Thesis (MSc (Genetics))--Stellenbosch University, 2008. / The vine mealybug, Planococcus ficus (Signoret), is a severe pest of grapevine in many grape and wine producing countries around the world. It is renowned not only for the considerable damage it infers to grapevine of its own accord, but in particular for its role in transmitting deleterious viral diseases such as grapevine leafroll disease, Kober stem grooving, Shiraz disease and corky bark. Incidentally, it is an exceptionally tenacious antagonist of grapevine, being resistant to both chemical and biological control mechanisms. As a result, finding an effective strategy for P. ficus control has become a main priority of viticultural industries worldwide. Possible implementation of biotechnological approaches to pest management has resulted in a need for P. ficus genetic data - of which there are currently very little available. The transcribed genes of an organism can be captured in a cDNA library, and the sequences of the various transcripts can then be characterized. In this study altogether five cDNA libraries were constructed from the transcribed sequences of Planococcus ficus (Signoret). Instrumental to their construction was the identification of an RNA extraction protocol that provided large quantities of high quality RNA from mealybugs. The five cDNA libraries were the result of a set of modifications to the Creator™ SMART™ cDNA Library Construction Kit (used for Primary Library construction), and differed mainly with regards to range of insert sizes they contain. Whereas an abundance of short fragments were found in the Primary Library (42% of screened inserts 60.5 kb, and 20% >1 kb), the Fractionated Libraries contained inserts of specific size ranges that were more-or-less equally represented. The broadest size range was found in Fractionated Library 4, for which a uniform distribution over the range 0.25 kb - 4 kb was observed. Average insert sizes of Fractionated Libraries 1 to 4 were estimated at 0.25 kb, 0.5 kb, 1 kb and 2 kb respectively. These results demonstrated the importance of using a protocol designed to circumvent the bias towards incorporation of shorter transcripts in cDNA libraries. Although the libraries were not exhaustively analyzed, the outcome of a pilot investigation indicated that 41% of the submitted sequences had matches in the non-redundant database of the National Center for Biotechnology Information (NCBI, E-value 6 10-5), and that approximately 82% of these were of insect origin. Moreover, two potential targets for an RNAi-mediated approach to P. ficus pest control were identified. With one exception, these sequences seemed to be unique to arthropods. Future research needs to investigate the efficiency by which these sequences are able to constrain P. ficus proliferation, and their suitability for grapevine transformation.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Grapevine trunk diseases are caused by invasive pathogens that are responsible for the slow decline of vines. In particular, Eutypa dieback of grapevine has had a devastating impact on vineyards worldwide, reducing growth and yield, eventually killing the grapevine. The causal organism of Eutypa dieback was first described as Eutypa armeniacae Hansf. & Carter, the pathogen that causes dieback of apricots, but since 1987 this species has been considered a synonym of Eutypa lata (Pers.:Fr.) Tul & C. Tul (anamorph Libertella blepharis A. L. Smith). Recently, it was proposed that at least two species that are capable of infecting grapevines are responsible for Eutypa dieback. Consequently, the molecular identification and characterisation of Eutypa dieback was used to delineate the species occurring on infected grapevines in South Africa. This involved the molecular analyses of three molecular markers, namely, the internal transcribed spacer (ITS) and large subunit (LSU) regions of the ribosomal DNA operon, and the -tubulin gene. The results obtained revealed the presence of a second species, namely, Eutypa leptoplaca (Mont.) Rappaz, that occurred together with E. lata on infected grapevines. Also co-habiting with these pathogens were related fungi form the Diatrypaceae family, Cryptovalsa ampelina (Nitschke) Fuckel and Eutypella vitis (Schwein.) Ellis & Everhart. Pathogenicity tests conducted on isolates representing C. ampelina, E. lata, E. leptoplaca, and E. vitis revealed that all were pathogenic to grapevine. Several species of Botryosphaeriaceae that commonly invade the woody tissue of grapevines are also pathogenic to grapevine. The symptoms in grapevine commonly associated with Botryosphaeriaceae are easily confused with the symptoms produced by Eutypa dieback which prompted the need for the development of a detection method that can correctly identify the presence of multiple pathogens. A reverse dot blot hybridisation (RDBH) method was subsequently applied to provide a rapid, accurate and reliable means of detecting the Eutypa species involved in the Eutypa disease complex, as well as those species of Botryosphaeriaceae known to cause disease in grapevines. The method involved the use of multiplex PCR to simultaneously amplify and label the regions of DNA that are used as pathogen specific probes. Consequently, membrane immobilised species-specific oligonucleotides synthesised from the ITS, - tubulin and LSU molecular data were evaluated during the application of this diagnostic method to detect Eutypa species. It was found that the species-specific oligonucleotides, designed from ITS sequence data, could consistently detect E. lata and E. leptoplaca. The application of the RDBH method for the detection of these Eutypa species, based on -tubulin and LSU sequence data, however, proved to be unsuccessful. Subsequently, a RDBH method, utilising species-specific oligonucleotides designed from elongation factor-1α sequence data, was successfully applied for the detection of Botyrosphaeria dothidea (Moug.:Fr.) Ces. & De Not., Neofusicoccum luteum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips, Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers, A.J.L. Phillips and Neofusicoccum ribis (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips. The method, however, was unsuccessful for the detection of Diplodia seriata De Not. In addition to the above-mentioned shortcomings, the RDBH was not amenable to the detection of pathogens directly from field or environmental samples, but required preparation of DNA from pure cultures. The method, however, allows for the identification of multiple pathogens in a single assay. As DNA extraction methods are amended, improved and honed to obtain DNA from environmental samples, so would it increase the usefulness of RDBH. / AFRIKAANSE OPSOMMING: Wingerd stamsiektes word veroorsaak deur patogene wat die vermoë het om wingerdplante te infekteer en dan stadige agteruitgang van dié wingerde te veroorsaak. Veral Eutypa terugsterwing het ‘n vernietigende effek op wingerde wêreldwyd deurdat dit groeikrag en oesmassa verlaag, maar ook omdat dit uiteindelik wingerdstokke kan dood. Die veroorsakende organisme is aanvanklik as Eutypa armeniacae Hansf. & Carter beskryf, die patogeen wat terugsterf by appelkose veroorsaak, maar sedert 1987 word hierdie spesies beskou as ‘n sinoniem van Eutypa lata (Pers.:Fr.) Tul & C. Tul (anamorph Libertella blepharis A. L. Smith). Dit is egter onlangs voorgestel dat ten minste twee spesies die vermoë het om wingerd te infekteer om Eutypa terugsterwing te veroorsaak. Gevolglik is molekulêre identifikasie- en karakteriseringstudies geloods om te bepaal watter spesies Eutypa terugsterwing in Suid-Afrikaanse wingerde veroorsaak. Dit het die molekulêre analise van drie molekulêre merkers behels, naamlik die interne getranskribeerde spasiëerderarea (“ITS”), die groot ribosomale subeenheid (“LSU rDNA”) en β-tubilien geen. Resultate van die filogenetiese analise dui daarop dat ’n tweede spesies, naamlik Eutypa leptoplaca (Mont.) Rappaz, saam met E. lata in geïnfekteerde plante voorkom. Saam met bogenoemde twee spesies het daar ook verwante spesies van die Diatrypaceae familie voorgekom, naamlik Cryptovalsa ampelina (Nitschke) Fuckel en Eutypella vitis (Schwein.) Ellis & Everhart. Patogenisiteitstudies wat uitgevoer is met verteenwoordigende isolate van C. ampelina, E. lata, E. leptoplaca, en E. vitis dui daarop dat almal patogene van wingerd is. Verskeie Botryosphaeriaceae spesies wat gereeld in houtagtige wingerdweefsel aangetref word, is ook patogene van wingerd. Interne simptome wat algemeen met Botryosphaeriaceae infeksies geassosieer word, kan baie maklik met dié van Eutypa terugsterwing verwar word en dit het die nood laat ontstaan om ‘n opsporingsmetode te ontwikkel wat akkuraat genoeg is om tussen veelvoudige infeksies te onderskei. ’n Omgekeerde-stippelklad-hibridisasie (OSH) metode is gevolglik aangewend om Eutypa spesies betrokke in die Eutypa-siektekompleks op ‘n vinnige, akkurate en betroubare manier op te spoor, sowel as die Botryosphaeriaceae species wat bekend is as patogene van wingerd. Die metode behels ’n saamgestelde PKR vir die vermeerdering en merk van DNS areas wat gebruik word as patogeen spesifieke peilers. Spesies-spesifieke oligonukleotiede ontwikkel vanaf die ITS, -tubilien en LSU molekulêre data is op ‘n membraan vasgeheg en gebruik om ’n diagnostiese toets te ontwikkel vir Eutypa species. Merkers ontwikkel vanaf die ITS kon E. lata and E. leptoplaca konsekwent opspoor. Die opspoor van Eutypa spesies met merkers vanaf die -tubulien en LSU gene met OSH was onsuksesvol. Die OSH metode met merkers vanaf die verlengingsfaktor-1α kon susksesvol gebruik word om Botyrosphaeria dothidea (Moug.:Fr.) Ces. & De Not., Neofusicoccum luteum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips, Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers, A.J.L. Phillips and Neofusicoccum ribis (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips op te spoor. Dié metode kon egter nie Diplodia seriata De Not. opspoor nie. Bykomend tot bogenoemde tekortkominge, kon die omgekeerde-stippelklad-hibridisasie metode ook nie aangepas word om patogene direk vanuit plantmateriaal op te spoor nie en word DNS afkomstig vanaf suiwer kulture benodig. Dié metode laat egter identifikasie van verskeie patogene in ‘n enkele toets toe. Soos DNS ekstraksie metodes aangepas, verbeter en verfyn word om DNS vanuit plantmateriaal te verkry, sal die bruikbaarheid van die omgekeerde stippelklad hibridisasie metode ook verbeter.

Thesis (MScAgric (Plant Pathology))--Stellenbosch University, 2009. / ENGLISH ABSTRACT: Sufficient fungicide deposition on the target site is an essential requirement for effective chemical management of fruit- and foliar diseases such as grey mould of grapevines. Control failure is often attributed to insufficient quantitative deposition on susceptible grapevine tissue. However, in high disease pressure situations control failure might also be attributed to poor qualitative deposition. The primary objective of spray technology is to optimise deposition, of which the plant surface is a critical component in the spray application process, specifically in the retention of spray droplets. Adjuvant technology is reported to improve the wettability and spread of droplets by surface-acting-agents on the target surface and thereby improve deposition and retention of the fungicide active ingredient. However, this relatively new spray technology on viticulture and horticultural crops, and possible effects of adjuvants on epicuticular wax affecting plant disease development, needs to be investigated. Moreover, the development of useful prescriptions for adjuvants by determining water volumes and adjuvant dosages is required for different pesticide tank mixes. The aims of this study were, firstly to determine the effect of selected adjuvants on quantitative and qualitative spray deposition on grapevine leaves and subsequent biological efficacy of a fungicide, and secondly to evaluate selected adjuvants under field conditions and determine the effects of adjuvant dosage and spray volume on deposition. Leaves were sprayed under similar laboratory conditions to pre-run-off with 1 mL of a mixture of fenhexamid (Teldor® 500 SC, Bayer) at recommended dose, a fluorescent pigment (SARDI Fluorescent Pigment, 400 g/L EC; South Australian Research and Development Institute) at 0.2 L/100 L, as well as 15 selected commercial adjuvants to manipulate the deposition quality of a given quantity of deposited spray. Spray deposition on leaves was illuminated under black light (UV-A light in the 365 nm region) and visualised under a stereo microscope (Nikon SMZ800) at 10× magnification. Photos of sprayed leaf surfaces were taken with a digital camera (Nikon DMX 1200). Digital images were quantitatively and qualitatively analysed with Image-Pro Discovery version 6.2 for Windows (Media Cybernetics) software, to determine spray deposition. The sprayed leaves were inoculated with 5 mg dry airborne conidia of Botrytis cinerea in a spore settling tower and incubated for 24 h at high relative humidity (≥ 93%). Leaf discs were isolated onto Petri dishes with paraquat-amended water agar and rated 11 days later for development of B. cinerea from isolated leaf discs. B. cinerea incidence on the upper and lower surfaces of water sprayed leaves averaged 90.4% and 95.8%, respectively. Despite full spray cover of leaves, applications with fenhexamid alone did not completely prevent infection and resulted in 34.6% and 40.8% B. cinerea incidence on the upper and lower surfaces of leaves, respectively. Through the addition of certain adjuvants, B. cinerea incidences were significantly lower (2.9-17.1% and 10.0-30.8%, respectively), while some adjuvants did not differ from the fungicide-only treatment, even though they might have improved spray deposition. The effects of Hydrosilicote and Solitaire alone and in combination with fenhexamid on germinating Botrytis conidia on leaf surfaces were studied in a histopathology study using epifluorescence microscopy. Distinct differences were observed in conidium mortality, germination and germ tube lengths between adjuvants alone and in combination with the fungicide, which might be attributed to indirect effects of the adjuvant mode of action on B. cinerea. The laboratory study clearly demonstrated the potential of adjuvants to improve the bio-efficacy of a fungicide directly through improved deposition on grapevine leaf surfaces. For the vineyard evaluations, the same fluorometry, photomicrography and digital image analysis protocol were used to assess quantitative and qualitative spray deposits under varying adjuvant dosage and volume applications. The Furness visual droplet-rating technique was initially included to determine optimum spray volume with a STIHL SR400 motorised backpack mistblower by assessment of pigment deposition on Chardonnay leaves under illuminated black light. Both assessment protocols showed that quantitative spray deposition increased with increasing spray volume applications of 40 L/ha to 750 L/ha, but decreased at 900 L/ha, possibly due to run-off. The addition of selected adjuvants at recommended dosage and at 600 L/ha demonstrated the potential of adjuvants to increase quantitative and qualitative deposition significantly on upper and lower leaf surfaces. Agral 90, BB5, Nu-film-P, and Solitaire significantly improved deposition on upper and lower leaf surfaces compared with the fenhexamid only and water sprayed control. Break-thru S 240 and Villa 51 did not improve quantitative deposition, although remarkably better qualitative deposition was obtained. An adjuvant dosage effect (within the registered dosage range) was evident, especially those retained on the upper leaf surfaces. Agral 90 and Nu-film-P affected significant improvement of spray deposition at the higher, but not at the lower dosage tested. Solitaire improved deposition at the lower dosage tested, whereas reduced deposition at the higher dosage was attributed to excessive spray run-off. No significant improvement of spray deposition was observed for both dosages tested with Villa 51. Spray mixtures with adjuvants Agral 90 and Solitaire yielded similar deposition values at 600 L/ha compared with the fenhexamid only control at 900 L/ha, but reduced deposition at the higher spray volume, possibly due to spray run-off. This study clearly demonstrated the potential of adjuvants to improve quantitative and qualitative deposition, but highlights the necessity to match adjuvant dosages and application volumes on the spray target to achieve maximum spray deposition. / AFRIKAANSE OPSOMMING: Effektiewe beheer van vrug- en blaarsiektes soos vaalvrot op wingerde benodig voldoende deponering van die swamdoder op die teikenoppervlak. Verlies aan beheer word gewoonlik aan onvoldoende kwantitatiewe deponering op vatbare wingerddele toegeskryf. Onder ‟n hoë siektedruk kan mislukte beheer ook moontlik toegeskryf word aan swak kwalitatiewe deponering. Die primêre doelwit van spuittegnologie is om deponering te optimaliseer met die plantoppervlak as ‟n belangrike komponent in die spuittoedieningsproses, spesifiek in die retensie van spuitdruppels. Byvoemiddel tegnologie het bewys dat oppervlak-aktiewe-agente verbeterde benatting en verspreiding van druppels op die teiken oppervlakte tot gevolg kan hê, en verder ook die deponering en retensie van die aktiewe fungisied bestanddele kan verbeter. Hierdie relatiewe nuwe spuittegnologie op wingerd- en hortologiese verbouing, asook die moontlike effekte van byvoegmiddels op epikutikulêre waks om siekte ontwikkeling te beïnvloed, moet ondersoek word. Verder word nuttige aanbevelings benodig vir byvoegmiddel toedienings by verskillende spuitvolumes en dosisse van die betrokke spuitmengsel. Die doelwit van hierdie studie was, eerstens om die effek van sekere byvoegmiddels op kwantitatiewe en kwalitatiewe spuitbedekking van wingerdblare te bepaal en dan te vergelyk met die biologiese effektiwiteit van ‟n fungisied, en tweedens om van die byvoegmiddels onder veldtoestande te evalueer, asook die effek van byvoegmiddel dosisse en spuitvolumes te bepaal. Blare is onder dieselfde laboratorium toestande tot net voor-afloop met 1 mL van ‟n spuitmengsel, bestaande uit fenhexamied (Teldor® 500 SC, Bayer) teen die aanbevole dosis, ‟n fluoreserende pigment (400 g/L EC; Suid Australiese Navorsing en Ontwikkeling Instituut) teen 0.2 L/100 L, sowel as 15 geselekteerde kommersiële byvoegmiddels gespuit om die kwalitatiewe deponering, vir ‟n gegewe kwantiteit van spuitdeponering, te manipuleer. Die fluoreserende pigment is op die blaaroppervlak belig met ‟n swart lig (UV-A ligbron in die 365 nm golflengte) en deponering is onder ‟n stereo mikroskoop (Nikon SMZ800) teen 10× vergroting waargeneem. Die gespuite blaaroppervlaktes is op die manier met ‟n digitale kamera afgeneem (Nikon DMX 1200), waarna die digitale foto‟s kwantitatief en kwalitatief deur die gebruik van „Image-Pro Discovery version 6.2 for Windows (Media Cybernetics)‟ sagteware geanaliseer is om spuitbedekking te bepaal. Na elke blaarspuit is die blare met 5 mg droë konidia van B. cinerea in ‟n inokulasietoring geïnokuleer en daarna vir 24 h onder hoë relatiewe humiditeit (≥ 93%) geïnkubeer. ‟n Aantal skyfies vanuit elke blaar is op Petri bakkies met paraquat medium geïsoleer en 11 dae later is die persentasie van B. cinerea ontkieming bepaal. Die gemiddelde voorkoms van B. cinerea op die blare wat slegs met water gespuit is, was 90.4% op die boonste en 95.8% op die onderste blaaroppervlaktes. Spuitbehandelings met slegs fenhexamied, ongeag goeie blaarspuitbedekking, kon nie die B. cinerea infeksie ten volle voorkom nie, en infeksie van gemiddeld 34.6% en 40.8% is onderskeidelik op die boonste- en op die onderste blaaroppervlaktes waargeneem. Met die byvoeging van sekere byvoegmiddels het die voorkoms van B. cinerea betekenisvol verminder (2.9-17.1% en 10.0-30.8%, onderskeidelik), terwyl ander byvoegmiddels nie van die fenhexamied behandeling verskil het nie, hoewel hierdie middels meestal wel spuitdeponering verbeter het. Die effek van slegs Hydrosilicote en Solitaire, en in kombinasie met fenhexamied op ontkiemende Botrytis conidia, is bestudeer in ‟n histopatologiese studie deur middel van die gebruik van epifluoresensie mikroskopie op die blaaroppervlak. Duidelike verskille in die aantal dooie konidia, ontkiemingpersentasies en kiembuislengtes is tussen die byvoegmiddels en in kombinasie met fenhexamied waargeneem, waar sommige waarnemings moontlik aan die indirekte effek van die byvoegmiddel op B. cinerea toegeskryf kan word. Hierdie laboratoriumstudie wys duidelik dat byvoegmiddels oor goeie potensiaal beskik om die bio-effektiwiteit van die fungisied te verbeter deur die direkte verbetering van deponering op die wingerdblaaroppervlak. Dieselfde fluorometrie, fotomikrografie en digitale foto-analise protokol is in ‟n wingerd evaluasie om die kwantitatiewe en kwalitatiewe spuitdeponering van verskillende byvoegmidel dosisse and spuitvolumes te bepaal, gebruik. Die Furness visuele druppel meting tegniek is aanvanklik ingesluit om die optimale spuit volume met ‟n „STIHL SR400 motorised backpack mistblower‟ te bepaal deur visuele meetings van gedeponeerde pigment op Chardonnay blare onder ‟n swart ligbron. Beide protokolle wys dat kwantitatiewe spuitbedekking met ‟n toename in spuit volumes 40 L/ha tot 750 L/ha verbeter het, maar afgeneem het teen 900 L/ha, moontlik as gevolg van druppel-afloop. Die byvoeging van ‟n byvoegmiddel teen die aanbevole dosis en 600 L/ha wys uitstekende potensiaal om kwantitatiewe en kwalitatiewe deponering betekenisvol op boonste en onderste blaaroppervlaktes te verbeter. Agral 90, BB5, Nu-film-P, en Solitaire het deponering betekenisvol op boonste en onderste blare in vergelyking met die fenhexamied alleen en die water kontrole verbeter. Break-thru S 240 en Villa 51 het nie kwantitatiewe deponering verbeter nie, alhoewel verbeterde kwalitatiewe bedekking met hierdie produkte waargeneem is. ‟n Byvoegmiddel dosis effek (binne die registreerde dosis reeks) was duidelik waarneembaar, veral vir druppel retensie op die boonste oppervlak van blare. Agral 90 and Nu-film-P verbeter die spuit deponering betekenisvol met die hoër getoetste dosis, maar nie teen die lae dosis nie. Solitaire verbeter egter die deponering teen die laer dosis, maar minder deponering teen ‟n hoër dosis kan moontlik toegeskryf word aan oormatige druppel-afloop. In die geval van Villa 51 was geen betekenisvolle verbetering van spuitdeponering vir beide die behandelingsdosisse waargeneem nie. Spuitmengsels met byvoegmiddels, Agral 90 en Solitaire, het soortgelyke deponerings gelewer teen 600 L/ha in vergelyking met die fenhexamied kontrole teen 900 L/ha, maar deponering neem af teen hoër spuitvolumes met byvoegmiddels moontlik as gevolg van druppel-afloop. Hierdie studie wys duidelik die uitstekende potensiaal van Byvoegmiddels om kwantitatiewe en kwalitatiewe deponering te verbeter, maar beklemtoon die noodsaaklikheid van die korrekte gebruik van byvoegmiddel dosis en volume om die maksimum spuitdeponering op die teiken te verkry.

Thesis (MScAgric )--Stellenbosch University, 2004. / ENGLISH ABSTRACT: In an attempt to combat some of the pathogens that are associated with trunk diseases and disorders of grapevines, research in this thesis focused on the taxonomy and pathological aspects of Coniellai/Pilidiella, Botryosphaeria and Phomopsis spp. Previously, conidial pigmentation was used to separate Pilidiella from Coniella. Recently, however, the two genera have been regarded as synonymous, with the older name, Coniella, having priority. The most important species in the Coniellai/Pilidiella complex of grapevines is C. diplodiella (Speg.) Petr. & Syd., the causal organism of white rot of grapevines. Previous studies found it difficult to distinguish between C. diplodiella and C. fragariae (Oudem.) B. Sutton, which is known to occur in soil and caused leaf diseases of Fragaria and Eucalyptus. Both these species have previously been reported from South Africa. None of the reports on C. diplodiella could be scientifically substantiated; therefore it is still a quarantine organism. However, this status has been questioned. Based on sequence analyses of the internal transcribed spacer region (ITS 1, ITS 2), 5.8S gene, large subunit (LSU) and elongation factor 1- α gene (EF l- α) from the type species of Pilidiella and Coniella, Coniella was separated from Pilidiella, with the majority of taxa residing in Pilidiella. Pilidiella is characterised by species with hyaline to pale brown conidia (avg. length: width >1.5), with Coniella having dark brown conidia (avg. length: width ≤1.5). Pilidiella diplodiella, previously C. diplodiella, causal organism of white rot of grapevines, was shown to be an older name for C. petrakii. This fungus is present in South Africa and is therefore no longer of quarantine importance. Based on analyses of the histone (H3) gene sequences of isolates in the P. diplodiella species complex, P. diplodiella was separated from a newly described species, P. diplodiopsis. A new species, P. eucalyptorum, is proposed for isolates formerly treated as C. fragariae, associated with leaf spots of Eucalyptus spp. This species clustered basal to Pilidiella, and may represent yet a third genus within this complex. Pilidiella destruens was newly described as anamorph of Schizoparme destruens, which is associated with twig dieback of Eucalyptus spp. in Hawaii. The genus Botryosphaeria Ces. & De Not. are known to be cosmopolitan, with broad host ranges and geographical distributions. Several saprotrophic species have been reported from grapevines, while others are severe pathogens of this host. These species include B. dothidea (Moug.: Fr.) Ces. & De Not., B. parva Pennycook & Samuels, B. obtusa (Schwein.) Shoemaker, B. stevensii Shoemaker, B. lutea A.J.L. Phillips and B. ribis Grossenb. & Duggar. Species reported from South Africa as grapevine pathogens are B. obtusa, B. dothidea, B. ribis and B. vitis (Schulzer) Sacco. In the present study, morphological, DNA sequence data (ITS 1, 5.8S, ITS 2 and EFI-α) and pathological data were used to distinguish 11 Botryosphaeria spp. associated with grapevines from South Africa and other parts of the world. Botryosphaeria australis, B. lutea, B. obtusa, B. parva, B. rhodina and a Diplodia sp. were confirmed from grapevines in South Africa, while Diplodia porosum, Fusicoccum viticlavatum and F. vitifusiforme were described as new species. Although isolates of B. dothidea and B. stevensii were confirmed from grapevines in Portugal, neither of these species, nor B. ribis, were isolated in this study. All grapevine isolates from Portugal, formerly presumed to be B. rib is, are identified as B. parva based on EF1-α sequence data. Artificial inoculations on grapevine shoots showed that B. australis, B. parva, B. ribis and B. stevensii are more virulent than the other species studied. The Diplodia sp. collected from grapevine canes was identified as morphologically similar, but phylogenetically distinct from D. sarmentorum, while D. sarmentorum was confirmed as anamorph of Otthia spiraeae, the type species of the genus Otthia (Botryosphaeriaceae). A culture identified as O. spiraeae clustered within Botryosphaeria, and is thus regarded as a probable synonym. These findings confirm earlier suggestions that the generic concept of Botryosphaeria should be expanded to include genera with septate ascospores and Diplodia anamorphs. The genus Phomopsis (Sacc.) Bubak contains many species that are plant pathogenic or saprotrophic. Ten species are known from grapevines. However, only two have been confirmed as being pathogenic, namely P. viticola (Sacc.) Sacc., causal organism of Phomopsis cane and leaf spot and P. vitimegaspora Kuo & Leu (teleomorph Diaporthe kyushuensis Kajitani & Kanem.), causal organism of swelling arm disease of grapevines. P. amygdali (Delacr.) 1.1. Tuset & M.T. Portilla, a known pathogen from Prunus sp., was shown to be a possible pathogen of grapevines in a previous study. D. perjuncta Niessl. causes bleaching of dormant canes only and is therefore of little importance as a grapevine pathogen. Recently a number of Phomopsis isolates were obtained from grapevines in the Western Cape province of South Africa. Isolations were made from Phomopsis-like symptoms, pruning wounds and asymptomatic nursery plants. These isolates showed great variation in morphology and cultural characteristics. Earlier taxonomic treatments of Phomopsis, based species identification on host specificity, cultural characteristics and morphology. Recent studies have indicated that these characteristics can no longer be used to distinguish species of Phomopsis due to wide host ranges and morphological plasticity of some species. The use of anamorph/teleomorph relationships in species identification is also untenable, since Diaporthe teleomorphs have only been described for approximately 20% of the known Phomopsis species. In this study morphological data, DNA sequences (ITS-I, 5.8S, ITS-2) and pathogenicity data were combined to distinguish Phomopsis spp. from grapevines. Fifteen species of Phomopsis were delineated by phylogenetic analysis of ITS sequence data. Diaporthe helianthi, a sunflower pathogen, was reported from grapevines for the first time, with a further six, unknown species also distinguished. Three different clades contained isolates previously identified as D. perjuncta. Based on type studies, it appeared that the name D. viticola was available for collections from Portugal and Germany, a new species, D. australafricana, was proposed for South African and Australian isolates, formerly treated as D. perjuncta or D. viticola. An epitype specimen and culture were designated for D. perjuncta. This species was distinguished from D. viticola and D. australafricana based on morphology and DNA phylogeny. Artificial inoculations of green grapevine shoots indicated that, of the species tested, P. amygdali, a known pathogen of peaches in the USA, and P. viticola were the most virulent. / AFRIKAANSE OPSOMMING: In 'n poging om sommige patogene geassosieer met stamsiektes en syndrome, te beveg, het die navorsing in die tesis gefokus op die taksonomie en patologiese aspekte van ConiellaiPilidiella, Botryosphaeria en Phomopsis spp Voorheen is konidium pigmentasie gebruik om Pilidiella (hialien tot ligbruin konidia) van Coniella (donkerbruin konidia) te skei. Onlangs is hierdie twee genera egter as sinoniem beskou met die ouer naam, Coniella, wat voorkeur gekry het. Die belangrikste spesies in die ConiellaiPilidiella kompleks van wingerd is C. diplodiella (Speg.) Petr. & Syd., die veroorsakende organisme van witvrot van wingerd. Vorige studies het dit moeilik gevind om te onderskei tussen C. diplodiella en C. fragariae (Oudem.) B. Sutton, wat bekend is dat dit in grond voorkom en ook blaarsiektes van Fragaria en Eucalyptus veroorsaak. Beide hierdie spesies is tevore in Suid-Afrika aangemeld. Geen van die aanmeldings van C. diplodiella is egter wetenskaplik bewys nie en daarom is dit steeds 'n kwarantyn organisme. Hierdie kwarantyn status is egter bevraagteken. Op grond van DNS volgordes van die interne getranskribeerde spasieerder area ("ITS 1", "ITS2"), die 5.8S rRNS geen, die groot ribosomale subeenheid ("LSU") en die verlengingsfaktor 1-α geen ("EF-lα") van die tipe spesies van Pilidiella en Coniella, is Coniella van Pilidiella geskei, met die meerderheid van die taxa wat binne Pilidiella resorteer. Pilidiella word gekarakteriseer deur spesies met hialien tot ligbruin konidia (gem. lengte: breedte > 1.5), in teenstelling met die donkerbruin konidia van Coniella (gem. lengte: breedte ≤ 1.5). Daar is verder bewys dat Pilidiella diplodiella, voorheen C. diplodiella, veroorsakende organisme van witvrot van wingerd, die ouer naam van C. petrakii is. Hierdie swam is teenwoordig in Suid-Afrika en P. diplodiella is dus nie meer van kwarantyn belang nie. Op grond van analises van die histoon (H3) volgordes van spesies in die P. diplodiella spesies kompleks, is P. diplodiella geskei van 'n nuut beskryfde spesie, P. diplodiopsis. 'n Nuwe spesie, P. eucalyptorum, is ook voorgestel vir isolate voorheen beskou as C. fragariae, geassosieer met blaarvlek van Eucalyptus spp. Hierdie spesie het basaal van Pilidiella gegroepeer en mag moontlik nog 'n derde genus binne hierdie kompleks verteenwoordig. Pilidiella destruens is nuut as anamorf van Schizoparme destruens beskryf, wat geassosieer word met loot terugsterwing van Eucalyptus spp. in Hawaii. Die genus Botryosphaeria Ces. & De Not. is bekend as kosmopolitaans met 'n wye gasheerreeks en geografiese verspreiding. Verskeie saprofitiese spesies is aangemeld vanaf wingerd, terwyl ander ernstige patogene van hierdie gasheer is. Laasgenoemde spesies sluit in B. dothidea (Moug.: Fr.) Ces. & De Not., B. parva Pennycook & Samuels, B. obtusa (Schwein.) Shoemaker, B. stevensii Shoemaker, B. lutea A.1.L. Phillips en B. ribis Grossenb. & Duggar. Spesies aangemeld in Suid-Afrika as wingerdpatogene, is B. obtusa, B. dothidea, B. ribis en B. vitis (Schulzer) Sacco In hierdie studie is morfologiese, DNS volgorde data ("ITSl", "ITS2", 5.8S en "EF-Iα") en plantpatologiese data gebruik om II Botryosphaeria spesies, geassosieer met wingerde in Suid-Afrika en verskeie ander werelddele, te onderskei. Botryosphaeria australis, B. lutea, B. obtusa, B. parva, B. rhodina en 'n Diplodia sp. is bevestig van wingerde in Suid-Afrika, terwyl Diplodia porosum, Fusicoccum viticlavatum en F. vitifusiforme as nuwe spesies beskryf is. AIhoewel isolate van B. dothidea en B. stevensii bevestig is van wingerde in Portugal, is geen van hierdie spesies en ook nie B. ribis geïsoleer nie. AIle isolate vanaf wingerd in Portugal, voorheen beskou as B. rib is, is as B. parva op grond van hul "EF-lα" volgordes geïdentifiseer. Uit kunsmatige isolasies gemaak op wingerdlote is die gevolgtrekking gemaak dat B. australis, B. parva, B. ribis en B. stevensii meer virulent is as die ander spesies wat bestudeer is. Die Diplodia sp. versamel vanaf wingerdlote is geïdentifiseer as morfologies eenders, maar filogeneties verskillend van D. sarmentorum, terwyl D. sarmentorum bevestig is as die anamorf van Otthia spiraeae, die tipe spesie van die genus Otthia (Botryosphaeriaceae). 'n Kultuur wat as 0. spiraeae geïdentifiseer is, het binne Botryosphaeria gegroepeer, en word dus as 'n moontlike sinoniem beskou. Hierdie bevindinge bevestig vroeëre voorstelle dat die generiese konsep van Botryosphaeria uitgebrei behoort te word om genera met gesepteerde askospore en Diplodia anamorwe in te sluit. Die genus Phomopsis (Sacc.) Bubak bevat verskeie spesies wat as of plantpatogenies, of saprofities, beskryf is. Tien spesies is bekend op wingerd. Slegs twee is as patogenies bevestig, naamlik P. viticola (Sacc.) Sacc., veroorsakende organisme van loot-en-blaarvlek ("streepvlek") en P. vitimegaspora Kuo & Leu (teleomorf Diaporthe kyushuensis Kajitani & Kanem.), veroorsakende organisme van geswelde arm van wingerd. In 'n vroeëre studie is bevind dat P. amygdali (Delacr.) 1.1. Tuset & M.T. Portilla, 'n bekende patogeen van Prunus sp., moontlik ook 'n patogeen van wingerd mag wees. D. perjuncta Niessl. veroorsaak egter net verbleiking van dormante lote en is dus van min belang as 'n wingerd patogeen. Gedurende die afgelope twee jaar is verskeie Phomopsis isolate van wingerde in die Wes-Kaap provinsie van Suid-Afrika verkry. Isolasies is gemaak van Phomopsis-agtige simptome, snoeiwonde en asimptomatiese kwekeryplante. Die isolate verkry uit hierdie materiaal het groot variasie ten opsigte van morfologie en kultuureienskappe getoon. Vroeëre taksonomiese verhandelings van Phomopsis het spesies-identifikasie op gasheerspesifisiteit, kultuureienskappe en morfologie gebasseer. Onlangse studies het egter getoon dat, weens wye gasheerreekse en morfologiese plastisiteit van somnuge spesies, hierdie eienskappe me meer gebruik kan word om Phomopsis spesies te identifiseer nie. Die gebruik van anamorflteleomorf verwantskappe in die identifikasie van Phomopsis spesies ook onbruikbaar omdat Diaporthe teleomorwe vir slegs ongeveer 20% van die bekende Phomopsis spesies beskryf is. Die huidige studie het dus morfologiese data, DNS volgordes ("ITS 1", 5.8S, "ITS2") en patogenisiteitsdata gekombineer ten einde Phomopsis spp. vanaf wingerd te identifiseer. Vyftien Phomopsis spesies is deur die filogenetiese analise van die interne getranskribeerde spasieerder area ("ITS") volgordes geskei. Diaporthe helianthi, 'n bekende patogeen van sonneblomme, is vir die eerste maal op wingerd aangeteken, terwyl 'n verdere ses, tans onbekende spesies van Phomopsis ook geidentifiseer is. Drie verskillende groepe het isolate bevat wat voorheen as D. perjuncta geidentifiseer is. Gebasseer op studies van tipes, het dit voorgekom dat die naam D. viticola beskikbaar is vir isolate uit Portugal en Duitsland. 'n Nuwe spesie, D. australafricana, is voorgestel vir Suid-Afrikaanse en Australiese isolate wat voorheen behandel is as D. perjuncta of D. viticola. 'n Epitipe monster en kultuur is vir D. perjuncta benoem. Hierdie spesie is van D. viticola en D. australafricana onderskei op grond van morfologie en DNS filogenie. Kunsmatige inokulasies van groen wingerdlote het getoon dat P. amygdali, bekende perske patogeen, en P. viticola die mees virulent was.

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)--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, 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.

Thesis (MScAgric)--Stellenbosch University, 1972. / ENGLISH ABSTRACT: no abstract available

Thesis (PhD(Agric)--Stellenbosch University, 1935. / No Abstract Available

Thesis (MSc)--Stellenbosch University, 1968. / Please refer to full text for abstract