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    Regulation of dothistromin toxin biosynthesis by the pine needle pathogen Dothistroma septosporum : a thesis presented in the partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatu, New Zealand
    (Massey University, 2014) Chettri, Pranav
    Dothistromin is a virulence factor produced by the fungal pine needle pathogen Dothistroma septosporum. It is similar in structure to a precursor of aflatoxin and sterigmatocystin. Unlike most secondary metabolite genes in fungi, the genes for dothistromin biosynthesis are not clustered but spread over six loci on one chromosome. Another characteristic feature of dothistromin synthesis is that dothistromin is produced mainly during the early exponential growth phase in culture. These unusual features have been proposed to be adaptations for the biological role of dothistromin in the disease process. It was therefore of interest to determine whether the regulation of dothistromin production in D. septosporum differs from the regulation of aflatoxin and sterigmatocystin in Aspergillus spp. and to address the question of whether genes in a fragmented cluster can be co-regulated. The availability of the D. septosporum genome facilitated identification of orthologs of the aflatoxin pathway regulatory genes aflR, aflJ and the global regulatory genes veA and laeA. These genes were functionally characterised by knockout and complementation assays and the effects of these mutations on the expression of dothistromin genes and the production of dothistromin were assessed. Inactivation of the DsAflR gene (?DsAflR) resulted in a 104 fold reduction in dothistromin production, but some dothistromin was still made. This contrasted with ?AflR mutants in Aspergillus species that produced no aflatoxin. Expression patterns in ?DsAflR mutants helped to predict the complete set of genes involved in dothistromin biosynthesis. AflJ was proposed to act as a transcriptional co-activator of AflR in Aspergillus spp. Disruption of DsAflJ resulted in a significant decrease in dothistromin production and dothistromin gene expression. Interestingly the expression of DsAflR was not affected by deleting DsAflJ, while conversely DsAflJ transcript levels increased significantly in a DsAflR mutant compared to the wild type. Heterologous complementation with A. parasiticus, A. nidulans and C. fulvum AflJ failed to revert the dothistromin level to wild type suggesting species-specific function of AflJ. VeA is an important regulator of secondary metabolism and development in fungi. Inactivation of the D. septosporum ortholog (DsVeA) resulted in reduced dothistromin production and showed the influence of DsVeA on the expression of other secondary metabolite backbone genes. Asexual sporulation was reduced but mutants were not compromised in pathogenicity. Overall, D. septosporum DsVeA showed functional conservation of the usual role in fungi. LaeA is a global regulator of secondary metabolism and morphogenetic development, first identified in Aspergillus nidulans. Unexpectedly, DsLaeA exhibited an unusual repressive function on the dothistromin pathway and DsLaeA mutants exhibited an extended period of dothistromin production compare to WT in vitro. The mutation of DsLaeA showed varied responses in expression of other secondary metabolite genes and had differences in sporulation and hydrophobicity compared to the wild type.
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    Investigation of dothistroma needle blight development on Pinus radiata : a thesis presented in the partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Microbiology and Genetics at Massey University, Manawatu, New Zealand
    (Massey University, 2014) Kabir, Md Shahjahan
    Dothistroma needle blight (DNB), caused by the fungi Dothistroma septosporum and Dothistroma pini, is an important foliar disease of pine species throughout the world and predictions of the future spread of this disease have been made using climate models. Although DNB infection is prevalent in many forests, attempts to achieve infection under controlled laboratory or glasshouse conditions are notoriously difficult. However, artificial infection is a very important tool for studying different aspects of plant-microbe interactions, such as pathogen life style and roles of virulence factors. D. septosporum was thought to have a hemi-biotrophic life style but this was not formally investigated in planta. The non-host selective toxin dothistromin produced by this fungus was shown not to be essential for pathogenicity but its role in pathogen virulence was unknown. The aims of this study were to improve the DNB pathogenicity assay and to use this system to test the hypotheses that D. septosporum is a hemi-biotrophic pathogen and that dothistromin plays a role in virulence. A new sporulation medium (pine needle medium with glucose) was used to obtain sufficient viable D. septosporum spores. The critical microclimatic component of leaf wetness was optimised to have a short (4-7 d) high wetness period followed by 'medium' wetness (continual misting), and using these conditions >80% needle infection was routinely achieved on Pinus radiata seedlings. A combination of microscopy, biochemical and molecular studies over a timecourse of infection of P. radiata by D. septosporum confirmed its hemi-biotrophic life style. Restricted mesophyll colonisation, shorter lesions and fewer spores from P. radiata needles infected with dothistromin-deficient mutants, compared to those with wild type D. septosporum, suggested that dothistromin has a role in virulence. Interestingly ‘green islands’ in which chlorophyll levels were maintained at higher levels than adjacent chlorotic and necrotic regions, surrounded early-appearing lesions caused by both wild-type and mutant isolates. At a later developmental stage of the lesion the green islands were still present in the mutant but appeared to be masked by the extended dothistromin-containing lesions in the wild type, which lead to the hypothesis that chloroplasts could be a site of action of dothistromin. The discovery that dothistromin is a virulence factor opens up new insights into the Dothistroma-pine interaction. This fundamental finding will be useful for management strategies for this important disease in the future.
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    Mode of action of dothistromin : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Massey University, New Zealand
    (Massey University, 1974) Harvey, A M
    Dothistromin is a bright orange-red pigment produced by the pine-needle pathogen Dothistroma pini Hulbary, the causal agent of a necrotic disease known as dothistromal blight. This compound, implicated as a fungal toxin, has been isolated both from laboratory cultures of D. pini and from infected Pinus radiata foliage. Detailed chemical investigation by Gallagher (1971) showed that dothistromin is a tri -α- hydroxyanthraquinone fused to a substituted tetrahydrobifuran ring system. The bifuran ring moiety is incorporated in other fungal metabolites including the extremely toxic and carcinogenic aflatoxin compounds. There is an increasing body of evidence to suggest that these fungal metabolites share a common biosynthetic origin. Bioassay has demonstrated the toxicity of dothistromin to the unicellular green alga, Chlorella pyrenoidosa, and to tissue cultures of P.attexuata. The very low level of solubility of the compound in aqueous solutions has precluded bioassay using pine seedlings. This thesis reports an investigation of the biochemical changes induced by dothistromin in microbiological systems. In the course of this investigation dothistromin has been shown to be toxic to a range of microorganisms in addition to its known toxicity to Chlorella pyrenoidosa. These studies have suggested possible ways of increasing the sensitivity of bioassays for dothistromin. It was found that the addition of dothistromin to liquid cultures of Chlorella as an ethyl acetate solution caused reproducible levels of inhibition, provided that the ethyl acetate concentration was less than 0.5% Batch culture techniques were used to establish the levels of dothistromin required for inhibition of growth of Chlorella. The ratio of dothistromin concentration to cell number was found to be an important factor in the inhibition response. Utilization of synchronous culture techniques permitted the study of biochemical changes induced by dothistromin throughout the cell cycle of Chlorella. Results showed a marked inhibition of the rate of increase of total protein and RNA over the cell cycle with no significant alteration of the rate of DNA increase. A dose-response curve for dothistromin inhibition of growth of Chlorella was established and a more detailed investigation of the action of dothistromin in inhibiting growth was undertaken using radioactive isotopes. By this means it was shown that 3H-uridine and 14C-phenylalanine incorporation into cell material is inhibited within 30 mins of exposure to the toxin. Difficulties encountered in attempts to obtain satisfactory incorporation of label into Chlorella DNA-fractions prevented further investigation of the effect of dothistromin on DNA synthesis in this organism. This led to the investigation of other microorganisms as more suitable experimental systems for this study. Bacillus megaterium KM, proved to be very sensitive to dothistromin and showed rapid incorporation of radioactive isotopes into protein and nucleic acid fractions. Growth curves established that the inhibitory ratios of dothistromin concentration to cell numbers for this organism were in the order of 0.25 μg/cell X 108 (as compared to 2.0 μg/cell X 108 for Chlorella). At this concentration, over the 30 min time course studied, dothistromin had no effect on the incorporation of 3H-thymidine into the DNA fraction. Inhibition of 3H-uridine incorporation was evident at 6 min and very marked by 10 min while the inhibition of 14C-phenylalanine incorporation into protein was not evident until considerably later. The effects of dothistromin in this system were compared with those of antibiotics with known sites of action. Dothistromin inhibition of 3H-uridine incorporation has a similar time course to that shown by actinomycin D, although marked inhibition by actinomycin D is evident at 3 min, whereas dothistromin inhibition is not noticeable until 6 min. On the basis of these results it is suggested that dothistromin interferes with RNA synthesis and that the observed inhibition of protein synthesis is a secondary effect of this impairment. Confirmation of dothistromin action 'in situ'by administration of the compound to pine seedlings is necessary before any definitive statement can be made concerning its role in dothistromal blight. However these results indicate the possible importance of dothistromin in pathology of dothistromal needle blight of pines. Impairment of the RNA synthetic capacity in pine needle tissue by the toxin could rapidly lead to cell death and to the necrosis of needle tissue observed in diseased foliage.
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    The biosynthesis of dothistromin : a thesis presented to Massey University in partial fulfilment of the requirements for the degree of Doctor of Philosophy
    (Massey University, 1975) Shaw, G John
    This thesis is concerned with the biosynthesis of dothistromin (2,3,3a,12a tetrahydro-2,3a,4,6,9 pentahydroxy-anthra[2,3-b]furo[3,2-d]furan-5,10 dione) by the fungus Dothistroma pini. The biosynthesis of related secondary metabolites is reviewed and as a working hypothesis it is proposed that dothistromin is solely acetate-derived. In the preliminary phases of the investigation strains of the organism giving high yields of the metabolite were sought and isolated from natural sources. Some growth media were tested for their ability to support growth, promote sporogenesis and sustain high yields of dothistromin. A medium containing malt and dried whole yeast was chosen. The growth characteristics of the organism in this medium were studied and the temporal relationship between growth and pigment production for a variety of cultural conditions was found. The findings of these experiments suggested times when it would be favourable to add possible precursors. Incorporation studies with [1-14C]-sodium acetate revealed that dothistromin incorporated isotope from this precursor and disclosed/that the lipids heavily incorporated the label. Subsequent experiments were concerned with examining the effects that precursor concentration, time of precursor addition and time of metabolite harvesting had on the isotope enrichment and yield of dothistromin. It was found that the optimisation of these two parameters were mutually exclusive processes and compromise conditions which were compatible with obtaining both reasonably good enrichments and yields of dothistromin had to be selected. Initially attempts were made to determine the distribution of isotope in dothistromin, which had incorporated isotopically labelled acetate, by chemical degradation. Potassium tertiary-butoxide/water cleavage of the anthraquinone ring of the pentamethyl derivative of dothistromin labelled by [1-14C]-acetate yielded 1,4-dimethoxybenzene which had a molar specific radioactivity that was 0.33 times that of the starting material. This finding was consistent with the formation of dothistromin from nine molecules of acetate. Subsequently 13C-NMR techniques were used to determine the distribution of isotope in dothistromin derived from [1-13C] and [2-13C]-acetates. Pulsed Fourier transform 13C-NMR spectra of the monoethyl acetal derivative of dothistromin were obtained using broad-band proton decoupling and off-resonance proton decoupling. By comparison with the 13C-NMR spectra of a number of model compounds the resonances in the spectrum of the dothistromin derivative were assigned in most cases to specific carbon atoms and in a few instances to two or three alternatives. The 13C-NMR spectra of the dothistromin derivatives which had been enriched by isotope from the carbon-13 labelled acetates showed nine resonances with intensities enhanced by enrichment from the carboxyl carbon of acetate, eight from the methyl carbon and one resonance of uncertain origin. The distribution of isotope in the anthraquinone moiety of the molecule was consistent with its formation from a polyketide precursor but this was not proven because of the equivocal assignment of some of the NMR signals. The distribution of isotope in the furo-furan ring moiety and its relation to the distribution in the anthraquinone part was the same as that reported by others for corresponding structures in aflatoxin B1, and sterigmatocystin.
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    Development of methods allowing correlation of Dothistroma and Dothistromin in planta : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Manawatu, New Zealand
    (Massey University, 2010) Owen, Timothy J.
    Dothistroma septosporum is a fungal pathogen of pines with a worldwide distribution. It is responsible for the disease red band needle blight, in which necrotic lesions appear on infected needles. The red colour of the disease is due to the presence of the mycotoxin dothistromin. This toxin is structurally related to the better characterised mycotoxins aflatoxin and sterigmatocystin. The function of these toxins is unknown, but dothistromin is hypothesised to act as a competition factor. While much work has been done on D. septosporum and dothistromin in broth culture, in planta work has been limited by the methods available. This work focused first on the development of a method for the reliable and high yield extraction of DNA from infected lesions, as previously used methods were found to be inadequate. It was found that the addition of an enzyme lysis step to the Qiagen DNeasy protocol and the replacement of its column purification with chloroform purification gave a greatly increased yield of DNA with an acceptable loss of purity. To allow quantification of dothistromin from the same lesion samples, previously used assay systems were optimised and compared in their accuracy and sensitivity. An HPLC-fluorescence method was found most effective, and was able to accurately quantify dothistromin at single lesion quantities. The developed methods were used to give a correlation between Dothistroma biomass and dothistromin in lesions at various stages of development. While this correlation was not found to be statistically significant, continuation of this work should allow valid conclusions to be drawn. To give insight into the evolution of dothistromin biosynthesis, the genomes of other dothideomycetes were examined for the presence of dothistromin biosynthesis gene homologs. While no homologs were conclusively identified, a number of genes were shown to have similarity to known toxin biosynthesis genes. In summary, while not all research hypotheses were able to be proven or disproven, this work sets a firm basis for future investigation in these areas using the methods developed, and strongly suggests the direction continued study should take.