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
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.