Neotyphodium unicinatum: Potential for improvement : A dissertation submitted in partial fulfilment of the requirements for the Degree of Bachelor of Agriculture and Science with Honours at Lincoln University

Abstract

Epichloe endophytes colonize cool-season grasses of the sub-family Pooideae, and comprise of sexual Epichloe species, and their asexual anamorphs, the Neotyphodium species.Notoriously, they synthesise a range of bioprotective alkaloids which provides their host withconsiderable protection against both insect and mammalian herbivory. Significantly, manyNeotyphodium species are hybrids of multiple Epichloe complexes, and resultingly haveaccumulated much larger genomes with repeated copies of gene loci. Consequently,Neotyphodium species have attained multiple secondary metabolite pathways and hence arecapable of producing a range of alkaloids which provide significant benefits to the symbiotum. Neotyphodium uncinatum is one such hybrid that has attained considerable attention in both the laboratory and in the agricultural community over the past few years because of its ability to synthesise high levels of the potent anti-insect compound lo line. However, highly contrary to other hybrid endophytes, no other detectable levels of alkaloids have been found in N uncinatum infected hosts. Peramine, another anti-insect compound, also has particularly high importance to the agricultural community and has been found to be conserved amongst many Epichloe and Neotyphodium complexes, however yet to be found in N uncinatum. Potentially, such endophytes which could synthesise high levels of both anti-sect compounds could provide superior protection to valuable grass species. Considering the recent sequencing of the gene responsible for peramine biosynthesis, this study investigated the peramine gene within N uncinatum and looked at approaches to restore its peramine production for commercial prospects. This study found multiple alleles of the gene (per A) responsible for peramine production in other endophytes, in a N uncinatum strain termed U2. Sequence analysis of the first 7 .8 kb of the 8.5 kb coding sequenced showed there to be no mutations which should account for the negative peramine phenotype observed in N uncinatum. However a number of novel insertions and deletions were found that were scattered through the gene. The major contributing factor to the negative phenotype of peramine in N uncinatum seems to be the loss of expression of the per A gene which catalyses all the steps in the biosynthesis of peramme. Approaches at restoring peramme production in N uncinatum were investigated, with protoplast fusion the being the most desirable considering the projects commercial objectives. Due to unsuccessful protoplast isolation attempts and the limited time frame of the project, protoplast fusion was not feasible. However, molecular marker tools were created in this study for future protoplast fusion studies