Ph.D. (Biochemistry) / Genetically modified (GM) crops were first introduced in the 1980s for the production of medicinal products. Since then, areas designated to GM crops have expanded drastically, with the GM crops grown to enhance agricultural productivity, improve agricultural practices, and as a tool to address potential pressures that will be faced by the agricultural sector and to address the issue of food security. Currently, cultivated GM crops include cotton, maize, rapeseed and soybean, carrying agronomic traits such as herbicide tolerance and insect resistance. Following the genetic modification of crops, three possible outcomes can be anticipated: these outcomes include the GM crop produced being equivalent to its untransformed counterpart, the GM crop differing from its untransformed counterpart with several well-defined characteristics, and the GM crop differing from its untransformed counterpart with a multitude of complex characteristics. In cases where the GM crop is equivalent to the untransformed counterpart, no further testing is needed. In instances where several well-defined and characterised differences are found between the GM crop and the untransformed counterpart, safety assessments are performed targeting these differences. The assessments will determine the impact of these unintended and unexpected alterations of the intended enhancement of the GM crops. However, methods currently used to assess GM crops have been found to be lacking, since they only focus on environmental and product-specific risks. Further evidence is essential, as part of GM crop safety assessment, on the molecular characterisation of these crops. This evidence is based on the potential impact of the transformation event, integration of the transgene into the host plant, as well as unintended alterations such as altered gene expression that may occur to the host plant. These events may assist in the further detection of potential dangers of the GM crop. As a result of these highlighted gaps, a project was formulated to study the unintended genomic alterations that may occur during and following the production of a transgenic plant...

Laurel wilt disease is a vascular wilt affecting the xylem and water conductivity in trees belonging to the family Lauraceae. The disease was introduced by an invasive species of ambrosia beetle, Xyleborus glabratus. The beetle, together with its newly described fungal symbiont Raffaelea lauricola (pathogenic to host trees), has lead to the devastation and destruction of over 300 million wild redbay trees in southeastern forests. Ambrosia beetles make up a very unique clade of beetle and share a co-evolved obligatory mutualistic relationship with their partner fungi. Rather than consuming host tree material, the beetles excavate galleries or canals within them. These galleries serve two purposes: reproduction and fungal gardening. The beetles house fungal spores within specialized sacs, mycangia, and essentially inoculate host trees with the pathogenic agent. They actively grow and cultivate gardens of the fungus in galleries to serve as their sole food source. Once the fungus reaches the xylem vessels of the host tree, it thrives and leads to the blockage of water flow, both because of fungal accumulation and to the host response of secreting gels, gums and tyloses to occlude vessels in an attempt to quarantine the fungus. This disease spreads rapidly, and as a result, once symptoms become visible to the naked eye, it is already too late to save the tree, and it has likely already spread to adjacent ones. The present study presents the first documented study involving the early detection of disease from deep within a tree through the use of scent-discriminating canines. In addition, the present study has lead to the development of a novel sample collection device enabling the non-destructive sampling of beetle galleries. Finally, a metabolomics approach revealed key biochemical pathway modifications in the disease state, as well as potential clues to disease development.

2 p. / Alfalfa varieties differ in fall dormancy, defined as growth during the fall. Nondormant alfalfa varieties are usually planted in mild winter areas for their ability to grow in the fall. However, fall growth of nondormant alfalfa may be undesirable in areas subject to repeated frosts or freezes. Nondormant, very nondormant, and extremely nondormant alfalfa varieties (fall dormancy class 8, 9, and 10) are adapted to elevations below 4000 feet in Arizona. Other dormancy classes not included in this publication are moderately nondormant varieties (fall dormancy class 7) which may be grown from 3000 to 5000 feet, and semi-dormant and dormant varieties (fall dormancy 6 and below) which are adapted to colder winter areas above 4000 feet.

Please read the abstract in the section 07back of this document / Dissertation (MSc (Plant Pathology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Fusarium oxysporum f.sp. cubense Schlecht (Foc), causal agent of Fusarium wilt of banana (Panama disease), is considered to be one of the most serious threats to banana production in the world. There is no effective control measure for Fusarium wilt, except for the replacement of susceptible with resistant banana varieties. However, resistant varieties are not always acceptable to producers and local consumer markets. A greater awareness of the detrimental effect of chemicals on the environment has stimulated research on biological control of plant pathogens. The use of indigenous microorganims, such as non-pathogenic F. oxysporum and the bacterium Pseudomonas fluorescens, therefore, offers not only an environmentally safe but also an economical approach to combat Fusarium wilt of banana as part of an integrated disease management strategy. Non-pathogenic F. oxysporum and P. fluorescens isolates have previously been isolated from the root rhizosphere in disease suppressive soils. These isolates have the ability to reduce the incidence of Fusarium wilt in greenhouse pathogenicity trials. In this study we had hoped to expand on existing knowledge on the biological control of Fusarium wilt of banana with non-pathogenic endophytic F. oxysporum and P. fluorescens. Isolates that significantly suppress disease development in greenhouse trials were tested under field conditions. Physiological and histological studies were also performed to understand the modes of action of putative biological control agents. For the histological investigations, non-pathogenic F. oxysporum isolates were modified with green and red fluorescent proteins. Chapter 1 depicts a general overview of the biological control of Fusarium wilt diseases of agricultural crops. This chapter addresses the biology and pathogenesis of F. oxysporum, before strategies to control Fusarium wilt are discussed. The application of biological control organisms was analysed in terms of potentially useful organisms, where they can be isolated, and their possible modes of action. Finally, factors that influence biological control of Fusarium wilt diseases are discussed. A good source of prospective biocontrol agents is suppressive soils. In Chapter 2, non-pathogenic F. oxysporum isolates were collected from healthy banana roots in disease suppressive soil. Random Fragment Length Polymorphisms of the intergenic spacer region were then applied to group the non-pathogenic F. oxysporum isolates into genotypes, from which candidates were selected for biological control studies. The selected endophytes were then inoculated onto banana roots to determine their ability to act as biocontrol agents against Foc. The isolates that protected banana best against Fusarium wilt in the greenhouse, together with P. fluorescens WCS 417, were tested in the field to determine whether these isolates could effectively reduce disease incidence in an uncontrolled environment. The ability of non-pathogenic F. oxysporum and P. fluorescens WCS 417 to induce systemic resistance in Cavendish banana plants against Foc was investigated in Chapter 3 with the use of a split-root technique. The putative biocontrol agents were inoculated, separately and in combination, on one half of the roots in a split-root experiment, while the other half was challenged by a pathogenic isolate of Foc. Five different phenolic acids were assayed which included total soluble phenolic acids, non-conjugated (free acids) phenolic acids, ester-bound phenolic acids, glycosidebound phenolic acids and cell wall-bound phenolic acids. The knowledge gained will contribute to the understanding of how the biocontrol agents may induce defense responses in banana roots against Foc. Non-pathogenic isolates of F. oxysporum were transformed with the green fluorescent protein (GFP) and DsRed-Express genes in Chapter 4. These isolates were used to visualise their interactions with a GFP-transformed Foc isolate on the banana root in a non-destructive manner by means of confocal laser scanning microscopy (CLSM) in Chapter 5. The ability of non-pathogenic F. oxysporum and P. fluorescens WCS 417 to induce structural changes was also investigated with a split-root system using the CLSM. Antibioses as a mode of action of the two potential biocontrol agents was tested in vitro. Understanding the modes of action of non-pathogenic F. oxysporum and P. fluorescens WCS 417 are important when considering strategies for the implementation of these isolates in an integrated disease management strategy against Fusarium wilt of banana. / Dissertation (MSc (Plant Pathology))--University of Pretoria, 2011. / Microbiology and Plant Pathology / unrestricted

Polygalacturonase-inhibiting proteins (PGIPs) are plant proteins believed to playa role in the defence against pathogenic fungi. In this study. it was hypothesized that apple PGIPI could be used to confer enhanced resistance against Verticillium-wilt. a major disease of potato caused by the fungus Verticillillm dahliae. Transgenic lines containing the apple pgip1 gene under control of the enhanced CaMV 35S (e35S) promoter had been generated previously. Stable integration of the transgene into the potato genome was shown by the polymerase chain reaction (PCR) and Southern blot with a DIG¬labelled apple pgip1 fragment as probe. Polygalacturonase (PG)-inhibiting assays (the agarose diffusion assay and reducing sugar assays) were employed to investigate the inhibiting activity of apple PGIP I extracts, prepared from the transgenic potato lines. on the PGs secreted by V. dahliae grown on pectin medium. Inhibition was successful for all but one of the transgenic lines. Active PGIPI was expressed in the leaves of in vitro- and glasshouse grown plants, as well as in roots of in vitro-grown plants. Due to the success of the in vitro inhibition results. it was anticipated that the apple pgip1 transgene would protect the transgenic lines against Verticillium-wilt in a subsequent glasshouse trial. The transgenic lines and untransformed BP I potato control were planted in soil inoculated with V. dahliae microsclerotia and control soil. Assessments of the visual symptoms of yellowing and wilt were made on a scale of 1-5. Colonisation of stem sections was determined by plating onto potato dextrose agar plates. Disease index values were calculated from the symptom and colonisation data. Analysis of variance indicated six lines to be significantly different from the rest when grown in the inoculated soil, but five of them also showed significantly slower senescence symptoms when grown in the control soil. It is proposed that the physiological effect of an extended juvenile phase resulted in the apparent increased disease resistance. This could be caused by transformation or tissue culture¬-induced somaclonal variation of the potato plants. The hypothesis that transformation of the apple pgip1 gene into potato would confer enhanced resistance against Verticillium-wilt was not supported by the data that was obtained. Expression of antifungal genes by pathogen-inducible promoters is a valuable strategy in the development of disease resistant crops of importance. A construct containing the apple pgipl gene under control of the pathogen-inducible gst1 promoter from Arabidopsis thaliana (L.) Heynh was generated. Agrobacterium tumefaciens GV31OI(pMP90RK) was transfonned with the plant transformation vector pCAMBIA2300 containing the gst1 and e35S promoter-pgip1 inserts. A. thaliana was transformed using the floral-dip method, and putative transgenic progeny were selected by kanamycin selection of the seeds. PCR verified the insertion of the transgene into the genomes of T2 and T3 lines. Gene expression from the two promoters was compared by performing PGIP extractions and the agarose diffusion assay. The gst1 promoter was active even without induction by methyl-salicylate. Both constructs led to the expression of active apple PGIP1 against V. dahliae PG in the heterologous plant A. thaliana. / Dissertation (MSc (Plant Biotechnology))--University of Pretoria, 2006. / Plant Science / unrestricted

Since the first report of Verticillium wilt of potato in 1950, the disease has been considered to be of minor importance in South Africa. Between 1995 and 2000, however, Verticillium spp. were isolated from 146 samples of symptomatic potato plant material received from 13 of the 14 potato production areas in the country. Of 93 Verticillium isolates that were obtained, 60% were identified as V. dahliae and 8 % V. nigrescens. V. dahliae was present in nine of the regions and V. nigrescens in seven. Unidentified Verticillium species were isolated from six of the regions. Both V. dahliae and V. nigrescens were pathogenic to potato in vivo, with V. dahliae the more virulent of the two species. Ten South African potato cultivars, eight of which have recently been released, were evaluated over two seasons in a greenhouse for resistance to V. dahliae. The cultivars Aviva, BP1, Bravo, Buffelspoort, Caren, Hoevelder and Ropedi were classified as susceptible to Verticillium wilt, whereas Calibra, Dawn and Devlin were rated as very susceptible. No resistance or tolerance was evident. The efficacy of broccoli volatiles on in vitro mycelial growth of Verticillium dahliae, and the effect of incorporation of fresh and dry broccoli residues on the survival of microsclerotia of V. dahliae and infection of potato, were determined in the laboratory and greenhouse. Volatiles emanating from freshly harvested macerated broccoli leaves were inhibitory to mycelial growth of V. dahliae on medium. Fresh and dry residues incorporated into soil artificially infested with V. dahliae, significantly reduced the viability of microsclerotia of the pathogen and the rate of infection of potato plants. Dry residues were more effective than fresh residues in reducing the viability of sclerotia, but suppression of infection was independent of the state of the residues. / Dissertation (MSc (Plant Pathology))--University of Pretoria, 2005. / Microbiology and Plant Pathology / unrestricted

Management of Fusarium wilt of banana, one of the most important diseases of agricultural crops, is complicated and involves the consideration of factors such as the biology, epidemiology and population structure of the pathogen, and genetic resources and production practices of the crop. The development of an integrated disease management programme, therefore, is of great importance in countries where the Fusarium wilt pathogen, Fusarium oxysporum f.sp. cubense Foc, has been introduced into banaria fields, and where resistant cultivars are not acceptable to local markets. To achieve this, it is important to investigate new management strategies and to review methods that have been less successful in the past. These management practices need to be practical and affordable. Since certain cultural practices have proven to be effective, management practices that could compliment them should be considered. This thesis has attempted to investigate such practices in order to develop an integrated disease management programme for Fusarium wilt of banana. One of the most important findings of this study, was that the surface sterilant previously used to prevent the introduction of the Fusarium wilt into uninfected areas in South Africa, are not effective. The sterilants Sporekill and Prazin proved to be highly effective, and are now recommended to replace the sterilants previously used. Several fungicides reduced mycelial growth of Foc in vitro, with the OMI fungicides and Benomyl found to be the most effective. The same fungicides reduced the disease severity of Fusarium wilt in the greenhouse significantly, especially when they were applied as root dip treatments. None of the fungicides found effective against Foc have been evaluated in the field against Foc before. The next step, therefore, would be to evaluate root dip treatments combined with drench treatment in the field. Although it is expected that these fungicides might have a negative effect on the microbial populations in the soil, this has yet to be investigated. Fungicides may even weaken or stress the pathogen, making it more vulnerable for the action of an effective biocontrol agent or agents. Chemical activators are probably one of the most attractive strategies to combat Fusarium wilt of banana, since it stimulate the plants' own defence system. Banana plantlets were found to be quite sensitive to the amount and method whereby chemical activators were applied. The activator benzo-(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester induced resistance against Foc on the susceptible Williams cultivar in the greenhouse, but not in the field. In field studies, environmental conditions were much more variable than in the greenhouse, which made it difficult to evaluate the effectiveness of chemical activators. Sodium nitroprusside and a product containing the harpin protein showed promising results on the Williams and DRSI cultivars, respectively. These activators need to be considered as part of an integrated disease management programme. Since they are not directly applied to the soil, they will not have a negative effect on the microbial populations in the soil. Several Fusarium isolates had been collected from banana fields with disease suppressive soils in Kiepersol, South Africa. Most of these isolates were F. oxysporum, and with the exception of one isolate, proved to be non-pathogenic to banana plants. A PCR-based restriction fragment length polymorphism (RFLP) analysis of the intergenic spacer region of the ribosomal RNA operon grouped the non-pathogenic F. oxysporum isolates into 12 distinct genotypes. A great diversity could be seen among the non-pathogenic isolates compared to the pathogenic Foc isolates. The known-biological control agent F047 grouped with three of the South African isolates, while the one pathogenic isolate grouped with the pathogenic Foc from diseased Cavendish bananas in South Africa By using PCR-RFLPs, we were able to rapidly characterize the structure of non-pathogenic isolates of F. oxysporum in disease suppressive soils in Kiepersol. This could assist us in our search for potential biological control agents for Fusarium wilt of banana. Representative isolates from the 12 genotype groups were selected for evaluation of Fusarium wilt suppressive properties in banana. These non-pathogenic F. oxysporum isolates appeared to be good biological control candidates and was compared to known biological control agents and commercial biological control products. Fourteen of the non-pathogenic isolates, the combination of two Trichoderma strains form suppressive soils in South Africa, and two Pseudomonas fluorescens isolates were found to significantly reduce Fusarium wilt development in the greenhouse. The commercial products Patostop, B-rus and a mixture of arbuscular mycorrhizae were also found to suppress the disease severity of Foc significantly. The well-know biological control agent F047 proved to be not effective. Results concluded that two of the non-pathogenic F. oxysporum isolates and the two P. fluorescens isolates, one of which was the well-known WCS 417, were the most effective of all the agents evaluated. Since combinations of biocontrol agents may provide even more consistent and effective control than a single agent, future research will include the combination of biocontrol agents found effective in this study. It would also be of great value to determine the mode of action of these isolates, so that isolates with different modes of action could be combined to enhance the suppression effect. Biological control can be a very useful component of an integrated disease management programme, since the effective agent or agents can easily be established on tissue culture banana plantlets before they are planted in the field. AFRIKAANS : Een van seker die mees belangrikste grondgedraagte siektes in lanbou, is Fusarium verwelksiekt van piesangs. In Suid-Afrika, is die siekte verantwoordelik vir emstige verliese in die piesang produksie. Aangesien daar geen weerstandbiedende kultivars beskikbaar is wat deur die mark aanvaar word nie, is dit van kardinale belang dat 'n geintegreerde siekte beheer program vir Suid-Afrika ontwikkel word. Voordat so 'n program saamgestel kan word, is dit belangrik dat verskeie faktore aangaande die patogeen en piesang poduksiepraktyke in ag geneem moet word. Beheermaatreëls moet prakties en bekostigbaar wees, en moet die reeds bestaande praktyke kan bevoordeel. Studies wat in hierdie tesis aangebied word, oorweeg beheermaatreëls wat gekombineer kan word met die huidige praktyke, nadat vorige praktyke ook in ag geneem is. Daar word gesoek na nuwe meer doeltreffende en ekonomiese metodes om siektes te beheer. Metodes wat doeltreffend aangewend kan word om die voorkoms van die siekte te vermirider. In vitro en in vivo studies het getoon dat die DMI swamdoders en Benomil die groei van die patogeen en die ontwikkeling van Fusarium verwelksiekte die meeste onderdruk. Die beste resultate is in die glashuis gevind nadat die wortels van plante in die middels geweek is. Positiewe resultate is ook verkry met die grondtoediening van Benomil 'n week nadat plante geplant is in Foc geïnfekteerde grond. Die chemiese beheer van Fusarium verwelsiekte kan verder ondersoek word deur die effek van die grondtoedienings en wortelbehandelings in die veld te ondersoek. Daar word egter verwag dat die swamdoders moontlik 'n negatiewe uitwerking op die mikrobiese aktiwiteit in die grond kan veroorsaak. Die gebruik van effektiewe ontsmettingmiddels is uiters belangrik vir die voorkomende beheer van Fusarium verwelkdiekte op piesangs. Die ontsmettingmiddel, koper oxichloried, wat tot onlangs in Suid Afrika gebruik was, is ondoeltreffend gevind vir ontsmettingsdoeleindes. Prazin en Sporekill, twee omgewingsvriendelike middels, is baie effektief gevind en word dus aanbeveel vir die ontsetting van voertuie, skoene en veld toerusing. Chemiese plant aktiveerders stimuleer plante om hulleself te beskerm deur middel van weerstandsmeganisms. Piesang plante het sensitiwiteit getoon toonoor die konsentrasie en die toedieningsmetode van hierdie chemiese aktiveerders. In die glashuisproewe het die aktiveerder benzo-(1,2,3)thiadiazole-7-carbothioic suur S-metiel ester weerstand gestimuleer in die Williams kultivar. As gevolg van veranderende toestande in die veld was dit moeiliker om die chemiese aktiveerders se werking te evalueer. Nogtans het die middels natrium nitroprussied en 'n produk wat die protein harpin bevat die voorkoms van siekte op die Williams en DRS 1 plante verlaag. Chemiese aktiveerders behoort sterk oorweeg te word as deel van 'n geintegreerde beheer program, aangesien chemiese aktiveerders nie direk tot die grond aangewend word nie, en geen negatiewe uitwerking op die natuurlike mikrobiese populasies in die grond uitoefen nie. Verskeie Fusarium isolate is geisoleer vanuit siekte onderdukkende gronde in die Kiepersol area van Suid-Afrika. Die meeste van die isolate is geidentifiseer as F. oxsysporum. 'n PKR-gebaseerde restriksie fragment lengte polimorfisme (RFLP) ontleding van die "intergenic spacer region" van die ribosomale DNS operon het die niepatogeniese F. oxysporum isolate in 12 verskillende genotypes opgedeel. 'n Groot diversiteit was sigbaar onder die nie-patogeniese isolate in vergelyking met die patogeniese foc isolate. Die bekende beheer agent, Fo47 het gegroepeer saam met drie van die Suid Afrikaanse nie-patogene. Hierdie tegniek het ons in staat gestel om die nie-patogeniese populasie van onderdrukkende gronde in Kiepersol vinnig te karakteriseer en potentiele biologiese agente te identifiseer. Verteenwoordigende isolate van die 12 genotipiese groepe wat geidnetifiseer is, is geselekteer vir verdere evaluasie. Dit is gevind dat die isolate goeie kandidate vir moontlike bio-beheer agente maak. Die onderdrukkingsvermoe van die nie-patogene is vergelyk met die van bekende bio-beheer agente en komersiele produkte wat beskikbaar is. Veertien van die nie-patogene, die kombinasie van twee Trichoderma spp., en twee Pseudomonas fluorescens isolate het die siekte ontwikkeling van Fusarium verwelking merkwaardig onderdruk in die glashuis. Die komersiele produkte Patostop®, B-rus en die kombinasie van twee mycorrhizae isolate is ook gevind om die voorkoms van siekte te verlaag. Die wel-bekende biobeheer agent Fo47 is oneffektief gevind teen Fusarium verwelksiekte van piesangs. Resultate van die studie het bewys dat twee van die nie-patogeniese F.Oxysporum isolate en twee P. Fluorescens isolate, waarvan een die welbekende WCS 417 is, uiters effektiewe beheer agente teen Foc is. Toekomstige studies sal fokus op die kombinasie van die bio-beheer agente wat die meeste potensiaal getoon het in die studie, asook hulle meganismes van werking. Biologiese beheer is van groot waarde vir 'n geïntegreerde beheer program. Dit kan maklik met bestaande beheer maatreëls gekombineer word en potensiële biologiese beheer agente kan vooraf op weefselkultuur plante in die kwekery gevestig word. / Dissertation (MSc)--University of Pretoria, 2011. / Microbiology and Plant Pathology / unrestricted

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13345号 / 論農博第2888号 / 新制||農||1079(附属図書館) / 学位論文||R2||N5252(農学部図書室) / (主査)教授 奥本 裕, 教授 土井 元章, 教授 冨永 達 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22787号 / 農博第2430号 / 新制||農||1081(附属図書館) / 学位論文||R2||N5307(農学部図書室) / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 井鷺 裕司, 教授 森 直樹, 准教授 大澤 直哉 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM