determine (i) infection and (ii) fungicide efficacy at specific sites on shoots of vinelets and bunches (table grape cultivar Dauphine and the wine grape cultivar Merlot) inoculated with dry, airborne conidia of B. cinerea. Vinelets, prepared from cuttings, and bunches obtained from the vineyards at full bloom, pea size, bunch closure, véraison and harvest stages, were sprayed in a spray chamber at the recommended dosages with iprodione, pyrimethanil, cyprodinil/fludioxonil and fenhexamid or were left unsprayed. After 24 h the vinelets or bunches were dusted with dry conidia of Botrytis cinerea in a settling tower and incubated for 24 h at a high relative humidity (±93%). Following incubation, both the vinelets or bunches were divided into three groups. Vinelets and bunches of the one group were surface-sterilised, the others were left unsterile. Vinelets and bunches of one unsterile group were placed in dry chambers, kept for 14 days at 22°C with a 12 h photoperiod daily and monitored for symptom expression and the development of B. cinerea. Vinelets and bunches of the sterile group, and from one unsterile group were used for isolation. From each of these vinelets leaf blades, leaf petioles, shoots and inflorescences were removed. Sites used for isolation in bunch parts were rachises, laterals and pedicels, and sites on berries were the pedicel-end, cheek and style-end. The different parts and segments were placed in Petri dishes on Kerssies' B. cinerea selective medium, or on water agar medium supplemented with paraquat and incubated for 14 days at 22°C with a 12 h photoperiod daily. Infection and fungicide efficacy was determined by observing intact vinelets and bunches for symptom expression, and by estimating the amount of B. cinerea at the various sites on the vinelets and bunches with isolation studies. No symptoms of B. cinerea decay developed on sprayed and unsprayed vinelets that were kept in dry chambers during the 2 wk observation period. The isolation and incubation studies showed that the different fungicides were highly and nearly equally efficient in reducing superficial B. cinerea inoculum and latent infection. .In the case of leaf blades, which showed a high amount of B. cinerea on unsprayed vinelets under the two sterility regimes, decay was significantly reduced by each fungicide on both cultivars. This was not the case for the other parts, which yielded B. cinerea at low incidences under the two sterility regimes. The study with bunches showed that dry, airborne conidia, and the fungicide sprays, penetrated loose and tight clustered bunches from bloom to harvest and evenly landed on the various bunch parts. At full bloom, the amount of B. cinerea in unsprayed bunches was high on the laterals and pedicels, but low on the embryos. Unsprayed intact bunches at full bloom were highly susceptible to B. cinerea and developed symptoms of grey mould. The fungicides inhibited symptom expression at full bloom, but could not prevent infection. Unsprayed bunches inoculated at the other stages remained asymptomatic. The amount of B. cinerea was generally high in the rachises and laterals at pea size and bunch closure stages, and in the pedicel end of berries at harvest. Infection was constantly low in the berry cheek. The fungicides had a differential effect on infection at the various sites. In the case of rachises, the amount of B. cinerea was at each growth stage drastically reduced by each fungicide. In laterals, it was effectively reduced at pea size and bunch closure. However, at these two sites, significant differences were found between the fungicides in efficacy at stages when the amount of B. cinerea was high. This study showed that if these fungicides are applied properly to vine in commercial vineyards between budding and prebloom, during flowering, and at bunch closure, they should effectively prevent infection and symptom expression and thus the development of B. cinerea epiphytotics.
AFRIKAANSE OPSOMMING: INFEKSIE DEUR DROË, LUGGEDRAAGDE BOTRYTIS CINEREA KONIDIA EN DIE EFFEK VAN FUNGISlEDE OP VERSKILLENDE SETELS BINNE WINGERDTROSSE EN OP LOTE: Evaluering van fungisieddoeltreffendheid in kommersiële wingerde word beïnvloed deur die sporadiese voorkoms van Botrytis cinerea op verskeie posisies van wingerddele, verskille in trosstruktuur tydens trosontwikkeling, en die feit dat simptoomuitdrukking in lote en trosse deur die weerstandsaksie van die verskillende morfologiese dele van lote en trosse beheer word. In die natuur speel infeksie deur enkel konidia 'n prominente rol in die epidemiologie van B. cinerea van wingerd. Die doel van hierdie studie was om (i) infeksie en (ii) die effek van fungisiede op verskillende posisies op lote en trosse (tafeldruif kultivar Dauphine, wyndruif kultivar Merlot), wat met droë, luggedraagde konidia van B. cinerea geïnokuleer is, te bepaal. Lote, verkry vanaf steggies, en trosse versamel vanuit die wingerde tydens blom-, ertjiekorrel-, trostoemaak-, deurslaan- en oesstadium, is teen aanbevole dosisse met iprodione, pyrimethanil, cyprodinillfludioxonil of fenhexamid in 'n spuitkas bespuit, of is onbehandeld gelaat. Na 24 h is die lote en trosse met droë konidia van B. cinerea in 'n inokulasietoring geïnokuleer en daarna vir 24 h onder hoë humiditeit [±93% RH] geïnkubeer. Na inkubasie is die lote en trosse in drie groepe verdeel. Die een groep lote en trosse is oppervlakkig gesteriliseer om die patogeen op die oppervlakte te elimineer, en die ander twee groepe is onbehandeld gelaat. Die lote en trosse van een nie-steriele groep is vir 14 dae in droë voghokke by 22°C met 'n 12 uur daaglikse fotoperiode geplaas, en daagliks vir siekteuitdrukking en die ontwikkeling van B. cinerea gemonitor. Lote en trosse van die ander twee groepe is vir isolasiestudies gebruik. Vanaf elke loot is blaarskywe, blaarstele, internodes en ongeopende blomtrossies verwyder. Vanaftrosse is ragisse, laterale en korreisteie verwyder, en vanaf korrels is skilsegmente aangrensend aan die korrelsteel, die stempel-end, en die wang verwyder. Die dele en segmente is op B. cinerea selektiewe medium, en op paraquat medium in Petri bakkies geplaas en vir 14 dae by 22°C met 'n 12 uur daaglikse fotoperiode geïnkubeer. Infeksie en die fungisiedeffek is bepaal deur die intakte lote en trosse vir siekte- uitdrukking te monitor, en deur die hoeveelheid B. cinerea op verskeie posisies op lote en trosse te bepaal. Geen simptome het op enige posisie op bespuite en onbespuite lote, wat in droë hokke gehou is, ontwikkel nie. Die isolasie- en inkubasiestudies het getoon dat die verskillende fungisiede hoogs effektief op lote was, en inokulumvlakke van die patogeen doeltreffend verlaag het. In die geval van blaarskywe, wat hoë vlakke van B. cinerea op onbespuite steggies onder die twee steriliteitskondisies getoon het, is verrotting op beide kultivars betekenisvol deur die fungisiedes verlaag. Dit het egter nie vir die ander dele, waarop daar 'n lae voorkoms van B. cinerea onder die twee steriliteitskondisies was, gegeld me. Die studie met trosse het getoon dat droë, luggedraagde konidia en fungisiednewels beide oop en kompakte trosse vanaf blomstadium tot oes penetreer en eweredig op die verskillende dele land. Met blomstadium was die hoeveelheid B. cinerea in onbespuite trosse hoog op laterale en korrelstele, maar laag op die embrios. Onbespuite, intakte trosse was hoogs vatbaar vir B. cinerea by blomstadium en het simptome van vaalvrot ontwikkel. Die fungisiede het siekte-uitdrukking by blomstadium voorkom, maar kon nie infeksie voorkom me. Onbespuite trosse wat op ander stadia geïnokuleer is, het geen siekte-uitdrukking getoon me. Die hoeveelheid B. cinerea was hoër in die ragi, asook in laterale by ertjiekorrel- en trostoemaak stadium, en hoër in korreisteie by oesstadium. Infeksie was konstant laag in die korrelskil. Die fungisiede het 'n differensiële effek op infeksie by die verskillende posisies gehad. In die geval van ragi was die hoeveelheid B. cinerea drasties deur elke fungisied by alle groeistadia verlaag. In laterale was dit effektief by ertjiekorrel- en trostoemaakstadium verminder. By hierdie twee posisies waar die hoeveelheid B. cinerea hoog was, is daar egter betekenisvolle verskille in die doeltreffendheid van fungisiedes gevind. Hierdie studie toon dat as fungisiede behoorlik in kommersiële wingerde tussen botvorming en blomstadium, en tydens blom- en trostoemaakstadium toegedien word, infeksie en siekte-uitdrukking, en dus ook die epifitotiese ontwikkeling van B. cinerea, voorkom behoort te word.

Thesis (MScAgric)--University of Stellenbosch, 2006.
ENGLISH ABSTRACT: Phaeomoniella chlamydospora, Eutypa lata, Phomopsis, Phaeoacremonium, and Botryosphaeria spp. are important trunk disease pathogens that cause premature decline and dieback of grapevine. Previous research has focused primarily on wine grapes and the incidence and symptomatology of these pathogens on table grapes were largely unknown. A survey was therefore conducted to determine the status and distribution of these pathogens and associated symptoms in climatically diverse table grape growing regions. Fifteen farms were identified in the winter rainfall (De Doorns, Paarl and Trawal) and summer rainfall (Upington and Groblersdal) areas. Samples were taken in July and August 2004 from Dan-ben-Hannah vineyards that were 8 years and older. Distal ends of arms were removed from 20 randomly selected plants in each vineyard. These sections were dissected and isolations were made from each of the various symptom types observed: brown or black vascular streaking, brown internal necrosis, wedge-shaped necrosis, watery necrosis, esca-like brown and yellow soft wood rot, as well as asymptomatic wood. Fungal isolates were identified using molecular and morphological techniques. Pa. chlamydospora was most frequently isolated (46.0%), followed by Phaeoacremonium aleophilum (10.0%), Phomopsis viticola (3.0%), Botryosphaeria obtusa (3.0%), B. rhodina (2.2%), B. parva (2.0%), Fusicoccum vitifusiforme (0.6%), B. australis, B. dothidea and an undescribed Diplodia sp. (0.2% each), while E. lata was not found. Most of these pathogens were isolated from a variety of symptom types, indicating that disease diagnosis can not be based on symptomatology alone. Pa. chlamydospora was isolated from all areas sampled, although most frequently from the winter rainfall region. Pm. aleophilum was found predominantly in Paarl, while P. viticola only occurred in this area. Although B. obtusa was not isolated from samples taken in De Doorns and Groblersdal, it was the most commonly isolated Botryosphaeria sp., being isolated from Upington, Paarl and Trawal. B. rhodina occurred only in Groblersdal and B. parva in Paarl, Trawal and Groblersdal, while B. australis was isolated from Paarl only. The rest of the isolates (33%) consisted of sterile cultures, Exochalara, Cephalosporium, Wangiella, Scytalidium, Penicillium spp. and two unidentified basidiomycetes, which were isolated from five samples with yellow esca-like symptoms from the Paarl area. These findings clearly illustrate that grapevine trunk diseases are caused by a complex of fungal pathogens, which has serious implications for disease diagnosis and management. Protection of wounds against infection by any of these trunk disease pathogens is the most efficient and cost-effective means to prevent grapevine trunk diseases. However, previous research on the effectiveness of chemical pruning wound protectants has mostly focused on the control of Eutypa dieback only. Fungicide sensitivity studies have been conducted for Pa. chlamydospora, P. viticola and Eutypa lata, but no such studies have been conducted for the pathogenic Botryosphaeria species from grapevine in South Africa. Ten fungicides were therefore tested in vitro for their efficacy on mycelial inhibition of the four most common and/or pathogenic Botryosphaeria species in South Africa, B. australis, B. obtusa, B. parva and B. rhodina. Iprodione, pyrimethanil, copper ammonium acetate, kresoxim-methyl and boscalid were ineffective in inhibiting the mycelial growth at the highest concentration tested (5 μg/ml; 20 μg/ml for copper ammonium acetate). Benomyl, tebuconazole, prochloraz manganese chloride and flusilazole were the most effective fungicides with EC50 values for the different species ranging from 0.36-0.55, 0.07-0.17, 0.07-1.15 and 0.04-0.36 μg/ml, respectively. These fungicides, except prochloraz manganese chloride, are registered on grapes in South Africa and were also reported to be effective against Pa. chlamydospora, P. viticola and E. lata. Results from bioassays on 1-year-old Chenin Blanc grapevine shoots indicated that benomyl, tebuconazole and prochloraz manganese chloride were most effective in limiting lesion length in pruning wounds that were inoculated with the Botryosphaeria spp after fungicide treatment. The bioassay findings were, however, inconclusive due to low and varied re-isolation data of the inoculated lesions. Benomyl, tebuconazole, prochloraz manganese chloride and flusilazole can nonetheless be identified as fungicides to be evaluated as pruning wound protectants in additional bioassays and vineyard trials against Botryosphaeria spp. as well as the other grapevine trunk disease pathogens.
AFRIKAANSE OPSOMMING: Phaeomoniella chlamydospora, Eutypa lata, Phomopsis, Phaeoacremonium, en Botryosphaeria spesies is die mees belangrikste stamsiekte patogene wat agteruitgang en vroeë terugsterwing van wingerd veroorsaak. Voorafgaande navorsing het hoofsaaklik gefokus op wyndruiwe en die voorkoms en simptomatologie van hierdie patogene op tafeldruiwe is dus grootliks onbekend. ‘n Opname is gevolglik gedoen in verskillende klimaaatsareas waar tafeldruiwe verbou word om die voorkoms en verspreiding, asook die simptome geassosieer met hierdie patogene, te bepaal. Vyftien plase is geïdentifiseer in die winter- (De Doorns, Paarl en Trawal) en somer-reënval (Upington en Groblersdal) streke. Wingerde (8 jaar en ouer) met die kultivar Dan-ben-Hannah is gekies vir opname en monsters is gedurende Julie en Augustus 2004 geneem. Die distale deel van ‘n arm is verwyder vanaf 20 lukraak gekose plante in elke wingerd. Hierdie dele is ontleed en isolasies is gemaak vanuit elke simptoomtipe wat beskryf is, naamlik bruin en swart vaskulêre verkleuring, bruin interne nekrose, wig-vormige nekrose, waterige nekrose, esca-geassosieerde bruin en geel sagte houtverrotting en asimptomatiese hout. Identifikasie van die swamagtige isolate is gedoen op grond van morfologiese eienskappe en molekulêre tegnieke. Pa. chlamydospora is die meeste geïsoleer (46.0%), gevolg deur Phaeoacremonium aleophilum (10.0%), Phomopsis viticola (3.0%), Botryosphaeria obtusa (3.0%), B. rhodina (2.2%), B. parva (2.0%), Fusicoccum vitifusiforme (0.6%), B. australis, B. dothidea en ‘n onbeskryfde Diplodia sp. (0.2% elk), terwyl E. lata nie geïsoleer is nie. Hierdie patogene is elk geïsoleer vanuit ‘n verskeidenheid simptoomtipes, wat daarop dui dat siektediagnose nie alleenlik op simptomatologie gebaseer kan word nie. Pa. chlamydospora is geïsoleer vanuit al die gebiede, alhoewel die patogeen opmerklik meer voorgekom het in die winter-reënval area. Pm. aleophilum het hoofsaaklik voorgekom in Paarl, terwyl P. viticola slegs in hierdie area voorgekom het. Alhoewel B. obtusa nie voorgekom het in die De Doorns en Groblersdal areas nie, was dit die mees algemeen geïsoleerde Botryosphaeria sp. en het in Upington, Paarl en Trawal voorgekom. B. rhodina het slegs in Groblersdal voorgekom, B. parva in Paarl, Groblersdal en Trawal en B. australis het slegs in Paarl voorgekom. Die res van die isolate (33%) het bestaan uit steriele kulture, Exochalara, Cephalosporium, Wangiella, Scytalidium, en Penicillium spesies asook twee onbekende basidiomycete isolate, geïsoleer vanuit vyf monsters met geel eska-geassosieerde simptome vanuit die Paarl area. Hierdie resultate illustreer dus die feit dat wingerdstamsiektes deur ‘n kompleks van swampatogene veroorsaak word, wat belangrike implikasies het vir die bestuur en diagnose van hierdie siektes. Wondbeskerming teen infeksie van enige van hierdie stamsiekte patogene is die mees doeltreffende en koste-effektiewe manier om wingerdstamsiektes te voorkom. Vorige navorsing aangaande die effektiwiteit van chemiese wondbeskermingsmiddels het egter slegs gefokus op die beheer van Eutypa terugsterwing. In vitro swamdoder sensitiwiteitstoetse is gedoen vir Pa. chlamydospora, P. viticola en Eutypa lata, maar geen studies is al gedoen ten opsigte van die patogeniese Botryosphaeria spesies op wingerd in Suid-Afrika nie. Tien swamdoders is dus getoets vir inhibisie van in vitro miseliumgroei van die vier mees algemene en/of patogeniese Botryosphaeria spesies wat in Suid-Afrika voorkom, naamlik B. australis, B. obtusa, B. parva en B. rhodina. Iprodione, pyrimethanil, koper ammonium asetaat, kresoxim-metiel en boscalid was oneffektief by die hoogste konsentrasies getoets (5 μg/ml; 20 μg/ml vir koper ammonium asetaat). Benomyl, tebuconasool, prochloraz mangaan chloried en flusilasool was die mees effektiewe swamdoders met EC50 waardes tussen 0.36-0.55, 0.07-0.17, 0.07-1.15 en 0.04-0.36 μg/ml, onderskeidelik vir die verskillende spesies. Hierdie fungisiedes, behalwe prochloraz mangaan chloried, is geregistreer op druiwe in Suid-Afrika en is ook effektief gevind teenoor Pa. chlamydospora, P. viticola en E. lata. Resultate van biotoetse op 1-jaar-oue Chenin Blanc wingerd lote het getoon dat benomyl, tebuconasool en prochloraz mangaan chloried die effektiefste was om die lengte van letsels in snoeiwonde, geinokuleer met Botryosphaeria spesies na die aanwending van swamdoder behandelings, te verminder. Die bevindinge was egter onbeslis as gevolg van die lae en variërende her-isolerings data. Benomyl, tebuconasool, prochloraz mangaan chloried en flusilasool kan egter geïdentifiseer word as swamdoders wat verder geevalueer kan word as snoeiwond beskermingsmiddels teen Botryosphaeria spesies asook ander wingerd stamsiekte patogene in verdere biotoetse en wingerdproewe.

Thesis (MScAgric)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: Leaf blackening, a postharvest disorder which is characterized by a dark brown to black discoloration, is found in most commercially important Protea cut flower species and cultivars. As this disorder is known to increase with storage time, it is a major concern to the South African industry as the use of sea freight is increasingly preferred due to lower transport costs and a more favourable carbon footprint. The cause of leaf blackening has been strongly linked to a carbohydrate stress exerted by the large inflorescence, thus requiring the utilization of sugar bound polyphenols in the foliage, which when removed, can oxidize enzymatically or non-enzymatically. A study where harvesting was done throughout the season as well as on selected days at 08:00, 12:00, 15:00 and 17:00, concluded that leaf blackening incidences in Protea cv. Sylvia stems varies significantly throughout the season, between years and even with the harvest time of day. Leaf blackening incidences increased from October onwards and remained high until February, before decreasing to acceptably lower levels towards March to May. Carbohydrate- and phenolic content together with water status of leaves at harvest was not able to accurately predict incidence of the associated leaf blackening. However, irrespective of the season of harvesting, leaf blackening was significantly lower when stems were harvested later in the day than compared to stems harvested in the morning. Low sucrose and high water content at these harvest times was positively correlated to high incidences of leaf blackening. In a next study where uptake dynamics of glucose pulsing was investigated, Protea cv. Sylvia was harvested at different times throughout the day, dehydrated to various levels and pulsed with an increasing range of glucose concentrations. Pulsing solution uptake per stem was found to be highly influenced by these factors, as dehydration of stems and a harvest time later during the day both decreased stem water potential, which then increased pulse-solution uptake within a certain time period. The daily harvest time influenced transpiration, whilst pulse-solution uptake decreased with an increase in glucose pulse concentration. When stems were pulsed pre-storage with an increasing range of glucose concentrations, not only did pulses of between 4.7 – 13.7% glucose significantly delayed the incidence of leaf blackening, but it also maintained a positive water balance longer in stems during vase life. Ethanol or acetaldehyde vapour did not provide a viable alternative for reducing leaf blackening incidence in Protea cv. Sylvia, although a synergistic effect was found when ethanol vapour or pulsing was used in combination with glucose. A commercial verification trial disclosed that Protea magnifica and Protea ‘Pink Ice’ reacted more beneficial to ethanol vapour than was observed in ‘Sylvia’. This study confirms that carbohydrate availability within the Protea cut stem remains a key factor in the control of leaf blackening. Factors which assist in maintaining high internal carbohydrate levels, such as enhanced glucose pulse uptake or effective vase solution utilization will contribute to providing an optimum control of leaf blackening during vase life following long-term cold storage.
AFRIKAANSE OPSOMMING: Loofblaarverbruining is ‘n na-oes defek wat gekarakteriseer word deur ‘n donker bruin na swart verkleuring wat voorkom in meeste kommersieël belangrike Protea snyblom spesies en kultivars. Hierdie defek is bekend daarvoor dat dit toeneem met stoortyd, dus is dit ‘n groot kommer vir die Suid-Afrikaanse industrie, met toenemende gebruik van seevrag as vervoer keuse wat laer vervoer kostes en meer gunstige ‘koolstof voetspoor’ bevoordeel. Die oorsaak van loofblaarverbruining word sterk gekoppel aan ‘n koolhidraat stres wat uitgeoefen word deur die groot bloeiwyse op die loofblare, waar suiker-gebonde polifenoliese verbindings ensiematies of nieensiematies geoksideer word met die verwydering van die suiker verbinding. 'n Studie waar geoes was regdeur die seisoen, sowel as op geselekteerde dae om 08:00, 12:00, 15:00 en 17:00, het bevind dat die voorkoms van loofblaarverbruining in stele van Protea kv. Sylvia aansienlik geskil regdeur die seisoen, tussen jare en selfs met die oes tyd gedurende die dag. Die voorkoms van loofblaarverbruining het toegeneem vanaf Oktober en het hoog gebly tot en met Februarie, voordat dit gedaal het tot aanvaarbare laer vlakke teen Maart, tot en met Mei. Koolhidraat-en fenoliese inhoud sowel as die water status van die blare by oes was onsuksesvol om die voorkoms van die gepaardgaande loofblaarverbruining akkuraat te voorspel. Loofblaarverbruining was egter aansienlik laer as stele geoes later in die dag teenoor stele geoes in die oggend, ongeag die seisoen van oes. Lae sukrose en 'n hoë water inhoud geassosieer met hierdie oes-tye was positief gekorreleerd met ‘n hoë voorkoms van loofblaarverbruining. In 'n volgende studie waar die opname dinamika van glukose pulsing ondersoek was, is Protea kv. Sylvia stele geoes op verskillende tye dwarsdeur die dag, gedehidreer tot verskillende vlakke en met 'n toenemende reeks van glukose konsentrasies gepuls. Pulsoplossing opname per steel is sterk beïnvloed deur hierdie faktore, aangesien dehidrasie van die stele asook stele geoes later gedurende die dag die afname van steel waterpotensiaal veroorsaak het, terwyl die puls-oplossing opname versnel het binne ‘n bepaalde tyd. Die tyd van oes beïnvloed ook transpirasie, terwyl vaas oplossing opname afgeneem met 'n toename in glukose puls konsentrasie. Wanneer ‘Sylvia’ stele gepuls was voor stoor met 'n reeks van toenemende glukose konsentrasies, het nie net die puls van tussen 4.7 – 13.7% glukose aansienlik die voorkoms van loofblaarverbruining vertraag nie, maar dit het ook ‘n positiewe water balans langer in stele gedurende die vaas lewe behou. Nie etanol of asetaldehied dampe is bevind as geskikte alternatief vir glukose pulsing om die voorkoms van loofblaarverbruining in Protea kv. Sylvia te verlaag nie, alhoewel ‘n sinergistiese effek waargeneem was wanneer etanol in kombinasie met glukose gebruik was. ‘n Kommersieële bevestigingstoetsing het bevind dat Protea magnifica en ‘Pink Ice’ meer voordeel uit ‘n ethanoldamp behandeling kon trek teenoor ‘Sylvia’. Hierdie studie het bevestig die belangrikheid van koolhidraat beskikbaarheid in die Protea snyblom, vir beheer van loofblaarverbruining. Faktore wat die handhawing van hoë interne koolhidrate vlakke, soos bevorderde glukose puls opname of effektiewe vaas oplossing benutting sal bydra tot ‘n optimal beheer van loofblaarverbruining tydens vaas lewe na langtermyn koueopberging.
National Research Fund (NRF) for their financial support in 2009; Protea Producers of South Africa (PPSA) and Productschap Tuinbouw (PT) as well as the Frank Batchelor Will Trust Grant for the financial support.

A number of experiments were carried out to study the potential of denitrifying bacteria and reduced nitrogen compounds for control of soil-borne damping-off pathogens. Measurement of the rhizosphere pH of growing soybean roots was carried out in soil adjusted to different pH states and packed into sheet microcosms. The results showed that the rhizosphere pH of soybean was lower than the bulk soil. Nitrate reductase activity and nitrite production was then characterised for the rhizosphere of intact 14 day-old soybean roots that were incubated in nitrate substrates adjusted to different pH values under water-logged conditions. The results showed that the rate and the quantity of nitrite production increased with increasing nitrate concentration and pH in the solution. A growth room experiment was carried out to determine root colonization by denitrifying bacteria in relation to disease caused by soil-borne pathogens, which are favoured by high soil moisture (approximately -5 KPa) and low oxygen levels. Nitrite producing bacteria were isolated from soybean roots grown in Grampian (Insch) soils which had not been cropped with soybean and Thai (Phitsanulok) soils which previously had been cropped with soybean. In the first pot experiment, the nitrite producing bacteria were isolated from different root sections of 12 and 19 day-old soybean plants after 8 weeks of continuous cropping of soils with soybean (a new crop was planted every week), and using different isolation media in order to determine the genus/species composition of the denitrifying bacteria on the rhizoplane. The results showed that continuous cropping of Thai soil and Insch soil with soybean increased pre-emergence damping-off disease and decreased fresh weight yields in seedlings that did emerge. ANOVA showed significant differences between root sections for most bacterial groups monitored {Bacillus spp., Pseudomonas and Enterobacteriaceae), with regression analysis generally showing densities increasing with root age or toward the shoot base. All nitrite producing bacterial isolates were screened for antifungal activity against Macrophomina phaseolina on agar plates and between 10 and 25% of nitrite producing bacteria were found to show in vitro antagonism. In a second pot experiment, the nitrite producing bacteria were isolated from root tissue below the crown (5 cm in length) every 2 weeks of continuous cropping of soils with soybean (a new crop was planted every 2 weeks). Plate-counting was carried out to determine the population of nitrite producing bacteria while a liquid culture MPN method was used for determination of NO, N2O and N2 producing bacteria. Linear regression analysis of the incidence of pre-emergence damping-off and soybean yields in seedling that did emerge showed a highly significant negative correlation between these parameters for both soils. ANOVA showed that there was a significant difference between soil type, with the Thai soil showing higher population densities of antagonistic bacteria on soybean roots. All nitrite producing bacterial isolates were screened for antifungal activity, but the plant pathogenic fungus, Pythium ultimum, was used in this experiment. The results showed that between 10 and 40% of nitrite producing bacteria showed in vitro antagonism. However, regression analysis showed that there was no significant increase or decrease in the nitrite producing antagonistic bacterial population with continuous soybean cropping. All 900 isolates of nitrite producing bacteria isolated from the soybean rhizoplane were screened for antagonistic activity towards Pythium ultimum based on a pot trial assay in the greenhouse. As expected, very low numbers of nitrite producing bacteria showed activity against P. ultimum and only one isolate gave a significant reduction in disease incidence in pot trials. The interactive effects of nitrite producing antagonist and an Arbuscular Mycorrhizal fungus (Glomus mosseae) and Bradryrhizobium japonicum, on control of the fungal pathogens, P. ultimum or M. phaseolina were investigated in the greenhouse. The results showed that improved plant growth was obtained with certain combined inocula involving nitrite producing bacterial antagonists, Glomus mosseae and Bradryrhizobium japonicum.

Thesis (PhD)--Stellenbosch University, 2001.
ENGLISH ABSTRACT: Polygalacturonase-inhibiting proteins (PGIPs) are present in the cell walls of a variety of plant species. These proteins have been shown to specifically inhibit endopolygalacturonases (endo-PGs) secreted by invading fungal pathogens as part of the induced disease resistance mechanism of plants. This is the first report on the isolation and characterisation of a pgip gene from Vitis vinifera L., designated grapevine pgip1. A single open reading frame encoding a deduced polypeptide of 333 amino acids with a predicted molecular mass of 37.1 kOa and a calculated isoelectric point of 8.61 was identified from a 5.6 kb subgenomic fragment of V. vinifera cv Pinotage. Nucleotide and derived amino acid sequence analysis of grapevine pgip1 showed significant homology with other characterised PGIP encoding genes and revealed features characteristic of PGIPs found in several other plant families. Genomic DNA analysis showed that grapevine pgip1 belongs to a small multigene family in Vitis cultivars. From Northern blot analysis it was evident that expression of the PGIP family is both tissue- and developmental stage specific. The grapevine pgip1 was transiently expressed in Nicotiana benthamiana L. with potato virus X (PVX) as a vector. Grapevine PGIP1 isolated from crude protein extracts of PVX-infected N. benthamiana were tested and showed inhibitory activity against polygalacturonases (PGs) from Botrytis cinerea. Grapevine PGIPs have not previously been purified and characterised. Molecular analyses have confirmed that PGIPs are typically encoded by multigene families and that the inhibitor specificities and kinetics of the isolated proteins differ within and among species. In this study, two PGIP isomers from V. vinifera berries were isolated. The one isomer, designated PGIP-A, was partially purified and had a molecular mass of 39 kOa, whereas the other PGIP, designated PGIP-B, was purified and had a molecular mass of 42 kOa as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SOS-PAGE) and Western blot analysis. Both proteins were cell wall-bound. Enzymatic deglycosylation confirmed that PGIP-B is a glycosylated protein. Grapevine PGIP-A showed strong inhibitory activity against a homogeneous PG from Aspergillus niger and to a lesser extent against PG from Fusarium moniliforme, but was unable to interact with a crude PG preparation from B. cinerea. Grapevine PGIP-B was able to strongly inhibit PGs from B. cinerea as well as from Colletotrichum gleosporoides, yet showed no inhibition towards PG from A. niger. The grapevine pgip1 gene was expressed under the control of the Cauliflower mosaic virus (CaMV) 35S promoter in tobacco plants via Agrobacterium tumefaciensmediated transformation. Transgenic tobacco plants expressing the grapevine PGIP (gPGIP1) were used to demonstrate the effectiveness of this inhibitor against fungal PGs and to investigate whether gPGIP1 influences disease development. Northern blot analysis identified 19 transgenic plants expressing pgip1 transcript levels. Crude PGIP extracts from the transgenic tobacco plants inhibited PGs from B. cinerea and C. gleosporoides, but not PG from A. niger. Leaves from untransformed tobacco plants, from transgenic tobacco lines showing high and low PG inhibition, and from transgenic plants that did not express pgip1, were inoculated with B. cinerea. Transgenic leaves showed a reduction in the size of necrotic lesions of macerated tissues of approximately 45% relative to control and non-expressing transgenic leaves. The results from the heterologous expression of gPGIP1, together with the results from the protein purifications and inhibition studies, indicate that the isolated grapevine pgip1 gene encodes the isolated PGIP-B isomer. This work has ; established a good model system to study certain aspects of plant-pathogen interactions in grapevine. Heterologous expression of gPGIP1 has demonstrated that PGIP inhibition of fungal PGs slows disease development of B. cinerea in planta.
AFRIKAANSE OPSOMMING: Sien volteks vir opsomming

Thesis (MSc)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Two genera of ophiostomatoid fungi occur in the seed-bearing structures of serotinous Protea species in the Cape Floristic Region. These fungi are dispersed by arthropods, including mites and beetles that visit the Protea host plants. Although the vectors of Proteaassociated ophiostomatoid fungi are known, their dispersal patterns remain unknown – especially the manner in which recently burnt fynbos vegetation is recolonized. Additionally, their reproduction strategy has not previously been investigated. The focus of this study was, therefore, to determine the extent of within- and between-plant dispersal of Proteaassociated ophiostomatoid fungi at the population level and to investigate their reproductive strategy. One Protea-associated ophiostomatoid fungus, Knoxdaviesia proteae, is found exclusively in the fruiting structures of P. repens and was the focus of this study. In order to interrogate natural populations of this fungus, 12 polymorphic microsatellite markers specific to K. proteae were developed with an ISSR-PCR enrichment strategy and pyrosequencing. These markers were amplified in two distantly separated populations of K. proteae. The genetic and genotypic diversities of both populations were exceptionally high and neither showed significant population differentiation. The lack of population structure in both populations implies that K. proteae individuals within a P. repens stand are in panmixia. As one of the sampling sites had burnt recently, the process whereby young fynbos is recolonized could be investigated. Compared to the adjacent, unburnt area, K. proteae individuals in the burnt area of this population had significantly less private alleles, suggestive of a young population that had experienced a genetic bottleneck. Knoxdaviesia proteae individuals that did not originate from the adjacent unburnt area were encountered within the burnt site and, additionally, isolation-by-distance could not be detected. The parsimony-based haplotype networks and the tests for linkage disequilibrium indicated that recombination is taking place within as well as between the two distantly separated populations. The observed panmixia in P. repens stands, widespread recolonization and the high genetic similarity and number of migrants between the two populations emphasizes long-distance dispersal and therefore the role of beetles in the movement of K. proteae. This cohesive genetic structure and connection across large distances is likely a result of multiple migration events facilitated by beetles carrying numerous phoretic mites.
AFRIKAANSE OPSOMMING: Twee genera ophiostomatoid swamme kom in die saad-draende strukture van bloeiende Protea spesies in the Kaapse Floristiese Streek voor. Hierdie Protea-verwante ophiostomatoid swamme word gekenmerk deur hul assosiasie met geleedpotige vektore – spesifiek die myt en kewer besoekers van die Protea gasheer plante. Alhoewel die geleedpotige vektore van Protea-verwante ophiostomatoid swamme bekend is, is die wyse waarop hierdie swamme versprei onbekend; veral die manier waarop onlangse gebrande fynbos geherkoloniseer word. Verder is die voortplantings-strategie van hierdie swamme nog nie voorheen ondersoek nie. Die fokus van hierdie studie was dus om die omvang van binne- en tussen-plant verspreiding van Protea-verwante ophiostomatoid swamme te bepaal op die populasie vlak en om hul voorplantings-strategie te ondersoek. Een Protea-verwante ophiostomatoid swam, Knoxdaviesia proteae, word uitsluitlik in die vrugdraende strukture van P. repens aangetref en was die fokus van hierdie studie. Om natuurlike populasies van hierdie swam te ondersoek is 12 mikrosatelliet-merkers spesifiek vir K. proteae ontwerp deur ‘n ISSR-PCR strategie en “pyro”-basisvolgorde bepaling te gebruik. Hierdie merkers is geamplifiseer in twee K. proteae populasies wat ver van mekaar geskei is. Die genetiese en genotipiese diversiteit van beide populasies was uitsonderlik hoog en nie een het beduidende populasie-differensiasie getoon nie. Die gebrek aan populasie struktuur in beide populasies veronderstel dat K. proteae individue binne ‘n P. repens stand in panmiksia is. Aangesien een van die steekproef terreine onlangs gebrand het, kon die herkolonisasie proses van jong fynbos ondersoek word. In vergelyking met die aangrensende, ongebrande area, het K. proteae individue in die gebrande area beduidend minder private allele gehad. Dit dui op ‘n jong populasie wat ‘n genetiese bottelnek beleef het. Knoxdaviesia proteae individue wat nie van die aangrensende, ongebrande area afkomstig is nie is ook binne die gebrande terrein aangetref. Verder is afsondering-deur-afstand nie aangetref nie. Die parsimonie-gebaseerde haplotiepe-netwerke en die toetse vir koppeling-onewewigtigheid het aangedui dat rekombinasie binne sowel as tussen die twee populasies plaasvind. Die panmiksia wat waargeneem is in P. repens populasies, wydverspreide herkolonisasie en die hoë genetiese ooreenkoms en hoeveelheid immigrante tussen die twee populasies beklemtoon lang afstand verspreiding en dus die rol van kewers in die beweging van K. proteae. Hierdie samehangende genetiese struktuur en die verband oor groot afstande is waarskynlik ‘n gevolg van verskeie migrasies gefasiliteer deur kewers wat talle foretiese myte dra.