Optimizing Dothistroma septosporum infection of Pinus radiata and the development of red-band disease : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North, New Zealand

The filamentous fungus Dothistroma septosporum infects pine species throughout the world causing red-band disease, one of the most serious diseases of conifer species. In NZ, a clonally derived asexual strain of D. septosporum was identified in 1964, and has spread throughout the country. There are conflicting accounts on the environmental conditions required for infection, which has lead to difficulties in optimizing a laboratory-based system for infection. The pathogen is spread naturally through rain-splashed inoculum of conidiospores from mature stromata that have erupted through the pine needle tissue. Diseased needles become necrotic, often with a red band due to the mycotoxin dothistromin produced by the hyphae. Dothistromin has the chemical structure of a difuranoanthraquinone and shows similarity to the aflatoxin precursor, versicolorin B produced by Aspergillus parasiticus. The role of dothistromin in pathogenicity has not yet been determined, although experiments have shown injecting toxin into pine needles results in the characteristic red band lesion. In this study it was found that fluctuating temperature (16°C/24°C), a 12 h diurnal cycle (white and ultraviolet light), high relative humidity and continuous moisture are conditions conducive to development of red-band disease on inoculated pine trees in an artificial environment. A higher rate of infection was obtained using pine seedlings as opposed to pine cuttings, and using a spore suspension containing a yeast extract. A dothistromin minus mutant was able to infect pine needles, indicating that dothistromin is not a pathogenicity factor, though it may be a virulence factor. The use of GFP-expressing isolates allowed the initial infection process to be monitored with both wild type and mutant isolates. Additionally, a PCR-based diagnostic procedure to confirm infection was developed. The production of aflatoxin by Aspergillus species is regulated by nutritional parameters and extracellular pH, which affect both growth and aflatoxin gene expression. D. septosporum similarly has enhanced growth at acidic pH, but it does not appear that pH has a strong influence on physiological processes as toxin biosynthesis and gene expression do not appear to be pH regulated. Different carbon and nitrogen sources also affect the morphology of D. septosporum

Early expression of aflatoxin-like dothistromin genes in the forest pathogen Dothistroma septosporum

The forest pathogen Dothistroma septosporum produces the polyketide dothistromin, a mycotoxin very similar in structure to versicolorin B, a precursor of aflatoxin (AF). Dothistromin is a broad-range toxin and possibly involved in red-band needle blight disease. As the role of dothistromin in the disease is unknown the expression of dothistromin genes was studied to reveal clues to its function. Although the genes of AF and dothistromin biosynthesis are very similar, this study revealed remarkable differences in the timing of their expression. Secondary metabolites, like AF, are usually produced during late exponential phase. Previously identified dothistromin genes, as well as a newly reported versicolorin B synthase gene, vbsA, showed high levels of expression during the onset of exponential growth. This unusual early expression was also seen in transformants containing a green fluorescent protein (GFP) gene regulated by a dothistromin gene promoter, where the highest GFP expression occurred in young mycelium. Two hypotheses for the biological role of dothistromin are proposed based on these results. The study of dothistromin genes will improve current knowledge about secondary metabolite genes, their putative biological roles, and their regulation