Thesis (M.S.)--University of Delaware, 2007. / Principal faculty advisor: Robert E. Lyons, Dept. of Plant & Soil Sciences. Includes bibliographical references.

Bibliography : leaves 92-99. / v, 99, [37] leaves, [14] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1992

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 )--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)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Petri disease and esca are devastating grapevine trunk diseases and compromise the sustainability of viticulture world-wide. Despite being extensively studied, knowledge of inoculum sources and mechanisms of spread of the causal pathogens is limited. Arthropods have been suspected to play a role in the spread of Petri disease and esca pathogens. However, little information is known about the extent to which arthropods are associated with these pathogens. This study aimed to determine whether arthropods occurring within or on declining grapevines, are associated with trunk disease pathogens and to identify arthropods associated with pruning wounds. The potential of selected arthropods to act as vectors of trunk disease pathogens was also investigated. Two vineyards exhibiting grapevine trunk disease infections were sampled weekly for two years for collection of arthropods. Arthropods were collected using pruning wound traps, visual searches as well as trunk and cordon traps. Fungal spores from surfaces of arthropods were collected in water. Samples were subjected to nested PCR using primers Pm1/Pm2 and Pch1/Pch2 to verify the presence of Phaeoacremonium spp. and Phaeomoniella chlamydospora, respectively. Water samples were also cultured and grapevine trunk disease pathogens obtained were identified by sequencing the internal transcribed spacers 1 and 2 and the 5.8S rRNA gene or the partial beta-tubulin gene. A total of 10 875 arthropod individuals, belonging to more than 31 families, were collected from declining grapevines. The most abundant arthropods included millipedes, ants, spiders and beetles. Portuguese millipedes and cocktail ants were associated with fresh grapevine pruning wounds. Thirty-three percent of the 5677 water samples analysed, contained propagules of pathogens associated with Petri disease and esca. Of these, 37 % were recovered from millipedes, 22 % from cocktail ants, 15 % from spiders and 10 % from beetles. All the major groups of grapevine trunk diseases were detected on the arthropods. Phaeoacremonium species were detected in 1242 samples while Phaeomoniella chlamydospora was identified from 855 samples. Other fungi isolated included members of the Botryosphaeriaceae, Diatrypaceae and Diaporthales. The potential of grapevine sap as a food source for Portuguese millipedes and cocktail ants was investigated, in vitro. Millipede individuals were offered a choice between water and grapevine sap while ants in nests were presented with grapevine sap, tuna and water and monitored for ingestion of sap. Both taxa preferred grapevine sap over the other food items, indicating close association with pruning wounds. Subsequently, the ability of both taxa to transmit a DsRed-transformed Phaeomoniella chlamydospora isolate to fresh pruning wounds of canes in polystyrene strips, floating in water, and potted vines was tested. Arthropods were exposed to the fungus for 24 hours and transferred to the base of the plants and canes and were removed after three days. Isolations after a month revealed that millipedes and ants were capable of transmitting the fungus onto wounds and cause infection. Millipede faecal pellets were also evaluated as potential sources of inoculum. Millipedes were fed on Phaeomoniella chlamydospora for 24 hours, surface sterilised and allowed to defaecate in sterile Petri dishes overnight. Faecal material was collected, macerated in water and plated onto potato dextrose agar. Propagules of Phaeomoniella chlamydospora survived passage through the gut of millipedes and were passed out in a viable state to form colonies of Phaeomoniella chlamydospora. This study concludes that a wide variety of arthropods can be a source of inoculum of trunk diseases in vineyards. The results of the dissemination trial provides evidence that millipedes and ants are able to disseminate and infect vines with Phaeomoniella chlamydospora. It is therefore, highly likely that other grapevine trunk disease pathogens are transmitted in the same manner. This knowledge highlights the need for control of certain arthropods to be taken into consideration when managing grapevine trunk disease pathogens. / AFRIKAANSE OPSOMMING: Petri siekte en esca is verwoestende wingerd stamsiektes en verhinder die volhoubaarheid van wingerdproduksie wêreldwyd. Hierdie siektes is al intensief bestudeer, maar kennis rakende die inokulum bronne en meganismes van verspreiding van die veroorsakende patogene is beperk. Arthropoda is al vermoed om ‘n rol te speel in die verspreiding van Petri siekte en esca patogene, maar weinig informasie is bekend oor die mate waartoe arthropoda geassosieer is met die patogene. Hierdie studie het ten doel gestel om die arthropoda wat op of in wingerdstokke wat terugsterf voorkom te identifiseer en te bepaal watter van die arthropoda geassosieer is met stamsiekte patogene. Daar is ook ten doel gestel om die arthropoda wat geassosieer is met vars snoeiwonde te identifiseer en ook die moontlike vektor status van die stamsiekte patogene deur arthropoda. Arthropoda is weekliks vir twee jaar gekollekteer vanaf twee wingerde met stamsiekte infeksies. Snoeiwond lokvalle, visuele soektogte en stam- en kordon lokvalle was gebruik om arthropoda te vang. Swamspore van die oppervlak van die arthropoda is afgewas met water. Van hierdie water monsters is gebruik om dubbelvoudige polimerase ketting reaksies (PKR) te doen met die inleiers Pm1/Pm2 en Pch1/Pch2 om vir die teenwoordigheid van Phaeoacremonium spp. en Phaeomoniella chlamydospora onderskeidelik te toets. Die oorblywende water monster is gekweek op medium om die swamme teenwoordig te bepaal. Die wingerd stamsiekte patogene is verder geidentifiseer deur die DNS volgordes te bepaal van die interne getranskribeerde spasies 1 en 2 en die 5.8S rRNS geen of ‘n gedeelte van die beta-tubulien geen. In totaal is 10 875 arthropoda, wat behoort tot 31 families, gekollekteer vanaf wingerde wat terugsterf. Die mees algemene arthropoda was duisendpote, miere, spinnekoppe en kewers. Die Portugese duisendpote en die wipstert mier is geassosieer met vars wingerd snoeiwonde. Van die 5677 water monsters wat geanaliseer is, het 33% propagules van die Petri siekte of esca patogene gehad. Van hierdie was 37 % afkomstig vanaf duisendpote, 22 % van wipstert miere, 15 % van spinnekoppe en 10 % van kewers. Al die hoofgroepe van wingerd stampatogene is opgespoor op die arthropoda. Phaeoacremonium species is opgespoor in 1242 monsters en Phaeomoniella chlamydospora is gevind in 855 monsters. Ander swamme wat ook geisoleer is sluit lede van die Botryosphaeriaceae, Diatrypaceae en Diaporthales in. Die potensiaal van wingerdsap as ‘n bron van voedsel vir Portugese duisendpote en wipstert miere is in vitro ondersoek. Duisendpoot invidue is ‘n keuse gegee tussen water en wingerd sap terwyl mierneste ‘n keuse gehad het tussen water, wingerd sap en tuna. Die duisendpote en miere is gemonitor vir die inname van wingerdsap in die teenwoordigheid van die ander bronne. Beide die duisendpote en miere het wingerdsap verkies wat aandui dat hulle ‘n noue assosiasie met wingerd snoeiwonde het. Vervolgens is beide taksons getoets vir hul vermoë om ‘n DsRooi-getransformeerde Phaeomoniella chlamydospora isolaat te vektor na vars snoeiwonde op lote gemonteer op polistireen stroke wat in water dryf en op wingerd plante in potte. Die duisendpote en miere is blootgestel aan die swam vir 24 uur en oorgedra na die basis van die plante en lote en is weer verwyder na drie dae. Na ‘n maand is isolasies gedoen wat gewys het dat die duisendpote en miere die swam suksesvol kon oordra na die snoeiwonde en infeksie veroorsaak. Duisendpoot uitwerpsels is geëvalueer vir die potensiaal as inokulum bron. Duisendpote het gevoed op Phaeomoniella chlamydospora vir 24 uur, daarna oppervlakkig gesteriliseer en toegelaat om oornag uitwerpsels te maak in steriele Petri bakkies. Uitwerpsels was gekollekteer, fyngemaak in water en op aartappel dekstrose agar uitgeplaat. Propagules van Phaeomoniella chlamydospora het die verteringskanaal van die duisendpote oorleef en het tipiese kolonies op die agar gevorm. Hierdie studie het vasgestel dat ‘n verskeidenheid van arthropoda ‘n bron van inokulum van stamsiektes in wingerd kan wees. Die resultate van die vektor proewe het gewys dat duisendpote en miere die vermoë het om Phaeomoniella chlamydospora te versprei na snoeiwonde wat die swam dan suksesvol geinfekteer het. Dit is daarom hoogs waarskynlik dat van die ander wingerd stamsiekte patogene ook versprei kan word op dieselfde manier. Hierdie kennis demonstreer dat die beheer van spesifieke arthropoda in ag geneem moet word in die bestuur van wingerd stamsiektes. / Winetech, Agricultural Research Council of South Africa and NRF for financial support

Thesis (Ph.D)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Copper oxychloride is a fungicide that is extensively used in vineyards in the Western Cape to treat and prevent fungal diseases. It is however not clear what the effects are on soil organisms, which play an important role in soil fertility, in South African soils. There is paucity of data linking results obtained in the laboratory to effects observed in the field, which will only become useful if a clear relation can be demonstrated. The aims of this study were to: ~ Determine the effects of copper oxychloride on field populations of earthworms and simultaneously monitor lysosomal membrane stability, measured as neutral red retention time (NRRT). ~ Validate experimental field studies by doing inventories of earthworm populations in long-term sprayed vineyards. ~ Determine the LC50 of copper oxychloride and simultaneously measuring NRRT, and linking them to the experimental field studies. ~ Conduct bioassays, burrowing activity- and soil-avoidance experiments to investigate their relations to earthworm population responses in the experimental field studies. Earthworms were sampled by hand-sorting in the field tests on treated and untreated field plots in the Western- (October 1998 - July 1999) and Northern Cape (April 1998 - October 1999). Soil samples and worms were analysed for copper contents and coelomocytes of live earthworms were used to conduct the neutral red retention assays. Acute toxicity tests were conducted over a period of 28 days during which the earthworms (Eisenia fetiday were exposed to different concentrations of copper oxychloride. Change in biomass and mortality were measured as endpoints, as well as NRRT. Bioassays, burrowing activity and soil-avoidance were conducted by exposing Aporrectodea caliginosa to grassland- and vineyard soil as well as grassland soil spiked with 60 J.1g.g-1copper in the form of copper oxychloride. Growth and mortality were recorded in the bioassays as well as copper concentrations In earthworm body tissues and substrates used over a period of28 days. Burrowing activity and soil-avoidance were determined by measuring the length of tunnels burrowed by A. caliginosa in soil profiles over a period of 4 days under different exposure regimes. Results from the field tests showed that spraying of copper oxychloride had a negative effect on earthworm populations at the prescribed application rates. NRR T in earthworms from the exposure plots was significantly (p<0.05) lower after just one spraying application. It was concluded that spraying copper oxychloride at prescribed application rates caused a decrease in field populations of earthworms and that NRRT was an early and reliable biomarker since it was indicative of later effects observed at the population level. Results obtained from the field inventory of earthworms in vineyards at Nietvoorbij, Robertson end Worcester confirmed data from the two field studies. The calculated LC50 of 882.78 I1g.g-1 for copper oxychloride and 519.40 I1g.g-1 for copper was ecologically relevant if a safety factor of 10 was applied. NRRT which manifested earlier than effects on biomass change in the acute toxicity tests, were significant when viewed against the background of responses of field populations of earthworms. From the bioassay experiments it was found that A. caliginosa exposed to copper oxychloride spiked soil had significantly (p<0.05) higher weight loss and mortality than those in grassland- and vineyard soil. This indicated that changes in biomass and mortality were indicative of population responses in the field and can be considered as ecologically relevant. Burrowing activity of A. caliginosa was significantly (p<0.05) lower in vineyard and copper oxychloride spiked soil than in grassland soil. Similarly in the soil avoidance experiments it was found that A. caliginosa avoided vineyard- and copper oxychloride contaminated soil. It is therefore concluded that burrowing activity and soil avoidance were ecologically relevant endpoints since they corresponded with population responses in the field. The study thus revealed that the long-term usage of copper oxychloride could have negative effects on earthworm populations. The spraying of copper oxychloride can have important implications on the sustainable use of agricultural soils since earthworms and other soil organisms play such an important role in soil fertility. The use of biomarkers and other ecotoxicological indicators can provide an early warning that soil organisms are under environmental stress. / AFRIKAANSE OPSOMMING: Die fungisied koperoksichloried word wyd gebruik in die Wes-Kaap om swamsiektes in wingerde te beheer en te voorkom. Dit is egter nie bekend wat die effek daarvan op Suid Afrikaanse grondbiota, wat 'n belangrike rol speel in grondvrugbaarheid, is nie. Daar is ook 'n tekort aan inligting wat die resultate van laboratoriumondersoeke in verband bring met veldstudies. Die doelstellings van die studie was om: ~ Die effek van koperoksichloried op erdwurmpopulasies in die veld te ondersoek en terselfdertyd membraanstabiliteit, as moontlike biomerker, gemeet as neutraal rooi retensietye (NRRT), te monitor. ~ Die geldigheid van eksperimentele veldstudies te toets deur ook grondanalises te doen in wingerde wat oor langtermyn met koperoksichloried bespuit is. ~ Die LC50 van koperoksichloried vir erdwurms te bepaal en terselfdertyd NRR T te meet asook om dié gegewens in verband te bring met die resultate van seisoenale veldstudies oor die uitwerking op erdwurmpopulasies. ~ Bio-evaluerings ("bioassays"), tonnelaktiwiteit- en vermydingseksperimente te onderneem en die verband tussen die toksiteitstoetse en populasieresponse, soos waargeneem in die veld, te ondersoek. Erdwurms is versamel deur handsortering tydens die veldtoetse in die Wes- (Oktober 1998 - Julie 1999) en Noord-Kaap (April 1998 - Oktober 1999) op kontrole en bespuite persele. Grondmonsters en erdwurms is spektrofotometries geanaliseer om koperinhoude te bepaal. Die selomosiete van lewende wurms is gebruik om NRR T te bepaal. Akute toksisiteitstoetse is uitgevoer oor 'n tydperk van 28 dae waartydens Eisenia fetida blootgestel is aan verskillende koperoksichloried konsentrasies. Veranderinge in biomassa en mortaliteit is bepaal asook NRRT. Bioevaluerings ("bioassays"), tonnelaktiwiteit- en vermydingseksperimente IS uitgevoer deur Aporrectodea caliginosa bloot te stel aan grasveld- en wingerdgrond asook grasveldgrond wat met koperoksichloried gekontamineer is. Groei en mortalitiet is bepaal in die "bioassays" asook koperkonsentrasies in die grond en erdwurm liggaamsweefsels oor 'n tydperk van 28 dae. Tonnelaktiwiteit en grondvermyding is bepaal deur die lengte van tonnels wat deur A. caliginosa gegrawe is te meet oor 'n tydperk van vier dae vir die verskillende blootgestelde groepe. Die resultate het aangedui dat koperoksichloriedbespuiting 'n negatiewe invloed het op erdwurmpopulasies teen die voorgeskrewe toedieningsprogram. NRRT in erdwurms van die blootstellingperseel, was beduidend (p<0.05) laer na 'n enkele bespuiting. Daar is verder bevind dat NRR T 'n betroubare en vroeë biomerker is, aangesien dit 'n aanduiding gegee het van latere effekte wat op populasievlak na vore getree het. Veldopnames in Nietvoorbij, Robertson en Worcester het die geldigheid van data verkry uit die veldstudies ondersteun. Die berekende LC50 van 882.78 ug.g" vir koperoksichloried en 519.40 ug.g" VIr koper was ekologies relevant indien 'n veiligheidsfaktor van 10 toegepas is. NRRT se ekologiese relevansie is bevestig deur dit te vergelyk met response wat in die veldtoetse waargeneem is. Deur bioevalueringseksperimente is bevind dat gewigsverlies en mortaliteit van A. caliginosa beduidend hoër was in koperoksichloried gekontamineerde grond as in die grasveld- (kontrole) en wingerdgronde. Veranderinge in biomassa en mortalitiet was aanduidend van populasieresponse soos waargeneem in die veldstudies en kan dus as ekologies relevante eindpunte beskou word. Tonnelaktiwiteit van A. caliginosa was beduidend (p<0.05) laer in wingerd- en koperoksichloried gekontamineerde grond as in grasveldgrond. Dieselfde is gevind in die grondvermydingstoetse waar A. caliginosa wingerd- en koperoksichloried gekontamineerde grond vermy het. Dit kan dus afgelei dat tonnelaktiwiteit en grondvermyding ook ekologies bruikbare eindpunte is aangesien dit verband hou met populasieresponse soos waargeneem in die veldstudies. Hierdie studie het getoon dat die herhaalde gebruik van koperoksichloried 'n nadelige invloed kan hê op erdwurmbevolkings. In die lig van die belangrike rol wat erdwurms en ander grondorganismes speel in grondvrugbaarheid kan die oormatige gebruik van hierdie fungisied ernstige implikasies inhou vir volhoubare benutting van landbougronde. Die gebruik van biomerkers en ander ekotoksikologiese eindpunte kan egter as vroeë waarskuwingsmetode dien dat die grondorganismes onder omgewingstres verkeer.

Upon detection of a pathogen, plants initiate specific signaling events designed to prevent host colonization and pathogen proliferation. Appearance of the hypersensitive response (HR), a type of programmed cell death signifies activation of active defenses in response to a one-to-one recognition of host, Resistance or R gene, and pathogen, avirulence or avr gene, encoded products. Turnip crinkle virus (TCV), however, has been shown to suppress the ability of Col-0 Arabidopsis thaliana plants to produce the HR in response to an avirulence factor. The extent of suppression was quantified by measuring cellular electrolyte leakage resulting from programmed cell death. Interestingly, cellular ion leakage levels were significantly lower in TCV-infected plants when challenged with bacteria expressing either of two bacterial effectors avrRpt2 or avrRpm1, suggesting that TCV can suppress the HR to a range of HR-inducing avirulence factors. In order to determine the viral component(s) responsible for mediating this suppression, each of the five TCV open reading frames (ORFs) was tested using an Agrobacterium tumefaciens-mediated transient expression assay in Nicotiana benthamiana. Though sequencing of the five TCV clones revealed mutations in the p28, p88, and p9 clones, Agro infiltration of an HR-inducing system in conjunction with individual TCV ORFs, or combinations of, was used to gather data to determine the role each may possess in the suppression phenotype. Full-length TCV was also expressed in the presence of AvrPto/Pto to establish suppression phenotype in Nicotiana. To assay for suppression of cell death in a heterologous system, both the mutant and wild-type clones were also tested in yeast for cell-death suppression induced by hydrogen peroxide exposure.

Thesis (M.S. in plant pathology)--Washington State University, December 2007. / Includes bibliographical references.

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