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Subject: search.filters.subject.Pinus radiata -- Diseases and pests

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    Further characterization of Dothistromin genes in the fungal forest pathogen Dothistroma septosporum : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Science in Molecular Genetics at Massey University, Palmerston North, New Zealand
    (Massey University, 2005) Jin, Hong Ping
    Dothistroma septosporum is a forest pathogen that causes a disease called Dothistroma needle blight. The symptoms are thought to be due to the accumulation of dothistromin toxin produced by D. septosporum. Dothistromin is characterized as a difuranoanthraquinone and shows remarkable similarity to the aflatoxin (AF) and sterigmatocystin (ST) precursor versicolorin B. The similar structure to AF/ST suggests that dothistromin biosynthesis shares biosynthetic steps with the AF/ST pathway. The AF gene cluster in Aspergillus parasiticus and ST gene cluster in A. nidulans have been well characterized. Nine putative dothistromin biosynthetic genes have been identified. One of them, dotA was previously characterized by gene disruption and shown to have a similar function to homologous genes in AF/ST biosynthesis. Two additional putative dothistromin biosynthetic genes, pksA and epoA, were characterized by gene disruption in this study. The inability of the pksA mutants to produce dothistromin indicated that the pksA is a key gene in dothistromin biosynthesis. The feeding of intermediates confirmed that pksA gene product is required for a very early step of dothistromin biosynthesis. The pksA mutants also showed reduced sporulation compared to wildtype, suggesting a relationship between dothistromin production and sporulation. The epoA gene replacements were also obtained successfully by homologous recombination. Both Southern blot and northern hybridization confirmed that the epoA gene was disrupted. However, the epoA mutants did not show any difference to the wild type in three analyses (growth rate, sporulation rate, dothistromin biosynthesis). However it was not possible to rule out a role for EpoA at a very late stage of dothistromin biosynthesis. RACE analysis of the nine identified dothistromin genes characterized the transcription start and stop sites of the genes. Analyzing the putative regulatory protein binding motifs in the untranscribed region of the genes provided clues about the regulation of dothistromin biosynthesis and suggested there might be an aflR-like gene that governs dothistromin biosynthesis. Both the pksA gene disruption and the RACE results suggested that the dothistromin biosynthetic pathway is homologous to that of AF/ST biosynthesis. Further work on the dothistromin gene cluster will help us to understand the evolution of fungal toxin gene clusters.
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    Further studies of dothistromin toxin genes in the fungal forest pathogen Dothistroma septosporum : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand
    (Massey University, 2007) Feng, Feng, Zhilun
    The fungal pathogen Dothistroma septosporum is the main causal agent of Dothistroma (red-hand) needle blight, which is a devastating foliar disease of a wide range of pine species. Dothistromin is a difuranoanthraquinone toxin produced by D. septosporum and is considered as a possible virulence factor for the disease. Based on the similarity of chemical structure between dothistromin and aflatoxin (AF) /sterigmatocystin (ST) precursors, nine putative dothistromin biosynthetic genes have been identified, which are homologous to their corresponding genes in the AF/ST gene clusters. However, in contrast to all 25 AF biosynthetic genes tightly clustered in one region (70-Kb) of the genome, the dothistromin gene clusters are located on a 1.3-Mb chromosome and separated into three mini-clusters along with non-dothistromin genes. The dotC gene, located in the mini-cluster 1, is predicted to encode a major facilitator superfamily (MFS) membrane transporter involved in secretion of dothistromin. In this work, by constructing DotC-eGFP fusion protein containing mutants, the subcellular localization of the DotC protein was determined to be mainly targeted to the plasma membrane. The biological function of the dotC gene was characterized by targeted gene disruption. The dotC gene disrupted mutants showed a significant reduction of dothistromin production in both the medium and mycelium. In addition, the exponential growth of dotC null mutants was inhibited when exogenous dothistromin was presented and these mutants also displayed more sensitivity than the wild type strain to exogenous dothistromin. The results indicated that the DotC protein is a membrane associated protein and might have a role in dothistromin production and be involved in secretion of exogenously supplied dothistromin toxin. Two novel dothistromin biosynthetic genes, norA/B and verB (partial sequence), were identified by using degenerate PCR and D. septosporum genomic library screening. The putative NorA/B and VerB are postulated to encode a dehydrogenase and a desaturase, respectively and are similar to AF/ST genes. These findings further confirmed that the dothistromin shares biosynthetic pathway steps with AF/ST.
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    Characterisation of a global collection of Dothistroma pini isolates : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular Biology at Massey University, Palmerston North, New Zealand
    (Massey University, 2000) Ganley, Rebecca Jayne
    Dothistroma pini is a filamentous fungus which infects Pinus radiata, New Zealand's predominant forest species. Dothistroma blight causes premature defoliation, a reduction in the rate of growth and, in extreme cases, death of the trees. This forest pathogen produces a toxin, dothistromin, which is implicated in the development of the disease symptoms. Only one strain of D. pini is thought to be present in New Zealand. However, world-wide there is a diverse range including the sexual form. A collection of D. pini strains from eight countries was collated in the UK. To prevent further introductions of 'foreign' D. pini to New Zealand and to assist in the identification and appropriate containment, should a new outbreak of needle blight occur, the D. pini isolates in this collection were characterised at both the species and individual strain level. Sequence analysis of the ribosomal internal transcribed spacer (ITS) and the production of dothistromin by the isolates in the collection confirmed all were D. pini. Quantification of the levels of dothistromin produced by the isolates, in culture, showed a large variation between the strains. Isolates MINI11, NEB8, GUA1 and, in particular, ALP3 produced significantly more dothistromin than NZE1. Changes in culture environment and media types were shown to affect the levels of dothistromin produced by the D. pini isolates. However, these changes were not sufficient to support the production of aflatoxin. To analyse the genetic diversity among the overseas D. pini isolates, a robust microsatellite-based DNA fingerprinting system was developed. Microsatellite loci were isolated. Primers designed to flank the microsatellite repeats were used for PCR amplification in the 'core' twelve D. pini strains. The unique fingerprint patterns obtained from these loci were used to distinguish the isolates to the individual strain level. This system of identification provides an effective tool for screening and prognosis of infected pine forest sites.