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.