Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Agrobacterium tumefaciens-mediated transformation technology has been used in a variety of applications throughout the fields of cellular and molecular plant biology as well as plant physiology. Research is conducted in order to extend this application range and overcome some of the intrinsic limitations of the Agrobacterium transformation system. Predominantly, these limitations can be attributed to the host range specificity of A. tumefaciens, as well as adverse effects induced on explant tissue by active plant defence mechanisms, triggered by the plant-pathogen-interaction. Typically, this active defence mechanism culminates in the hypersensitive response (HR), characterised by localised cell death and necrosis. Not all Agrobacterium species, however, share the same host range and some have evolved the ability to infect plant species not normally considered hosts of A. tumefaciens. This host range specificity can be exploited to extend the application of existing Agrobacterium transformation systems. In an attempt to establish an efficient transformation system for Vitis vinifera which, has proven very difficult to transform with A. tumefaciens, indigenous A. vitis strains have been evaluated as possible host-specific transformation agents. Strains of Agrobacterium vitis should be suitable for this type of endeavour, since they have evolved several unique characteristics directly linked to the infection of their hosts. These include the ability to utilise, tartrate, a host abundant carbon source, as well as the production of an acid polygalacturonase that could play a role during the infection process. The proposition that the evolution of A. vitis is a fairly recent event is also confirmed by the relatively little divergence observed between A. tumefaciens and A. vitis. In this study, a selection of A. vitis strains were evaluated in screenings designed to accentuate desirable traits in strains such as good infectivity of grapevine material (presumably an indicator of an efficient mechanism of gene transfer to be exploited in an engineered transformation system) as well as a favourable reaction (causing no necrosis) on grapevine somatic embryos. Two strains produced large tumours on grapevine cuttings and caused little necrosis on the somatic embryos. Significant variation in infectivity as well as callus necrosis was observed between the strains as well as in a genotype-specific manner on the host material. This genotypic-specific effect of either host or pathogen could be an indication of the degree of specialisation developed by plant pathogens to infect specific hosts. On the basis of these results, it was possible to select an A. vitis strain for further biochemical and genetic characterisation. Simple biochemical analysis classified the strain as an octopine strain. DNA-DNA hybridisation techniques combined with a plasmid walking technique resulted in the partial characterisation of the T-DNA of the selected A. vitis strain. A partial restriction enzyme map of the T-DNA was constructed and the T-DNA and flanking areas were cloned. Significant differences, most notably, the absence of a TB-area as well as the absence of the agrocinopine (aes) gene from the 5' area of the T-DNA, were observed. Partial sequencing data indicated the presence of at least four conserved T-DNA genes located on the TA-DNA, as well as the presence of three bacterial insertion (IS-)elements flanking the region. Two of these IS elements, both related to the IS 110 family of IS elements have not yet been reported in A. vitis. In fact, these two elements seem to be the 5' and 3' ends of a disrupted element and could therefore have played an evolutionary role in the development of this strain. This study provides fundamental background for the development of a more efficient transformation system specific for grapevine, exploiting same of-the unique characteristics of one of its pathogens, A. vitis. / AFRIKAANSE OPSOMMING: Agrobacterium tumefaciens-gebaseerde transformasiesisteme word in "n wye reeks van toepassings in die velde van sellulêre- en molekulêre plantbiologie asook plantfisiologie aangewend. Navorsing word voortdurend onderneem om die inherente beperkinge van die Agrobacterium-transformasiesisteem te oorkom en sodoende die toepassingsveld van die sisteem verder te verbreed. Die beperkinge tipies aan dié sisteem kan hoofsaaklik toegeskryf word aan die gasheerspesifisteit van A. tumeteciens, asook die negatiewe reaksies op eksplantmateriaal wat deur die plant se aktiewe verdedigingsmeganisme, soos ontlok deur die plant-patogeen interaksie, veroorsaak word. Hierdie aktiewe verdedigingsmeganisme lei gewoonlik tot In hipersensitiewe respons (HR) in die plant, wat deur gelokaliseerde selafsterwing en nekrose gekenmerk word. Alle Agrobacterium-spesies het egter nie almal dieselfde gasheerreeks nie en sommige rasse het as gevolg van evolusionêre ontwikkelings die vermoë verkry om plantspesies wat normaalweg buite die gasheerreeks van A. tumefaciens val, te infekteer. Hierdie tipe gasheerspesifisiteit kan uitgebuit word om die toepassingsmoontlikhede van bestaande Agrobacterium-transformasiesisteme te verbreed. In In poging om In effektiewe transformasiesisteem vir Vitis vinifera, In moeilik transformeerbare gewas, te ontwikkel, is inheemse rasse van Agrobacterium vitis ondersoek as moontlike gasheerspesifieke transformasie-agente. Rasse van A. vitis behoort uiters geskik te wees vir so "n toepassing, aangesien hulle verskeie unieke eienskappe, wat direk aan die infeksie van die gasheer gekoppel is, vertoon. Van hierdie eienskappe is onder meer die vermoë om tartraat, In koolstofbron volop in druifplante, te benut. A. vitis produseer verder ook In suur poligalaktorunase wat vermoedelik In rol in die infeksieproses speel. Die voorstel dat die evolusionêre ontwikkeling van A. vitis In redelike onlangse gebeurtenis is, word onderskryf deur die betreklike homogenisiteit met A. tumefaciens. In hierdie studie is "n groep A. vitis-rasse met behulp van siftingsprosedures wat daarop gemik is om gesogte eienskappe in rasse uit te wys, beoordeel. Die vermoë om druifplantmateriaal te infekteer (wat vermoedelik "n aanwyser van "n effektiewe meganisme van geenoordraging is wat in "n gemanipuleerde transformasiesisteem benut kan word), sowel as 'n gunstige reaksie (d.w.s geen nekrose) op druifplant somatiese embrio's is van die gesogte eienskappe waarvoor gesoek word. Twee rasse het groot tumors op druifplant-stingelsegmente veroorsaak terwyl hulle bykans geen weefselskade op somatiese embrio's geïnduseer het nie. Betekenisvolle verskille in infektiwiteit en in kallusnekrose is tussen die rasse sowel as in 'n genotipe-spesifieke-verhouding waargeneem. Hierdie genotipe-spesifieke effek, kenmerkend van óf die gasheer óf die patogeen, kan aanduidend wees van die vlak van spesialisasie wat heers by die infeksie van spesifieke gashere. Na aanleiding van bogenoemde resultate was dit moontlik om 'n A. vitis-ras te selekteer wat verder aan biochemiese en genetiese analises onderwerp kon word. Eenvoudige biochemiese analises het dit moontlik gemaak om die ras as oktopien te klassifiseer. DNA-DNA hibridisasietegnieke gekombineerd met 'n unieke plasmiedwandeltegniek het gelei tot die gedeeltelike karakterisering van die geselekteerde A. vitisras. In Gedeeltelike restriksie-ensiem (RE) kaart van die T-DNA kon gevolglik opgestel word. Die T-DNA en die aangrensende gedeeltes is boonop gekloneer. Betekenisvolle verskille, spesifiek die afwesigheid van In TB area, sowel as die afwesigheid van die agrosinopien-sintasegeen (acs) aan die 51-kant van die T-DNA, is waargeneem. Gedeeltelike basispaaropeenvolgingsdata het egter die teenwoordigheid van minstens vier gekonserveerde T-DNA-gene, asook die teenwoordigheid van drie bakteriese invoegingselemente (IS) aan weerskante van die area, geïdentifiseer. Twee van hierdie elemente, wat beide homologie vertoon met die IS110 familie van IS elemente, is nog nie vantevore in A. vitis aangetref nie. Dit wil boonop blyk of dié twee elemente die 51 - en 31 - areas van In onderbroke element vorm, wat dus In moontlike aanduiding is van hul potensiële rol in die evolusionêre ontwikkeling van die ras. Hierdie studie verskaf basiese inligting wat daartoe kan lei dat 'n doeltreffender transformasiesisteem spesifiek vir druifplante ontwikkel word deur van die unieke kenmerke van een van sy patogene, A. vitis, uit te buit.

Dissertation (PhD Agric)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Chrysanthemoides monilifera ssp. monilifera is a shrub indigenous to South Africa, which has become a serious weed of native vegetation in Australia. Endophyllum osteospermi is a microcyclic, autoecious, rust fungus that induces witches' brooms on C. monilifera ssp. monilifera. This rust is considered as a candidate biocontrol agent for use against C. monilifera ssp. monilifera in Australia. The vegetative growth and reproductive output of healthy branches on bushes with different levels of E. osteospermi infections were measured at three sites. The growth of healthy branches on infected bushes was 26- 81% less than that of healthy branches on uninfected bushes. The number of buds, flowering capitulae, fruiting capitulae, and cypselas on healthy branches of infected bushes was 35-75%, 45-90%, 15-99%, and 15-90% less, respectively, than those on uninfected bushes. At five sites, the infection levels and number of witches' brooms were determined every two months. The increase in number of witches' brooms per bush ranged between o and 282 within one year, with an average increase per bush of28 (SE ± 4.8) and 39 (SE ± 9.2) during two years. The average simple interest rate (rs) increase of infection levels for all bushes was 0.015 month-I (s.e. ± 0.0041, n = 72) and 0.0098 month" (s.e. ± 0.0073, n = 43) during two years. Aecidioid teliospores germinated between 10 and 20oe, with 15°e as optimum. Light, and particularly near-uv light, stimulated germination. A period of 6 to 8 hours of light was needed to obtain optimum germination levels. The temperature requirements for basidiospore development differed from that of aecidioid teliospore germination. Optimum was at 15°e, but a rapid decrease in basidiospore production occurred at higher temperatures, few developed at 19°e. Two nuclear divisions occurred within 12 hours of germination to produce a metabasidium with three or four nuclei. A third nuclear division occurred in the basidiospores between 24 and 48 hours. Plants inoculated under controlled conditions took 5 to 24 months before witches' brooms began to develop. A Geographic Information System (GIS) approach was used to model the potential distribution of E. osteospermi in South Africa, based on monthly average climate surfaces with parameters derived from the above experiments. The same model was applied to Australia to suggest a potential distribution of the rust if released in Australia. This potential distribution was similar to one generated using the climate matching computer programme CLIMEX©, but gave greater spatial accuracy. Both approaches indicate that E. osteospermi should establish in temperate Australia. Chrysanthemoides species, as well as other South African asteraceaus plants, were monitored for E. osteospermi between 1992 and 2003. Endophyllum osteospermi was recorded on C. monilifera ssp. monilifera, C. monilifera ssp. pisifera, C. monilifera ssp. rotundata, C. monilifera ssp. canescens, C. monilifera ssp. subcanescens, C. incana, an undescribed taxon of Chrysanthemoides, Osteospermum ciliatum, 0. polygaloides and 0. potbergense. Endophyllum dimorphothecae sp. nov. is described on Dimorphotheca cuneata. Aecidium elytropappi, which was recorded on Elytropappus rhinocerostis and Stoebe plumose, is transferred to Endophyllum as E. elytropappi comb. nov. Germination of aecidioid teliospores and penetration by basidiospores were observed on the surface of excised leaves of 32 plant species at 4 days after inoculation. Germinating aecidioid teliospores aborted on 14 plant species, whilst no penetration was attempted on a further 12. Penetration only occurred on 9. Therefore only these 9 plant species need to undergo traditional host specificity testing. Pending these results, E. osteospermi could be safely released in Australia for the biological control of C. monilifera ssp. monilifera. / AFRIKAANSE OPSOMMING: Chrysanthemoides monilifera ssp. monilifera 'n meerjarige wat inheems in Suid Afrika is, het 'n belangrike onkruid in Austalië geword. Endophyllum osteospermi 'n mikrosikliese, autoecious roesswam, induseer heksebesems op C. monilifera ssp. monilifera. Hierdie roesswam word as 'n potensiële biologiese beheeragent teen C. monilifera ssp. monilifera in Austalië beskou. Die vegetatiewe groei en voortplanting van gesonde takke op struike met verskillende vlakke van E. osteospermi infeksies is by drie lokaliteite gemeet. Groei van gesonde takke op geinfekteerde bosse was 26-81 % minder as die van gesonde takke op ongeïnfekteerde bosse. Die aantalokselknoppe, blommende capitulum, vrugdraende capitulum en pitvrugte op individuele gesonde takke van geïnfekteerde bosse was onderskeidelik 35-75%, 45-90%,15-99%, en 15-90% minder, as die op ongeïnfekteerde bosse. By vyf lokaliteite is die infeksievlakke en die aantal heksebesems elke twee maande vasgestel. Die toename in heksebesems van elke plant was tussen 0 en 282 binne eenjaar, met 'n gemmidel van 28 (SE ± 4.8) en 39 (SE ± 9.2) geduurende twee jaare. Die gemiddelde eenvoudige rentekoers (rs) toename in infeksievlakke van al die struike was 0.015 maand" (s.e. ± 0.0041, n = 72) en 0.0098 maand-1 (s.e. ± 0.0073, n = 43) gedurende twee jaare. Ontkieming van aecidioidteliospore het tussen 100e en 200e met 15°e as die optimum. Lig en veral naby-uv lig het ontkieming gestimuleer, terwylontkieming relatief swak was onder donker toestande. 'n Periode van 6 tot 8 uur lig was nodig vir optimale ontkiemingsvlakke. Die temperatuurvereistes vir basidiospoor ontwikkeling het verskil van die van aecidioid teliospoor ontkieming. Optimale was by 15°e, maar 'n vinnige afname in basidiospoorproduksie het by hoër temperature voorgekom, min het by 19°e voorgekom. Twee kernverdelings het binne 12 ure van die begin van ontkieming voorgekom om 'n metabasidium te produseer met drie of vier kerne. 'n Derde kern verdeling het in die basidiospore tussen 24 en 48 uur voorgekom. Plante wat onder beheeerde toestande geïnokuleer is het heksebesems 5 tot 24 maande na inokulasie ontwikkel. 'n Geografiese Inligtings Sisteem (GIS) benadering is gebruik om 'n model vir die potensiële verspreiding van E. osteospermi in Suid Afrika te ontwikkel, gebasseer op die maandelikse gemiddelde klimaatoppervlaktes met parameters wat vanaf bogenoemde eksperimente verkry is. Dieselfde model is in Austalië toegepas om 'n potensiële verspreiding van die roesswam voor te stel. Hierdie potensiële verspreiding was soortgelyk aan 'n program wat met die klimaats vergelykende rekenaarsprogram CLIMEX© ontwikel is, maar dit het groter ruimtelike akkuraatheid gemaak. Beide benaderings het aangedui dat E. osteospermi in Austalië behoort te vestig. Chrysanthemoides spesies, asook ander inheemse plante van die Asteraceae, is tussen 1992 en 2003 vir die voorkoms van E. osteospermi in Suid Afrika waargeneem. Endophyllum osteospermi is op C. monilifera ssp. monilifera, C. monilifera ssp. pisifera, C. monilifera ssp. rotundata, C. monilifera ssp. canescens, C. monilifera ssp. subcanescens, C. incana, 'n onbeskryfde taxon van Chrysanthemoides, Osteospermum ciliatum, a. polygaloides en a. potbergense waargeneem. Endophyllum dimorphothecae sp. nov. is beskryf op Dimorphotheca cuneata. Aecidium elytropappi, wat op Elytropappus rhinocerostis en Stoebe plumosa voorkom, is by Endophyllum ingesluit as E. elytropappi comb. nov. Ontkieming van aecidioidteliospore en penetrasie deur basidiospore op die oppervlak van verwyderde blare van 32 plant spesies 4 dae na inokulasie is waargeneem. Ontkiemende aecidioidteliospore het op 14 toets plant spesies ge-aborteer, terwyl geen penetrasie op 'n verdere 12 gepoog is nie. Penetrasie het slegs op 9 voorgekom. Derhalwe hoef slegs die 9 plant spesies tradisionele gasheer spesifisiteitstoetse te ondergaan. Afhangende van die resultate kan E. osteospermi dus met veiligheid in Austalië vir die biologiese beheer van C. monilifera ssp. monilifera vrygelaat word.

published_or_final_version / Botany / Master / Master of Philosophy

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Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 103-124).

Thesis (Ph. D.)--Rutgers University, 2008. / "Graduate Program in Entomology." Includes bibliographical references (p. 132-139).

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