Doctor of Philosophy / Department of Plant Pathology / James Stack / The trade of food, plant, and animal products has increased the worldwide movement and establishment of exotic pathogens with dramatic negative impacts on plant systems. Fusarium proliferatum is a broad host-range pathogen and among the most common maize pathogens globally. It is often seed-borne and symptomless in maize, making it a high risk for introduction in maize and other grains. Considering the global distribution of maize and the wide host range and production of mycotoxins by F. proliferatum, a better understanding of its life history is needed. To provide markers for tracking F. proliferatum in laboratory experiments, strains of F. proliferatum were transformed to express a green fluorescent protein (GFP). Active dispersal (at least 1.5cm at 25°C and -50mb soil matric potential) and colonization of organic matter in nonsterile field soil was demonstrated in soil microcosms. Fusarium verticillioides is commonly isolated from maize seed also colonized by F. proliferatum. A red fluorescent (mRFP) F. verticillioides transformant was developed to study competition with F. proliferatum. For quantification in host tissues, a TaqMan multiplex qPCR protocol was developed using primer and probe sets targeting fragments of the green and red fluorescence genes to detect F. proliferatum and F. verticillioides, respectively. Prior colonization of maize tissues by F. verticillioides (p=0.6749) and other seed-borne microorganisms (p=0.1910) did not affect subsequent colonization by F. proliferatum. Genotyping-by-sequencing (GBS) was used to identify genetic markers in F. proliferatum. Primer sets based GBS markers were designed to allow detection of specific isolates in field experiments. F. proliferatum populations were characterized from maize seed prior to planting and again after harvest. End-point PCR identified F. proliferatum isolates containing the GBS marker. AFLP-fingerprinting indicated that 23 of the 817 F. proliferatum isolates contained the molecular marker and were genetically related to the original isolate. Based on the subclade and percentage similarity in UPGMA phylogenetic trees, and the population grouping observed in STRUCTURE and Principal Coordinate Analysis, these isolates could have a single origin and be clonal. Understanding the life cycle of F. proliferatum is critical for learning more about the risk of introducing seed-borne exotic isolates into new environments.

A survey involving 71 farmers from rural communities in Mpumalanga, South Africa was conducted to gather information regarding the importance and utilisation of cowpea. Cowpea was rated third most important in contributing to household security, preceded by maize and vegetable production. Cowpea was mainly produced for own consumption, as a source of income and as fodder for livestock to a lesser extent. The crop was used by 8.5% of the farmers for medicinal purposes. Results indicated that 20% of the farmers encountered problems with mouldiness during storage, with insect infestation to a lesser degree. Cowpea seed samples from South Africa and Benin, West Africa were analysed for seed mycoflora and various fungal genera, particularly Aspergillus, Phoma and Lasiodiplodia were recorded. The results indicated an array of Fusarium spp. including F. equiseti, F. chlamydosporum, F. graminearum, F. proliferatum, F. sambucinum, F. scirpi, F. semitectum and F. subglutinans. The seed samples and the F. proliferatum isolates, cultured on maize patty media, were analysed for fumonisin production. Samples were extracted with methanol/water (70:30) and cleaned-up on strong anion exchange solid phase extraction cartridges. High-performance liquid chromatography with pre-column derivatisation using o-phthaldialdehyde was used for the detection and quantification of FB1, FB2 and FB3. The cowpea cultivars from South Africa had levels of FB1 ranging between 0.12 – 0.61 µg/g. All the F. proliferatum isolates produced FB1, FB2 and FB3 with total fumonisin concentration levels between 0.80 - 25.30 µg/g. The highest level of FB1 detected was 16.86 µg/g. Surface-disinfected seeds were imbibed in sterile distilled water amended with FB1 to yield final concentrations of 10, 25, 50 and 100 ppm. Percentage germination was determined in paper towels according to the International Seed Testing Association (ISTA) rules. Root and shoot length was measured after 9 days. All the toxin concentrations significantly decreased seed germination whilst root and shoot elongation was inhibited by the 50 and 100 ppm concentrations. Embryonic seed tissue treated with FB1 indicated compaction of the protoplasm and separation of the plasma membrane from the cell wall. Lipid bodies accumulated and seemed to line the cell wall. Acetone and ethanol extracts of the leaves of two cowpea cultivars exhibited significant inhibition of the growth of fungal plant pathogens at 5.0 mg/ml, with the exception of Fusarium equiseti. The growth of some fungi, in particular Alternaria alternate, was also reduced by lower concentrations of certain extracts. Acetone extracts of the Bechwana White cultivar inhibited growth of Staphylococcus aureus and Enterococcus faecalis at 2.5 mg/ml and Bacillus cereus, B. subtilis and Enterobacter cloacae at 5.0 mg/ml. Ethanol extracts of the same cultivar showed antibacterial activity against E. faecalis and E. cloacae at 5.0 mg/ml. This study represents the first report on the natural occurrence of fumonisins on cowpea seed and the potential of F. proliferatum isolates from cowpea seed to produce fumonisins. The phytotoxic effects of FB1 on cowpea seeds as well as the antimicrobial potential of cowpea leaf extracts were demonstrated for the first time. / Thesis (PhD)--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Stink bugs, including native brown stink bug (Euschistus servus) and invasive brown marmorated stink bug (Halyomorpha halys), cause damage to a variety of crops including field corn (Zea mays). Frequency and size of stink bug infestations have increased in corn fields in the Mid-Atlantic U.S., and there are growing concerns that these infestations may contribute to reductions in grain quality including increased mycotoxin concentrations. Prior research on native and invasive stink bugs has focused on understanding their biology, the damage they cause, and elucidating effective and economic management strategies. However, few studies examined the potential for stink bugs to facilitate fungal infection and mycotoxin contamination of corn grain. Thus, the objectives of this research were to: 1) assess the relationship between invasive brown marmorated stink bug (H. halys) feeding injuries and fumonisin contamination of field corn in the Mid-Atlantic U.S., 2) determine if stink bugs are a vector for mycotoxigenic Fusarium spp. in corn, and 3) evaluate the efficacy of pesticides for mitigating stink bug feeding injury and associated mycotoxin contamination in field corn. A correlation between H. halys feeding injury and fumonisin concentrations was identified, and the ability of H. halys to increase F. verticillioides infection and fumonisin concentrations in corn was demonstrated in field experiments. Fusarium species including fumonisin-producing F. verticillioides and F. proliferatum were isolated from field-collected stink bugs, and in laboratory experiments, E. servus was able to transmit F. verticillioides to non-infected corn ears after feeding on F. verticillioides-infected corn. In field studies, both fungicide and insecticide reduced stink bug-associated mycotoxin concentrations in corn, but levels of control were inconsistent. Thus, additional tactics that target both the stink bug and Fusarium should be implemented to mitigate risks of mycotoxin contamination in corn. / Doctor of Philosophy / Native and invasive stink bugs can severely damage crops including field corn. Frequency and size of stink bug infestations in Mid-Atlantic U.S. corn fields have increased, and there is growing concern that this may contribute to reductions in grain quality. Insect feeding injury is a risk factor for fungal infection and mycotoxin contamination in corn. Mycotoxins are toxic chemicals produced by certain fungi that have detrimental health effects on animals including livestock and humans. The relationship between stink bug feeding injuries and mycotoxin contamination in corn grain is not well understood, and management strategies to minimize the risk of mycotoxin contamination in corn need to be identified. The main goal of this research was to characterize interactions between stink bugs and mycotoxin-producing fungi and identify tactics for controlling both the insect pest and pathogen. Specific objectives were to: 1) assess the relationship between invasive brown marmorated stink bug (H. halys) feeding injuries and fumonisin contamination of field corn in the Mid-Atlantic U.S., 2) determine if stink bugs are a vector for mycotoxin-producing Fusarium spp. in corn, and 3) evaluate the efficacy of pesticides for mitigating stink bug feeding injury and associated mycotoxin contamination in field corn. Results from this work indicated that stink bugs have the ability to cause feeding injuries which facilitate invasion of mycotoxin-producing Fusarium species, leading to increases in mycotoxin concentrations in corn grain. Studies also demonstrated that stink bugs can vector Fusarium species during feeding and increase Fusarium infection of corn resulting in subsequent mycotoxin contamination. Field studies indicated that pesticide applications targeting both the stink bugs and mycotoxigenic fungi may be needed to minimize risk of mycotoxin contamination in corn. However, under low pest pressure, application of pesticides is unlikely to be profitable.

Au cours de ce travail, différents glucosylamines et aminodésoxyglucoses ont été synthétisés et caractérisés par différentes méthodes spectroscopiques dont l’IRTF, la RMN 1H, 13C et MALDI-Tof MS. L’étude des propriétés biologiques de ces molécules réalisée, d’une part, avec deux champignons du bois, Coriolus versicolor et Poria placenta, et d’autre part, avec trois microorganismes potentiellement rencontrés dans des aliments, Listeria innocua, Salmonella typhimurium et Fusarium proliferatum ont indiqué une contribution positive de la N-alkylation, du degré de N-substitution et de la quaternisation sur l’inhibition de leur croissance. Par ailleurs, l’impact sur la bioactivité, de la position du groupe amine sur le sucre, a été étudié. Il a été montré que la position du groupe amine sur le C-1 du glucose conduisait à une activité antifongique contre C. versicolor et P. placenta plus prononcée alors que la position C-3 du glucose était favorable à une activité antimicrobienne contre L. innocua et S. typhimurium. / In this study different glucosylamines and amino desoxyglucoses were synthesized and characterised using various spectroscopic methods including IRFT, both 1H and 13C NMR spectroscopy and MALDI-Tof MS. Biological assessment of these compounds realised with two wood decay fungi, Coriolus versicolor and Poria placenta on one hand, and with three food microorganisms Listeria innocua, Salmonella typhimurium and Fusarium proliferatum on other hand, indicated a positive impact of both N-alkylation and degree of N-substitution and quaternisation on their growth inhibition. Furthermore, a biological impact of the amine position on sugar was studied. It was found that amine function attached to the C-1 of glucose conducted to the best antifungal activity against both C. versicolor and P. placenta while that fixed on the C-3 of glucose was indicated for antibacterial activity against L. innocua and S. typhimurium.

Doctor of Philosophy / Department of Plant Pathology / John F. Leslie / Members of the genus Fusarium are widely distributed in many geographic regions of the world. This genus includes plant pathogens of many important cereal crops, e.g., wheat, maize, rice and sorghum, and of other native and economically important plants. From culture collections at Kansas State University and Universiti Sains Malaysia, strains from Southeast Asia, primarily from Malaysia and Thailand, associated with mango malformation disease, bakanae disease of rice, and stalk rot of sorghum were analyzed in sexual crosses and molecular diagnostics, e.g., Amplified Fragment Length Polymorphisms (AFLPs). Fusarium proliferatum was recovered from all three crops, with each crop also yielding some species unique to the crop, e.g. F. fujikuroi from rice, F. thapsinum from sorghum, and F. mangiferae from mango. These results are consistent with hypotheses that F. proliferatum has a wide host range while other species have much more limited host preferences. The absence from our samples of species associated with these diseases in other parts of the world suggests policies should be developed to reduce the chances of introduction of novel pathogens into Southeast Asia. Fusarium fujikuroi and F. proliferatum are closely related. They usually can be separated by sexual cross-fertility and DNA sequence analysis. However, some strains can cross irregularly and with poor fertility to produce viable interspecific hybrids. From a laboratory cross between F. fujikuroi FGSC8932 and F. proliferatum FGSC7615, 533 progeny were collected. These progeny were characterized for their AFLP genotype, mating type, gibberellic acid production, and pathogenicity on rice, onions, and apples. A recombination-based map from this interspecific cross was constructed. QTLs associated with gibberellic acid production, rice pathogenicity, and onion pathogenicity were identified. Gene segregation amongst the progeny of the F. fujikuroi × F. proliferatum cross was distorted towards F. proliferatum. Both novel and transgressive pathogenicity phenotypes were detected. Overall, this research demonstrates the potential threats that can result from an interspecific cross. These threats include pathogens with novel toxin profiles, new pathogenicity phenotypes, and more virulent strains. The variation observed among the progeny may enable isolation and characterization of genetic factors that have a role in pathogenicity, toxin production, and host specificity.

Magister Scientiae (Biodiversity and Conservation Biology) / Wheat is a key global commodity in terms of acreage and tradeable value and as a staple in household diets. Many factors including biotic stress conditions have detrimental effects on global wheat production and yield. The increasing prevalence of biotic stress inflicted by fungal species such as Fusarium has significantly reduced yield and quality of cereal crops thus, threatening sustainable agriculture and food security. Interactions between wheat and Fusarium spp. such as Fusarium proliferatum triggers the accumulation of reactive oxygen species (ROS) to levels toxic to the plant thus leading to oxidative damage and ultimate cellular death. In order to maintain redox homeostasis, plants rely on ROS-scavenging antioxidants (enzymatic and non-enzymatic) to control ROS molecules to levels less toxic to plants. This study investigated the impact of F. proliferatum on the physio-biochemical responses of two wheat cultivars (SST 015 and SST 088). Changes in seed germination, growth, biomass, chlorophyll and mineral contents were monitored. Furthermore, changes in ROS accumulation and antioxidant enzyme activity was measured in the shoots of both wheat cultivars.

<p>  </p> <p>Plant-parasitic nematodes are amongst the most important pathogens impacting crops. Potatoes and soybean are vital crops for rural livelihoods and essential for food security, but their cyst nematode parasites remain a significant constraint globally. <em>Globodera</em> and <em>Heterodera </em>species are amongst the most damaging and internationally recognized quarantine pests of these crops, causing up to 80% yield loss. Their second stage juveniles (J2s) penetrate the host plant root tips and establish a residence close to the vascular bundle from where they extract nutrients as they complete their life cycle. Restrictions of control chemicals have led to an urgent need for alternative control strategies for cyst nematodes. Biological control is a promising alternative control measure, and fungi possess many characteristics that could make them great biological control agents of cyst nematodes. Most nematode populations are thought to be regulated by their natural enemy community. It is unclear which fungi are best adapted as natural enemies of these parasites and how they might do this. This project aimed to (a) characterize and compare the mycobiome diversity of cyst nematode species; (b) isolate and characterize fungi associated with potato and soybean cyst nematodes (c) screen isolated fungi for their efficacy as biocontrol agents against cyst nematodes; and (d) describe any isolated fungi identified as new and having potential for cyst nematode antagonism. Cyst populations were collected from different regions and screened for fungi using culture-based methods. For our next-generation sequence data analysis, we found differences in fungal community assemblages between center of origin of the potato cyst nematode (Peru; South America) and the regions where these nematodes were introduced such as Europe and North America.  There was no significant difference in fungal community assemblages of cysts collected between the years 2019 and 2020. We characterized fungi associated with the cyst nematodes and found that the most frequently isolated genera were <em>Fusarium, Penicillium, Cylindrocarpon, Phoma, Aspergillus </em>and<em> Verticillium</em>. Filtrates from <em>Trichoderma</em> sp. 2, <em>Alternaria alternata, </em>and <em>Fusarium acaciae-mearnsii </em>were toxic on SCN eggs while <em>Purpureucillium lilacinum, Fusarium proliferatum</em> and <em>Aureobasidium</em> <em>ellingtonae </em>sp. nov. were toxic on PCN eggs and juveniles. A new species of <em>Aureobasidium</em> isolated from the potato cyst nematode (<em>Globodera ellingtonae</em>) for the first time, and having biocontrol potentials against this nematode, was also identified, and described. </p>

No description available.