Thesis (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Grapevine leafroll-associated virus-3 is one of ten members of the C/osteroviridae that are known to infect grapevine. Nine of these viruses are associated with grapevine leafroll disease, of which GLRaV-1 and GLRaV-3 are the most important and widespread. Members of the C/osteroviridae are unique amongst the viruses, as it is the only known family whose members encode a heat shock protein 70 kOa homolog (Hsp70h). The Hsp70h is a movement protein (MP) that is required for the active translocation of the virion structure through the plasmodesmata into adjacent cells. Broad-spectrum resistance to unrelated viruses can be obtained by a pathogen-derived resistance (POR) strategy that is based on the expression of a dysfunctional MP in plants. The Hsp70h has two distinct domains. The N-terminal two thirds of the protein is an ATPase domain and shares high homology with the ATPase domains of all Hsp70h proteins from the C/osteroviridae and Hsp70 proteins from the prokaryote and eukaryote kingdoms. Conserved amino acids are found in the ATPase domain and are required for the positioning of the ATP at the catalytic site for ATP hydrolysis. The C-terminal domain is variable and the function of this domain in the Closteroviridae is not known. In prokaryote and eukaryote Hsp70 proteins, the C-terminal domain is required for protein-protein interactions. The American NY-1 isolate of GLRaV-3 has been sequenced and POR strategies have been attempted with the coat protein, divergent coat protein and replicase genes, but not with a dysfunctional form of the hsp70h gene. In this study, double-stranded RNA was isolated from a commercial vineyard with unknown virus status, but with distinct grapevine leafroll symptoms, and from two grapevine sources of known virus status, one with mild and one with severe symptoms. The GLRaV-3 hsp70h gene was amplified by RT-PCR from the dsRNA and the gene sequence was analysed. The hsp70h gene from the three virus sources contained more than 94% nucleotide sequence homology to the NY-1 isolate and the conserved amino acids required for ATPase activity were present. The hsp70h gene isolated from GLRaV-3 from a commercial Stellenbosch vineyard showing clear leafroll symptoms was selected for further work and was subjected to site-directed mutagenesis to engineer four point mutations in the gene. These four mutations resulted in the substitution of Asn for Asp", Gly for Thr1O, Lys for Glu 174 and Asn for Asp 197. The wild type (WT) and mutated (Mut) forms of the hsp 70h genes were cloned into a bacterial expression vector. Expression of both the WT- and Mut-Hsp proteins was achieved, and the protein was expressed in the insoluble inclusion bodies. All attempts to refold and isolate active proteins from the inclusion bodies were unsuccessful. Attempts to increase the concentration of soluble protein within the expressing bacteria were unsuccessful. Due to the lack of active protein, biochemical tests on the ATPase activity of the WT- and Mut-Hsp proteins could not be conducted. The wt- and mut-hsp genes were cloned into a plant expression vector for transformation into tobacco plants. These transformations were successful and gave rise to 22 Km' and 18 Km' plants from the WT- and Mut-Hsp constructs respectively. Two plant lines, M5 and M10, transformed with the mut-hsp transgene construct, appeared to have a high level of resistance to the challenging potato X potexvirus, whereas all the other tested plants were susceptible to the challenging virus. It was thus shown that a dysfunctional form of the GLRaV-3 Hsp70h could provide resistance to an unrelated virus in tobacco. / AFRIKAANSE OPSOMMING: Wingerdrolblaar-geassosieerde virus 3 (GLRaV-3) is een van 10 lede van die Closteroviridae wat wingerd kan infekteer. Nege van die virusse is met wingerdrolblaar geassosieer. Die GLRaV-1 en GLRaV-3 is die belangrikste en mees wyd verspreide lede van die rolblaar-geassosieerde Closteroviridae. Lede van die Closteroviridae is uniek in die opsig dat die virusse vir 'n 70 kDa-homoloë hitteresponsproteïen (Hsp70h) kodeer. Die Hsp70 is 'n bewegingsproteïen (MP) wat belangrik is vir die translokasie van die virus deur die plasmodesmata na die naasliggende sel. Breë-spektrum weerstand teen onverwante virusse kan behaal word deur 'n patogeen-afgeleide weerstandstrategie (POR), wat op die uitdrukking van 'n disfunksionele MP wat in plante uitgedruk word, gebaseer is. Die Hsp70hproteïen het twee gebiede. Die N-terminale gebied is In ATPase-gebied en toon hoë homologie met ander ATPase-gebiede van Hsp70h-proteïene van die Closteroviridae, asook die prokariotiese en eukariotiese koninkryke. Gekonserveerde aminosure wat belangrik is vir die posisionering van ATP in die katalitiese domein vir ATP-hidrolise is in die ATPase-gebied gevind. Die C-terminale gebied is variërend en die funksie van die gebied in die Closteroviridae is onbekend. In prokariotiese en eukariotiese Hsp70h-proteïene is die C-terminale gebied belangrik vir proteïenproteïen interaksies. Die nukleotiedvolgorde van die Amerikaanse NY-1-isolaat van GLRaV-3 is al bepaal en POR-strategieë is ook op die kapsiedproteïen, uiteenlopende kapsiedproteïen en die replikasie-proteïen uitgevoer, maar nog nie op 'n disfunksionele vorm van die Hsp70h-geen nie. In hierdie studie is dubbelstring-RNA (dsRNA) van 'n kommersiële wingerd met onbekende virusstatus wat rolblaarsimptome toon, geïsoleer, asook van twee wingerde met 'n bekende virusstatus, een met ligte en een met strawwe simptome. Die GLRaV-3 hsp70h-geen is met hulp van die polimerasekettingreaksie-metode (PKR) vanaf die dsRNA geamplifiseer en die geen se nukleotiedvolgorde is bepaal. Die hsp 70-gene van drie verskillende wingerde het meer as 94% homologie met die NY-1-isolaat getoon. Die gekonserveerde aminosure wat vir ATPase-aktiwiteit belangrik is, was teenwoordig. Die hsp70h-geen van GLRaV-3, wat uit 'n kommersiële wingerd met duidelike rolblaarsimptome in die Stellenbosch-gebied geïsoleer is, is vir verdere navorsing gekies en dit is aan setel-gerigte mutagenese blootgestelom vier mutasies van die geen te bewerkstellig. Die gevolg van hierdie vier mutasies was die verandering van Asn na Asp", Gly na Thr1o, Lys na Glu174 en Asn na Asp197. Die wilde (WT) en veranderde (Mut) vorms van die hsp-gene is in 'n bakteriese uitdrukkingsvektor gekloneer. Uitdrukking van beide die WT- en die Mut-Hspproteïene is behaal, maar die proteïene was in die onoplosbare fraksie geleë. Pogings om die onoplosbare proteïene te isoleer en in 'n aktiewe oplosbare vorm te verkry, was onsuksesvol. Verdere pogings om die proteïene in die oplosbare fraksie van die bakteriese ekspressiesisteem uit te druk, was ook onsuksesvol. As gevolg van die gebrek aan aktiewe proteïen kon biochemiese toetse nie op die ATPaseaktiwiteit van die WT- en Mut-Hsp proteïne gedoen word nie. Die wt- en mut-hsp-gene is ook in In plantekspressievektor gekloneer vir transformasie in tabakplante. Hierdie transformasies was suksesvol en het aanleiding gegee tot 22 kanamisienbestande (Km') en 18 Km' plante vanaf die WT- en Mut-Hspkonstrukte onderskeidelik. Twee plantlyne, M5 en M10, wat met die mut-hsptransgene getransformeer is, het 'n hoë vlak van weerstand teen die infekterende aartappelvirus X getoon in vergelyking met ander plante wat met die virus geïnfekteer is. Daar is dus bewys gelewer dat 'n disfunksionele vorm van die GLRaV-3 Hsp70h weerstand kan bied teen 'n onverwante virus in tabak.

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

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

Thesis (DSc (Biochemistry))--Stellenbosch University, 2009. / The fumonisin mycotoxins are known to be the causative principle for several animal diseases and are associated with the development of liver and oesophagus cancer and neural tube defects in humans. The thesis focuses mainly on the characterisation of the compounds from maize cultures of the fungus Fusarium verticillioides, isolated from maize, the toxicological effects in animals, mechanism involved in hepato- and nephrocarcinogenicity and discussing the major differences and contradictions in the literature together with their impact on setting relevant risk assessment parameters to safeguard human health. Controversies include the importance of non-genotoxicity vs genotoxicity in the development of cancer, the role of threshold effects in carcinogenesis and the establishment of realistic risk assessment parameters that will also be applicable in developing countries. Recent approaches suggest that thresholds should also apply for genotoxic carcinogens as interaction with the DNA is only one event in the multi-step process of cancer development and therefore could not be taken as the basis for applying a no-effect threshold for genotoxins. It would appear that a carcinogen such as fumonisin, whether it is labeled genotoxic or non-genotoxic per se, exhibits some degree of risk at any level due to additive or synergistic interactions with other xenobiotics and/or dietary constituents. The underlying mechanisms of fumonisin-induced carcinogenicity includes the disruption of sphingolipid, phospholipids and fatty acid metabolism, which plays a major role in the modulation of apoptotic and cell proliferative pathways related to cancer development. Interactive responses between arachidonic acid and ceramide affect downstream cell signal transduction pathways and depending on the cell type the disruption of these pathways could either stimulate or inhibit cell proliferation which eventually will determine the induction of apoptosis and hence affect cell survival. The modulating roles of dietary constituents such as vitamins, protein and the South African herbal teas are also highlighted as they affected the outcome of toxicological assays, thus determining thresholds of the adverse effects in specific target organs that will impact risk assessment parameters. Regulation of the fumonisins in food and the associated risk are debated from many perspectives. In developing countries there is a lack of quality control implying that maize highly contaminated with mycotoxins may directly enter the food chain of adults and children as control of mycotoxins is difficult or in some cases totally absent. The interaction of politics, economy and technology will eventually determine the impact on health as the regulation of fumonisin in food differs between countries. Knowledge about the biological effects of the fumonisins is currently playing an important role in the development of simple and inexpensive methods to reduce the levels of the fumonisin in maize by targeting specific populations at risk.

No description available.

No description available.

Kelpak®, a seaweed concentrate (SWC) prepared from the brown seaweed Ecklonia maxima (Osbeck) Papenfuss, improves overall plant mass and fruit yield in a variety of crops. The main active principals isolated from Kelpak® are cytokinins and auxins. Although these compounds are partly responsible for the growth promoting effect observed with Kelpak® application, they do not fully account for the complete effect of Kelpak® treatment. For this reason the focus has turned to polyamines (PAs) which are found in all cells of plants, animals and microorganisms, including eukaryotic algae. Polyamines also have growth promoting effects in plants. A study was carried out to investigate the PA levels in E. maxima and Kelpak® through a biennial cycle and to investigate if the PAs present in Kelpak® may have an effect on root growth, alleviating nutrient deficiency and the transport and accumulation of PAs in plants. To determine the amount of PA in the stipes, fronds and SWC prepared from E. maxima, samples were collected monthly over a two-year period (June 2009-June 2011). Extracts were benzoylated and quantified using a Varian HPLC. Putrescine concentrations ranged from 15.98-54.46 μg.g⁻¹, 6.01-40.46 μg.g⁻¹ and 50.66-220.49 μg.g⁻¹ DW in the stipe, fronds and SWC, respectively. Spermine concentrations ranged from 1.02-35.44 μg.g⁻¹, 1.05-26.92 μg.g⁻¹ and 7.28-118.52 μg.g⁻¹ DW in the stipe, fronds and SWC, respectively. Spermidine concentrations fell below the detection threshold. This is the first report of PAs being detected in a SWC. The seasonal pattern established for the stipe, frond and SWC followed the same trend over a biennial cycle. Polyamines accumulated in the seaweed tissue during periods of active growth and as a stress response elicited by rough wave action. This PA trend was similar to the cytokinin trend reported by MOONEY and VAN STADEN (1984b) for Sargassum heterophyllum which suggests that PAs play an important role in the hormone cascade during active growth. Routine monthly screening of Kelpak® carried out in the Research Centre for Plant Growth and Development indicated that Kelpak® consistently resulted in more rooting in the mung bean bioassay than the IBA control. The potential root promoting effect of PAs were investigated. Individually applied PAs did not increase rooting in the mung bean bioassay, but a synergistic relationship was observed between Put (10⁻³ M) and IBA (10⁻⁴ M). When applied together, rooting increased significantly above Put (10⁻³ M) and IBA (10⁻⁴ M) applied separately. The Put-auxin combination produced a similar number of roots to those treated with Kelpak®. It is possible that the PAs present in Kelpak® have a synergistic effect with auxins present in Kelpak® to promote root development and growth. Several physiological effects of Kelpak® and PAs on plant growth were investigated in a series of pot trials. Kelpak® significantly improved the growth of P- and K-deficient okra seedlings and masked the detrimental effects exerted by P- and K-deficiency. The application of PAs (10⁻⁴ M) significantly improved the seedling vigour index (SVI) of okra seedlings subjected to N-deficiency. The statistical difference was attributed to the N-containing growth regulators and polyamines being degraded and metabolized by the okra seedlings. Polyamine application did not alleviate P- and K-deficiency but increased root growth significantly in seedlings receiving an adequate supply of nutrients. It is likely that the additional PAs supported auxin-mediated root growth. A pot trial with okra plants was conducted to establish if the PAs in Kelpak®, applied as a soil drench or foliar application, are absorbed and translocated in a plant. Plants were also treated with Put, Spm, Spd to establish if PAs can be absorbed and translocated. Once the fruit had matured, plants were harvested and the endogenous PA content quantified by HPLC in the roots, stems and fruits. Applying PAs as a soil drench was not as effective as a foliar spray at increasing the PA content in the different plant parts. Kelpak® treatment (0.4%) did not contribute more PAs in any plant part. Spermidine concentrations were higher, in the various plant parts, than Put or Spm, irrespective of the mode of application. The application of Put, Spd and Spm increased Spd concentrations in the roots. Considering that Spd is the main PA produced in the roots and that exogenously applied PAs are readily converted to Spd, it seems evident that Spd is the preferred PA for long-distance transport in plants. The cytokinins and auxins in Kelpak® play an important role in stimulating growth in plants. It is, however, the totality of different compounds in Kelpak® that gives it its unique growth stimulating ability. Polyamines, occurring within the seaweed contribute to this activity, having an active role in root production and thus increased plant growth. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

No description available.

Shuk-man Chow. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 171-186). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgement --- p.viii / General abbreviations --- p.x / Abbreviations of chemicals --- p.xi / List of figures --- p.xii / List of Tables --- p.xiii / Table of contents --- p.xv / Chapter 1. --- Literature review / Chapter 1.1. --- General introduction to rice disease --- p.1 / Chapter 1.1.1. --- Pathogenesis of Bacterial Leaf Blight (BLB) --- p.1 / Chapter 1.1.2. --- Pathogenesis of rice blast --- p.2 / Chapter 1.1.3. --- Control of rice diseases --- p.3 / Chapter 1.2. --- Plant defense mechanisms --- p.4 / Chapter 1.2.1. --- Basal resistance in plants --- p.4 / Chapter 1.2.2. --- Wound induced defense response --- p.5 / Chapter 1.2.3. --- Pathogen induced host defense response --- p.6 / Chapter 1.3. --- Structure of R gene products --- p.7 / Chapter 1.4. --- Recognition between R and Avr proteins in rice --- p.8 / Chapter 1.5 --- Current knowledge on Xa resistance and AvrXa avirulence protein --- p.9 / Chapter 1.6 --- Current knowledge on Pi resistance and AvrPi avirulence protein --- p.10 / Chapter 1.7 --- Pathogen induced signal transduction cascade --- p.12 / Chapter 1.7.1. --- R gene mediated signal transduction cascade --- p.12 / Chapter 1.7.2. --- Signal events of G-protein activation --- p.12 / Chapter 1.7.3. --- Signaling events for the accumulation of Ca2+ in cytosol --- p.13 / Chapter 1.7.4. --- Signaling events for oxidative burst --- p.14 / Chapter 1.7.5. --- MAPK cascade in defense signaling --- p.15 / Chapter 1.7.6. --- Transcriptional regulation of disease resistance related genes --- p.16 / Chapter 1.7.7. --- Translational regulation of disease resistance related genes --- p.17 / Chapter 1.8. --- Defense responses and defense related genes --- p.19 / Chapter 1.8.1. --- Pathogenesis related (PR) proteins --- p.20 / Chapter 1.8.2. --- Phytoalexins --- p.21 / Chapter 1.9. --- Disease resistance related genes common between rice blast and BLB resistance --- p.22 / Chapter 1.10. --- SA induced signal transduction pathway in rice --- p.23 / Chapter 1.11. --- Important tools facilitating the identification of disease resistance related genes from BLB resistant rice lines --- p.24 / Chapter 1.12. --- Hypothesis --- p.26 / Chapter 1.13. --- Project objective --- p.26 / Chapter 2. --- Materials and Methods --- p.27 / Chapter 2.1. --- Plant Materials --- p.27 / Chapter 2.2. --- Pathogen Inoculation --- p.27 / Chapter 2.3. --- RNA extraction --- p.29 / Chapter 2.4. --- Denaturing gel electrophoresis --- p.29 / Chapter 2.5. --- Subtraction libraries construction --- p.30 / Chapter 2.5.1. --- Cloning of disease resistance related genes --- p.32 / Chapter 2.5.1.1. --- pBluescript II KS (+) T-vector preparation --- p.32 / Chapter 2.5.1.2. --- Ligation --- p.32 / Chapter 2.5.1.3. --- Transformation --- p.32 / Chapter 2.5.1.4. --- Colony picking --- p.33 / Chapter 2.5.1.5. --- PCR amplification of DNA inserts --- p.33 / Chapter 2.5.1.6. --- Purification of PCR products --- p.34 / Chapter 2.6. --- Gene chips printing --- p.34 / Chapter 2.7. --- Probes synthesis and gene chips hybridization --- p.35 / Chapter 2.8. --- Standard-RNAs synthesis --- p.35 / Chapter 2.9. --- Data collection and analysis --- p.36 / Chapter 2.10. --- Sequencing --- p.36 / Chapter 2.11. --- cDNA synthesis --- p.37 / Chapter 2.12. --- RT-PCR --- p.38 / Chapter 2.13. --- DNA gel electrophoresis --- p.39 / Chapter 3. --- Results --- p.58 / Chapter 3.1. --- Construction of BLB gene chips --- p.58 / Chapter 3.1.1. --- Preparation of cDNA clones for gene chips construction --- p.58 / Chapter 3.1.2. --- Purification of PCR products on microtiter plate --- p.59 / Chapter 3.1.3. --- Gene chips construction --- p.59 / Chapter 3.1.4. --- DNA immobilization --- p.62 / Chapter 3.1.5. --- Probe synthesis --- p.62 / Chapter 3.1.6. --- Gene chip analysis --- p.65 / Chapter 3.1.6.1. --- Scanning --- p.65 / Chapter 3.1.6.2. --- Data analysis --- p.65 / Chapter 3.2. --- "Identification of disease resistance related genes commonly regulated by Xa2, Xal2 and Xal4 BLB resistance loci" --- p.70 / Chapter 3.2.1. --- "Signal perception, transduction and regulatory elements" --- p.71 / Chapter 3.2.1.1. --- Proteins involved in reversible phosphorylation cascade --- p.71 / Chapter 3.2.1.2. --- Proteins potentiate signal transduction through specific protein-protein interaction --- p.72 / Chapter 3.2.1.3. --- Other signal transduction components --- p.73 / Chapter 3.2.2. --- Transcriptional and translational regulatory elements --- p.74 / Chapter 3.2.2.1. --- Proteins involved in transcriptional regulation --- p.74 / Chapter 3.2.2.2. --- Proteins involved in post-transcriptional regulation --- p.75 / Chapter 3.2.2.3. --- Proteins involved in translational regulation --- p.76 / Chapter 3.2.3. --- "Oxidative burst, stress, apoptotic related genes" --- p.77 / Chapter 3.2.3.1. --- Stress related proteins --- p.77 / Chapter 3.2.3.2. --- Proteins involved in induction of oxidative burst --- p.78 / Chapter 3.2.3.3. --- PR proteins --- p.79 / Chapter 3.2.3.4. --- Proteolysis related proteins --- p.79 / Chapter 3.2.4. --- Cell maintenance and metabolic genes --- p.80 / Chapter 3.2.4.1. --- Antioxidant --- p.80 / Chapter 3.2.4.2. --- Metabolic genes --- p.81 / Chapter 3.2.4.3. --- Molecular chaperone --- p.82 / Chapter 3.2.4.4. --- Cell cycle regulators --- p.82 / Chapter 3.2.4.5. --- Cell wall maintenance --- p.83 / Chapter 3.2.4.6. --- Proteins involved in protein transport --- p.83 / Chapter 3.2.5. --- Unclassified/others --- p.84 / Chapter 3.3. --- Expression analysis of disease resistance related genes --- p.88 / Chapter 4. --- Discussion --- p.141 / Chapter 4.1. --- Differential expression of disease resistance candidates --- p.141 / Chapter 4.2. --- Disease resistance signal transduction components --- p.143 / Chapter 4.2.1. --- Reversible phosphorylation cascade --- p.143 / Chapter 4.2.2. --- Signal transduction potentiated by protein-protein interaction --- p.144 / Chapter 4.3. --- Other signaling molecules --- p.145 / Chapter 4.3.1. --- PRL1-interacting factor G --- p.145 / Chapter 4.3.2. --- Vacuolar-type H+-ATPasen subunit G --- p.146 / Chapter 4.4. --- Regulation of expression of disease resistance candidates --- p.146 / Chapter 4.4.1. --- Transcriptional regulation of disease resistance related genes --- p.146 / Chapter 4.4.1.1. --- G-box binding protein --- p.147 / Chapter 4.4.1.2. --- MYB TF --- p.147 / Chapter 4.4.2. --- Post-transcriptional modification of disease resistance candidates --- p.148 / Chapter 4.4.2.1. --- RNA splicing factor --- p.148 / Chapter 4.4.2.2. --- Glycine rich RNA binding proteins --- p.149 / Chapter 4.4.3. --- Translational regulation of disease resistance related genes --- p.149 / Chapter 4.5. --- Induction of oxidative burst --- p.150 / Chapter 4.6. --- PR proteins --- p.151 / Chapter 4.7. --- Cell maintenance --- p.152 / Chapter 4.7.1. --- Protein folding --- p.152 / Chapter 4.7.2. --- Protein degradation --- p.153 / Chapter 4.7.3. --- ROS scavenging --- p.154 / Chapter 4.7.4. --- Regulation of cell cycle --- p.154 / Chapter 4.8. --- "Confirmation and profiling of disease resistance related candidates commonly regulated in Xa2, Xal2 and Xal4 BLB resistance NILs at different time points" --- p.155 / Chapter 4.8.1. --- Basal resistance related genes --- p.156 / Chapter 4.8.2. --- General disease resistance related genes --- p.161 / Chapter 4.8.3. --- Pathogen responsive genes --- p.164 / Chapter 4.8.4. --- Prediction of novel genes functions --- p.168 / Chapter 4.9. --- Future prospect --- p.169 / Chapter 4.10. --- Conclusion --- p.169 / References --- p.171 / Appendix --- p.187

Orientador: Antonio Carlos Maringoni / Banca: Maria José de Marchi Garcia / Banca: Margarida Fumiko Ito / Banca: Marcelo Agenor Pavan / Banca: Tadeu Antonio Fernandes da Silva Júnior / Resumo: A cultura do pimentão (Capsicum annuum L.) tem elevada importância econômica e social para o Equador. Vários fatores têm contribuído para a baixa produtividade dessa cultura no país e dentre eles destacam-se as doenças de diversas etiologias. Poucas informações sobre a ocorrência de doenças nessa cultura estão disponíveis nas para as condições equatorianas. Neste trabalho foi realizado levantamento da ocorrência da doença "pata seca" e de viroses na cultura do pimentão, em plantios comerciais, durante anos de 2013 e 2014; a identificação dos agente(s) causal(ais) da "pata seca"; avaliações do eficácia da pulverização da aplicação de produtos fitossanitários (sulfato de cobre + fosetil alumínio; iprodione + clorotalonil; Trichoderma harzianum + T. koingii) em condições de campo, no controle da doença nos híbridos Nathalie e Quetzal e a sensibilidade in vitro do(s) patógeno(s) dessa doença a fungicidas (tebuconazole, benomil, clorotalonil, fosetil alumínio, iprodione, metalaxil + propanocarb e tebuconazole). A "pata seca" foi constatada em um 79,2% das propriedades, com incidência entre 5% a 53,6%, em quanto as viroses, foram detectada em 62,5% das propriedades, com incidência entre 9,6% a 61,2%. Os fungos S. clerotium rolfsii e Fusarium spp. foram isolados com maior freqüência das plantas com sintomas de "pata seca". Entretanto, S.rolfsii foi mais virulento, sendo considerado o principal agente causal da doença "pata seca". Não foi observada diferença dos produtos fitossanitários, aplicados no colo das plantas, na incidência de "pata seca", nos dois ensaios realizados em campo. A aplicação de iprodione + clorotalonil propiciou uma maior quantidade de frutos comerciais nos dois ensaios e uma maior produção em um dos ensaios. Os Isolados de S. rolfsii apresentaram maior sensibilidade aos fungicidas tebuconazole, benomil e iprodione, ... / Abstract: Sweet pepper (Capsicum annuum L.) has high economic and social importance for Ecuador. Several factors have contributed to the low productivity of this crop in the country and among them there are the diseases of different etiologies. Little information on the occurrence of diseases in this culture is available to the Ecuadorian conditions. This study was carried out survey of the occurrence of the disease "pata seca" and viruses in sweet pepper in commercial plantations, during the years 2013 and 2014; the identification of the agent (s) causal (es) of "pata seca"; evaluations of the effectiveness of spray application of pesticides (copper sulfate + fosetyl aluminum, iprodione + chlorothalonil; Trichoderma harzianum + T. koingii) in field conditions in controlling the disease in hybrid Nathalie and Quetzal and in vitro sensitivity ( s) pathogen (s) of the disease to fungicides (tebuconazole, benomyl, chlorothalonil, fosetyl aluminum, iprodione, metalaxyl + propanocarb and tebuconazole). The "pata seca" was found in a 79.2% of the properties, with an incidence of 5% to 53.6%, as in the viruses were detected in 62.5% of the properties, with an incidence of 9.6% to 61.2%. The fungus Sclerotium rolfsii and Fusarium spp. were isolated more frequently plants with symptoms of "pata seca". However, S. rolfsii was more virulent, being considered the main causative agent of the disease "pata seca". There was no difference of pesticides applied in the base of the plants, the incidence of "pata seca" in the two trial carried out in the field. The application of iprodione + chlorothalonil provided a larger amount of fruits in the two ... / Doutor