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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
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.