Current Role: Research Scientist with Agriculture Victoria

PhD title: Development of screening assays for determining toxicity of grass-associated fungal endophytes

Perennial ryegrass is the major forage in south east Australia and New Zealand grazing systems. Genetic improvement of perennial ryegrass has led to elite cultivars, that have greatly improved persistence, these improved cultivars are then combined with asexual Epichloë endophyte strains as the endophyte provides both abiotic and biotic stress tolerances that enhance host plant persistence. However, endophyte-infected perennial ryegrass is capable of producing secondary metabolites that are detrimental to grazing animals. The indole diterpenoid mycotoxin lolitrem B is found in endophyte-infected perennial ryegrass and is present in around 90% of ryegrass pastures. Ingestion causes a neurological syndrome in grazing livestock called “ryegrass staggers disease” where clinical signs include hyperexcitability, muscle tremors, ataxia (“staggers”) and, in severe cases, clonic seizures and death.Endophytes are selected for progression into a breeding program based on their toxin production profiles. However, the negative effect of the grass-endophyte associations (e.g. animal toxicity) are generally not tested until very late in the breeding program. Furthermore, endophytes that do not produce these known toxins can still produce intermediates in the biosynthetic pathway that can also cause animal toxicity. Some of these intermediates are known toxins but the degree of toxicity is largely undefined.

To better characterise toxicity, I isolated and purified lolitrem B and several intermediates from endophyte-infected perennial ryegrass using large scale extraction and isolation techniques. Using a time-series analysis, the effects on movement and tremor in dosed mice were quantified for lolitrem B and terpendoles B, C and E. Animals exposed to lolitrem B showed significant and prolonged tremors and decreased stability during accelerated rotarod testing. Terpendole C induced severe short-term tremors, whereas terpendole B and E were inactive.

To determine metabolic effects of the major toxin lolitrem B on the central nervous system, mice were exposed with lolitrem B at two key time points reflecting peak and post-peak tremors. A targeted approach was employed to determine the distribution of lolitrem B in the body and brain regions of toxin exposed mice and also to analyse the metabolic variation elicited in the brain. At peak tremor high levels of lolitrem B was found in the cerebral cortex and although all brain regions show metabolic perturbation, the cerebral cortex showed the most profound effect with respect to activation of amino acids and neurotransmitters.

Characterisation of clinical signs and metabolic changes associated with exposure to indole diterpenoid toxins present in perennial ryegrass have provided useful information for generation of animal safe grass varieties for the dairy and meat industries.

Science outcomes:

The research conducted resulted in a unique body of work that demonstrated the use of behavioural assays and metabolomics to assess compounds produced by endophytes. The distribution of the toxin in the body and brain as well as its effects on metabolism are the first to be conducted in the field.

Behavioural platforms coupled with metabolic profiling provide a sensitive approach for detecting sub-clinical toxicosis in animals. This is a new concept in assessing naturally occurring toxins in a rodent model.

Three journal articles from my PhD have been accepted for publication in reputable journals. I am also currently drafting two more journal publications that are associated with my PhD.

Industry impact:

This study provides recommendations to the plant breeding program, on the safety of novel endophytes and the optimum levels of possible bioprotective compounds in pasture. This will enable the delivery of safe, persistent and robust pasture for the dairy and meat industries.

Education program:

The Dairy Futures CRC and DairyBio education program provided opportunities for personal and professional development that enabled me to participate in competitions such as the CRCA (Co-operative Research Centre Association) where I was one of five finalists from across Australia to present at the Australia 2040 Symposium.

Particular highlights were attendance at international conferences, training in intellectual property, and learning about the broader science happening within the DFCRC from other PhD students. Also with the support of the Dairy Futures CRC and DairyBio I was able to present my research at the AOMSC (Asia Oceania Mass Spectrometry Conference) in Singapore. The mentoring program in the DFCRC also provided me the skills to network with research leaders and peers in the field.