Post-hail associated die-back of Pinus radiata is associated with the opportunistic pine pathogen Sphaeropsis sapinea. This disease has a serious impact on forestry; especially in regions such as South Africa where Pinus spp. are planted as exotics. Current management strategies are based on the selection of disease tolerant Pinus spp. and the planting of these pines in regions less affected by adverse environmental conditions. A possible management strategy for S. sapinea is biocontrol through dsRNA- mediated hypovirulence. Improvement of current management strategies and the implementation of biocontrol, however, require a re-evaluation of the taxonomic status of the pathogen and the assessment of genetic van ability in populations. Chapter One of this thesis reviews knowledge on S. sapinea in order to obtain the ultimate goal of formulating an integrated management strategy for S. sapinea, especially in South Africa and other regions dependent on exotic pines for commercial forestry. Successful management of S. sapinea in exotic forestry plantations has not, as yet, been fully achieved. This is probably due to a lack of knowledge of the current taxonomy of the fungus, the genetic status of S. sapinea populations and the association with dsRNA viral fragments that could be exploited for biocontrol. In Chapter Two, three morpho types (A, B and C) of S. sapinea are reported from diverse geographical areas. This also includes the discovery, for the first time, of a third morpho type of the fungus. Isolates of the C morphotype are characterised based on RAPD analysis and ITS sequences, as well as cultural and conidial characteristics. Isolates of the C morphotype were found to be more similar to those of the A morphotype based on cultural characteristics, texture of their conidial walls and ITS sequences. The three morphotypes are, however, clearly distinguishable based on their RAPD banding profiles and conidial size. Chapter Three represents the characterisation of the C morphotype of S. sapinea based on morphology and pathogenicity. Based on cultural and conidial characteristics, as well as growth rate the A and C morphotypes are very similar. Isolates of the C morphotype were, however, found to be much more virulent than either those of the A or B morphotype. This could have a great impact on the management of S. sapinea die¬back, as well as on quarantine practices. In Chapter Four, I present an assessment of the diversity for S. sapinea populations from various regions. The diversity in the different populations was found to be highly variable, despite the fact that only an asexual state is known for this fungus. The diversity in presumed native populations from Mexico, California and Switzerland was relative low. In the introduced Malagasy population the diversity was very high, probably due to many introductions of the fungus with seed imports. Due to the high variability of the fungus in the Malagasy population, management of S. sapinea from this region will be extremely difficult. In Chapter Five, dsRNA fragments of different sizes were commonly found to occur in isolates of S. sapinea. Two previously described dsRNA viruses (SsRVI and SsRV2) from South Africa were found to also occur in isolates from Madagascar and Colombia. It shows that these dsRNA fragments have been introduced with S. sapinea into new environments. The type and number of dsRNA fragments was found to have some effect on the growth rate and pathogenicity of the fungus. These. alterations in the normal phenotype of the fungus could, however, not be correlated to the presence of dsRNA fragments. The presence of dsRNA fragments does, however, still have the potential to be implemented in future management programmes for S. sapinea. / Dissertation (MSc)--University of Pretoria, 2006. / Microbiology and Plant Pathology / Unrestricted

Diplodia tip blight is an important disease of pines, especially Scots pine Christmas trees in Kentucky. The hypothesis for my thesis work was that D. pinea could be acquired and transmitted on the tools during annual shearing of the Christmas trees. Samples taken from tools after shearing on two different Christmas tree farms in Kentucky in 2005 and 2006 yielded D. pinea colony forming units, but in very low quantities; typically less than 10 CFUs per collection. Diplodia-associated dieback from the sheared tips was never found in the field, suggesting that transmission and subsequent infections were not occurring via these sheared tips. Controlled infections indicated that a minimum of 100 spores was necessary to create symptomatic infections on sheared tips. Lysolandamp;reg; Disinfectant Spray did not remove D. pinea from tools when sprayed on them after shearing, but it did effectively prevent spore germination in vitro. Observations of Diplodia lesion development on one Scots pine Christmas tree farm in Kentucky during the springs of 2006 and 2007 suggested that D. pinea infections occurred primarily via the bases of needle bundles on elongating shoots. The most likely source of inoculum was dead infested pine tissues within and beneath the canopy. The use of a protectant fungicide may have resulted in an observed dramatic decrease of disease on this farm.

Sphaeropsis sapinea é conhecido como um importante patógeno de várias espécies de Pinus, causando a seca de ponteiros e a morte de árvores em plantios comerciais. Esse patógeno foi introduzido no Brasil, provavelmente, durante as introduções do gênero Pinus. Seu primeiro relato ocorreu na década de 1940, com os primeiros plantios de P. radiata no estado de São Paulo, os quais foram dizimados. Um projeto de reintrodução dessa espécie florestal no Brasil foi delineado para a seleção de famílias de P. radiata resistentes a S. sapinea. Desse modo, este estudo objetivou a caracterização morfológica, molecular e patogênica de isolados de S. sapinea, para escolher os isolados mais agressivos para uso na seleção de material resistente. Quatro isolados da região Sul do Brasil foram obtidos e a caracterização morfológica e patogênica indicou que os isolados estudados pertencem ao morfotipo “A” de S. sapinea. Houve diferenças na agressividade e na diversidade genética dos isolados em todos os testes in vitro e in vivo. A maior herdabilidade de P. radiata para tamanho das lesões foi obtida com o isolado SS1.3 (H2 M = 0,3357870), enquanto que para secamento dos ponteiros a maior herdabilidade foi obtida com o isolado SS2.4 (H2 M = 0,256347). Os resultados mostraram a possibilidade de seleção precoce de material resistente à seca de ponteiros em mudas de P. radiata

Diplodia pinea causes Diplodia tip blight on more than 30 different pine species. During the past 10 years, Diplodia tip blight has emerged as a serious problem in landscape and Christmas tree farms in this region. Surveys of diseased and symptomless Austrian pines revealed that latent infections of symptomless shoots by D. pinea were common. Latent infections may account for the recently observed rapid decline of mildly diseased pines in our region. To investigate the colonization habits of D. pinea within its host, molecular cytology was attempted and traditional histology was performed on naturally infected, diseased and asymptomatic Austrian pine tissues. I devoted much effort to developing a transformation system for D. pinea. Ultimately I did not succeed in this goal, but I was able to develop a highly efficient protocol for Agrobacterium tumefaciens-mediated transformation of another pathogenic fungus, Colletotrichum graminicola, in the process. The work that I did should help in future efforts to transform D. pinea, something that will be essential if it is to become a tractable system for the study of fungal latency. Traditional histological methods were more successful, and provided important information about the nature of latent infections. Very sparse epiphytic and subcuticular fungal growth was observed in healthy shoots, however, no fungal tissues were present within the shoots. In diseased and latently infected shoots, crevices created between the needle bundles and the shoots were filled with fungal material, and hyphae were observed colonizing the needle sheaths. Hyphae were also observed breaching the shoot epidermal layer in these crevices and colonizing the underlying periderm. D. pinea colonization was extensive in all tissues of diseased shoots early in symptom development. In contrast, localized pockets of degradation were observed in the periderm and adjacent cortical cells located around areas of needle attachment in asymptomatic, latently infected shoots. The mechanism that operates to prevent expansion of these infected pockets in the latently infected shoots is still unclear. Obvious signs of pine defense mechanisms were only observed in 2 shoots. My observations were consistent with the idea that colonization progresses into the vascular tissues, and that this results in symptom development. Vascular colonization may occur more readily if the host is stressed. My research lays the groundwork for future efforts to understand the nature of the transformation from latent to pathogenic infection.