Fusarium circinatum is the causal agent of the disease known as pitch canker of pine. The fungus causes resinous cankers on stems and branches of mature trees, dieback of female flowers and cones, as well as root rot and pre- and post emergence damping off of seedlings. Little is known regarding the epidemiology and biology of F. circinatum in South African pine seedling nurseries, where the fungus has been causing major economic losses since its introduction into the country in the early 1990s. The objectives of this study were, therefore, to study the infection biology and epidemiology of F. circinatum on pine seedlings, the organism’s saprophytic biology. I also considered approaches to rapidly diagnose plants infected by F. circinatum and its relatedness to other species. Much research has been done on the pitch canker disease and the causal agent F. circinatum. Chapter 1 of this thesis aimed to summarize the available knowledge on the pitch canker fungus and its biology, ecology and epidemiology. Trials to screen for resistance of Pinus spp. to the pitch canker fungus have been conducted by many research groups and also by industries that rely on Pinus spp. for pulp and wood production. In Chapter 2, parameters for such trials, including optimal wounding methods, spore concentrations, plant physiological considerations and time elapsed between wounding and inoculation, were investigated. Temperature and ambient humidity are considered important factors in plant disease epidemiology. The effect of these factors on pitch canker epidemics has not yet been studied. In Chapter 3, a survey of disease incidence in pine nurseries from different geographic areas in South Africa with different climates is presented. This was done by correlating disease incidence data from the nurseries with temperature and humidity measurements. The saprophytic biology of a plant pathogen is of great importance in its epidemiology. The extent of any plant pathogen’s saprophytic survival determines the initial inoculum levels at the onset of an epidemic. In Chapter 4, I investigated the saprophytic growth and survival of F. circinatum in various substrates, temperatures and moisture regimes. Fusarium circinatum is morphologically similar to fungi referred to as Fusarium subglutinans sensu lato. Distinguishing F. circinatum from other species in this group has in the past required pathogenicity tests and sexual crosses, which are labour intensive and time consuming. In Chapter 5, a molecular diagnostic technique, based on real-time PCR, with which identification of Fusarium spp. commonly occurring in South African nurseries is possible, was developed. Fusarium circinatum and other fungi referred to as F. subglutinans sensu lato are members of the Gibberella fujikuroi species complex. Molecular taxonomic studies have shown that F. subflutinans sensu lato is a polyphyletic taxon. The objective of the study presented in Chapter 6 was to resolve the taxon phylogenetically with the use of multiple loci. The studies in the individual chapters of this thesis present individual aspects of the biology, ecology, epidemiology and molecular ecology of Fusarium circinatum. Each chapter represents an independent entity and consequently repetition between chapters has been unavoidable. / Dissertation (MSc)--University of Pretoria, 2007. / Microbiology and Plant Pathology / Unrestricted

There are many examples of associations between insects and fungi. Where the fungi involved are pathogens, such associations may be of economic importance. Insects of no economic concern alone can also become important pests because of their association with fungal pathogens. Insects may assist in the spread of pathogens by carrying them on or in their bodies. Insects may also predispose plants to infection by creating wounds during feeding, oviposition or other behavioural activities. Knowledge of associations between insects and fungal pathogens often form a crucial component in the management strategy of these pathogens. The pitch canker fungus, Fusarium circinatum, causes severe disease symptoms on mature pines in the USA. Various insects have been implicated as vectors of this disease. In South Africa, F. circinatum is reported only to cause disease on pine seedlings, where it results in severe losses in nurseries. Various insects are present in the nursery that could possibly be associated with the spread of the fungus or the infection of its hosts. Amongst these insects, fungus gnats are the prime suspects due to their history of association with fungal pathogens in other nurseries. The presence of fungus gnats in South African pine nurseries and their possible association with F. circinatum and other pathogens has never been investigated critically. The objective of this study was to expand the base of knowledge of fungus gnats in South African pine nurseries, and to consider their possible association with F. circinatum and their population structure within and between nurseries. The literature review provides a summary of fungus gnats in the nursery environment. This includes their description, biology and association with fungal pathogens. Information from these studies is used to evaluate the possible association between fungus gnats and F. circinatum in South African pine nurseries. In nurseries around the world where fungus gnats are considered pests, various control options have been used, and these are further discussed. The first research aim of this study was to determine whether fungus gnats are present in the major pine nurseries of South Africa. Thus, in Chapter 2, surveys were undertaken in four of the major pine nurseries. All fungus gnats collected were identified to species level. Other diptera collected were identified to family level. Furthermore, all diptera collected were isolated on general and selective growing medium to examine for the presence of F. circinatum. Results from Chapter 2 showed that only one species of fungus gnats was present in the nurseries and it was present in all four of the nurseries surveyed. This raised interesting questions regarding the phylogeographic structure of these populations and the diversity within and between populations. These questions are addressed in Chapter 3 using analysis of mitochondrial COI sequence data from fungus gnats collected in the four nurseries. Of particular importance was the interpretation of these results as it pertains to the movement of fungus gnats between populations, together with their associated fungi. Using general and specific growing medium to isolate fungal pathogens from insects is not necessarily an accurate method. Pathogens may be overgrown by faster growing fungi before they are noticed, especially if they are present only in small amounts. Chapter 4 examined the use of DNA-based methods as a tool to detect fungal pathogens on fungus gnats. Fungus gnats were collected from the same four nurseries as in Chapter 2. Species-specific primers for F. circinatum and Botrytis cinerea were used to detect these fungi. Dilution series were done to examine the sensitivity of the primers. General primers were used to detect other fungi. This dissertation includes some of the first studies ever undertaken on fungus gnats in South African pine nurseries. Their association with the very virulent pitch canker fungus is also considered in some detail. It is my hope that these studies will form a foundation for future research on fungus gnats in South Africa. Copyright / Dissertation (MSc)--University of Pretoria, 2006. / Zoology and Entomology / unrestricted

Spontaneous mutagenesis can be divided into three main steps: the introduction of DNA damage and lesions, damage recognition and DNA repair. All sources of spontaneous mutagenesis originate from within the cell itself, e.g., polymerase errors cause DNA mismatches and reactive oxygen species alter the chemical composition of DNA bases. The combined effects of all these processes influence spontaneous genomic mutation rates, which are thought to be a characteristic of individual species and/or groups of species. Although much is known about different mutagens and how they cause mutations the sequence context of these mutations are less well understood. The results of this MSc study on mutation in the filamentous fungus Fusarium circinatum showed that the 5ʹ and 3ʹ neighbouring bases of a single nucleotide polymorphism can significantly influence the type of substitution that occurred leading to the formation of mutational motifs. This was the case for both sets of genes examined (core housekeeping and non-ribosomal protein synthetase genes), whose evolution is known to differ. The fact that none of the identified motifs are shared between the two sets of genes could indicate that the cellular mutagens and/or repair machinery function differently for the two gene groups. Furthermore, none of the mutable motifs that have been identified for the well-known mutagens in model organisms could be detected in the fungus, which suggests that mutagens and/or DNA repair mechanisms of this fungus are unique. Although limited information is available for non-model eukaryotes, an estimate for the rate at which mutations arise across the genome of F. circinatum could be a good starting point for comparisons of its evolutionary rate to those of its close relatives. This was accomplished using a fluctuation analysis involving nitrate non-utilizing mutation reversion. Although mutation rate determined in this study is probably not precisely accurate, it represents a good starting point for future comparative studies on the evolutionary rate of Fusarium species. As a whole this study laid the foundation for a better understanding of spontaneous mutagenesis at specific sites in certain groups of genes as well as across the genome of the economically important plant pathogen F. circinatum. Restricted until August 2017 / Dissertation (MSc)--University of Pretoria, 2013. / gm2013 / Microbiology and Plant Pathology / Unrestricted

The future resilience of Pinus radiata D. Don (Monterey pine) is dependent upon the development of a silviculture program inclusive of either preventative or management techniques for the potentially fatal pitch canker disease (Fusarium circinatum Nirenberg and O’Donnell [=F.subglutinans (Wollenw. & Reinking) Nelson et al. f. sp. Pini). As an ecologically and commercially valued species, a myriad of factors threaten the genetic resources of the geographically limited natural range. This study evaluated the effectiveness of uneven-aged forest management for regeneration success in the native, pitch canker infected Año Nuevo stand at Swanton Pacific Ranch in Davenport, California. Seedling survival and growth averages were used to evaluate the impact of the variables of gap size (0.20-hectare, 0.10-hectare, and 0.05-hectare), site-preparation treatment (pile and burn, lop and scatter), and parent tree (13 local seed sources). Pitch canker symptoms were quantified to conclude if there is indeed a range of expressed resistance according to parent tree. Statistical models (i.e. ‘mixed effect’) yielded moderately significant differences in odds of survival between site treatments, with pile and burn plots producing higher survival rates (p=0.066). No significant difference in survival was detected between gap-sizes (p=0.936 and 0.803, 0.05- and 0.10-hectare gaps respectively to 0.20-hectare reference). Significant variation was expressed (estimate=0.1219, SE=0.057) between the 13 parent trees, implicating usefulness in predicting seedling survival. ‘Mixed effect’ models only detected a significant effect from gap-size on growth measures: 0.20-hectare gaps yielded height and diameter measures significantly larger than 0.05-hectare gaps (p=0.027 and 0.0081 respectively). Contingency tables detected significant association between gap-size and number of pitch canker branch symptoms. The range of infection rates produced by the 13 parent trees did not prove significant by analysis. Pile and burn site preparation treatment increases the probability of survival for artificially regenerated seedlings and 0.20-hectare gaps yield significantly higher growth averages over 0.05-hectare gaps.

Thesis (MScAgric )--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Pine pitch canker, caused by Fusarium circinatum, is a major constraint to the production of high quality timber in the Western Cape Province of South Africa. Under nursery conditions the pathogen causes a serious root and collar rot disease, and in plantations it causes stem cankers and branch die-back. In this thesis, sources of inoculum within a forestry nursery were investigated and novel approaches developed to eradicate the pathogen. Plant material was evaluated for plantation establishment in the Western Cape Province, and epidemiological studies conducted to better understand the infection, disease development, spore release and dissemination of F. circinatum in pine plantations. Fusarium circinatum was first reported in a forestry nursery in the Mpumalanga Province of South Africa in 1990, and now causes significant economic losses to most forestry nurseries in the country. The irrigation system, planting tray inserts, seeds and air at Karatara nursery were screened as potential sources of F. circinatum. Both the irrigation water and planting tray inserts were found to serve as sources of inoculum. An oxidation reduction potential (ORP)-based system, using hydrogen peroxide, was tested to sanitize irrigation water and planting tray inserts. An ORP level of 400 mV for 6 hours was effective in killing F. circinatum spores in irrigation water without being phytotoxic to pine seedlings. An ORP value of >500 mV cleaned planting tray inserts of all inoculum. Pinus radiata is the principal pine species grown in the Western Cape Province, but this species is also highly susceptible to F. circinatum. There are numerous different varieties or families of P. radiata available that can be planted, and it is known that tolerance levels between these varieties and families vary. Pinus radiata families, as well as other Pinus spp. that could potentially be planted in the Western Cape Province in future, were evaluated for tolerance to the pitch canker fungus. The P. radiata families were moderately to highly susceptible to F. circinatum. Pinus taeda was the Pinus spp. most tolerant, while P. elliottii also had good tolerance to the pathogen. None of the P. radiata families screened showed tolerance comparable to that of either P. elliottii or P. taeda. Pine pitch canker was first reported from the Tokai plantation in the Western Cape Province in 2005. In order to develop a disease management strategy for the disease in pine plantations, we investigated the effect of pruning wounds as potential entry sites for the pathogen, and how fast the disease spread under South African climatic conditions. The amount and time of sporulation was also investigated. Pitch canker increased slowly over a 3-year period, and pruning wounds appeared not to serve as primary entry points for F. circinatum. Spore quantification over a 12-month period using quantitative real-time PCR showed that inoculum was produced throughout the year, but that significantly more spores were released during the cold, rainy winter months. The role of insects in pathogen transmission and spread should be investigated in the Tokai plantation. / AFRIKAANSE OPSOMMING: Fusarium circinatum veroorsaak “pine pitch canker”. Hierdie siekte is een van die grootste beperkinge op die Suid Afrikaanse bosbou industrie. In kwekerye veroorsaak die patogeen wortelvrot en wortelkraagvrot. In plantasies veroorsaak F. circinatum kankers op die hoofstam van dennebome en die terugsterwing van sytakke. In hierdie tesis was bronne van inokulum in ‘n bosbou kwekery ondersoek. Nuwe tegnieke was gebruik om die bronne uit te wis. Plantmateriaal wat in die Wes-Kaap geplant kan word, was geëvalueer vir toleransie teen F. circinatum. Epidemiologiesie studies was uitgevoer om die infeksieproses, siekte-ontwikkeling, spoorvrystelling, en verspreiding van F. circinatum binne denneplantasies beter te verstaan. Die eerste verslag van F. circinatum in ‘n bosboukwekery was in 1990 in die Mpumalanga Provinsie. Verliese word tans veroorsaak deur die patogeen in die meeste bosbou kwekerye in Suid Afrika. Die besproeiingstelsel, planthouers en saad was getoets as potensiële bronne van inokulum. Daar was gevind dat beide die besproeiingstelsel en die planthouers dien as bronne van F. circinatum inokulum. ‘n Oksidasie reduksie potensiaal (ORP)-gebaseerde sisteem waar waterstofperoksied gebruik was, was getoets of dit die besproeingwater en planthouers kan ontsmet. ‘n ORP-vlak van 400 mV vir ‘n blootstellingstydperk van 6 ure is effektief om al die F. circinatum spore dood te maak, sonder dat dit fitotoksies is teenoor die dennesaailinge. ‘n ORP-vlak van >500 mV het die F. circinatum in die planthouers uitgewis. Pinus radiata is die belangrikste dennespesie wat geplant word in die Wes-Kaap Provinsie, maar hierdie spesie is hoogs vatbaar vir F. circinatum. Verskillende variteite van P. radiata met verskillende tolleransie vlakke bestaan. Pinus radiata families en ander spesies wat in die toekoms potensieel geplant kan word in die Wes-Kaap was geevalueer vir toleransie teen F. circinatum. Die P. radiata families was matig to hoogs vatbaar vir F. circinatum. Pinus taeda het die hoogste toleransie vlak gehad en P. elliottii was matig tolererant. Nie een van die P. radiata families wat getoets was het toleransie vlakke gehad wat vergelykbaar was met die toleransie vlakke van P. taeda of P. elliottii nie. In 2005 was F. circinatum vir die eerste keer geraporteer in die Tokai plantasie op volwasse dennebome in die Wes-Kaap Provinsie. Die rol van snoeiwonde in die infeksieproses van F. circinatum was bestudeer asook hoe vinning die siekte ontwikkel en versprei. Die hoeveelheid en tyd van sporulasie was ook bestudeer. “Pitch canker” het stadig toegeneem oor die 3-jaar moniteringsperiode en dit blyk asof snoeiwonde nie die primêre ingangspunt vir die patogeen is nie. Kwantifisering van die sporulasie van F. circinatum deur middel van qPCR het aangedui dat spore reg deur die jaar geproduseer word. Meer spore word geproduseer in die koue nat wintersmaande. Die rol van insekte in die infeksieproses en verspreiding van F. circinatum in die Tokai plantasie moet ondersoek word.