Insect bioactive capabilities of Epichloë festucae var lolii AR48 infected Lolium perenne: a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand

As the modern world expands and develops, new innovative methodologies for more efficient and environmentally friendly agricultural practices are required. Loss of crops through abiotic (e.g. drought) and biotic (e.g. herbivory) stresses has a major effect on the success of an agricultural industry. For animal production pasture crops are a key aspect of animal husbandry and directly affects yield and health. Symbiotic fungi belonging to the genus Epichloë form associations with cool season forage grasses and have been exploited as a new innovative method for insect pest management. Ryegrass infected with the asexual E. festucae var lolii strain AR48 has insect bioactivity against both the stem boring fly (SBF-Ceradontha australis) and cutworm moth caterpillar (CC -Agrotis ipsilion). The bioactive/s targeting both insects is currently unknown. The aim of this thesis was to identify the gene/s and/or bioactive/s present in AR48 infected ryegrass that have bioactivity against the SBF and/or CC. Two approaches were taken; the known insect bioactive secondary metabolite pathways in Epichloë were investigated in AR48 through bioinformatics and mass spectrometry, and the gene ‘makes caterpillars floppy’ (mcf), encoding an insect toxin like protein, was investigated through reverse genetics and insect bioactivity trials. A new indole diterpene compound (IDT) was identified in AR48 infected plant material and this compound was absent in other Epichloë strains that do not have SBF and CC bioactivity. The same mcf gene allele as that present in the E. typhina mcf model, previously identified as having CC bioactivity, is present and predicted to be functional in AR48. The other Epichloë strains also have mcf genes predicted to be functional, however the mcf allele is different to the bioactive E. typhina mcf model. Overall, this project was able to identify a new IDT compound with potential insect bioactivity as well as identify two Epichloë mcf gene alleles that potentially have differing insect bioactivities.