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Bioactive Chemicals of Importance in Endophyte-Infected Grasses

Janthitrems are believed to be involved in the observed sporadic cases of AR37- infected perennial ryegrass staggers. Investigations into the role of janthitrems in perennial ryegrass staggers are difficult as isolation of the compounds from the ryegrass is hindered by the inherent instability of these compounds. Therefore attempts were made to isolate janthitrems from an alternative source, allowing these janthitrem analogues to be used as surrogates for endophyte produced janthitrems. Analysis of a series of Penicillium janthinellum cultures revealed the presence of janthitrems in a number of strains, including janthitrem B, janthitrem C and two novel janthitrem compounds. Detailed one- and two-dimensional NMR and mass spectral techniques identified the two novel compounds as 11,12- epoxyjanthitrems B and C, which were subsequently given the trivial names janthitrems A and D, respectively. Janthitrems B and C were isolated and identified by NMR and revisions of some previously reported chemical shift assignments were proposed. In addition to the janthitrems, penitrems were also identified in two strains of P. janthinellum. The isolated janthitrem B was utilised for the development of efficient extraction procedures, and for the determination of ideal storage conditions for janthitrem compounds. A method for the extraction and isolation of janthitrem B from a P. janthinellum culture was developed and optimised to yield 6 mg of janthitrem B from 900 mL of fungal culture in two days. Stability studies of janthitrem B indicated the ideal storage condition which minimised degradation was dry at −80 C where only 7% sample loss was observed over 300 days. Bioactivity studies of janthitrems A and B found these compounds to be tremorgenic to mice, with janthitrem A (an epoxyjanthitrem) inducing more severe tremors than janthitrem B. Insect testing also showed that both janthitrems A and B displayed anti-insect activity to porina larvae. Since the epoxyjanthitrems, which are associated with AR37 endophyte-infected ryegrass, were also shown to be tremorgenic and to display anti-insect activity, the insect resistance and the sporadic cases of ryegrass staggers displayed by AR37 may be related to the presence of epoxyjanthitrem compounds. LC-UV-MS analysis of janthitrems A-D, penitrems A-F, lolitrem B, paspalinine, paxilline and terpendole C found these indole-diterpenoids to be more sensitive by analysis using an APCI source as opposed to an ESI source. APCI negative ion LC-UV-MS required source induced dissociation in combination with increased collision energy to suppress an acetate adduct peak, sourced from the acetic acid buffer. Negative ion MS2 and MS3 data produced more informative fragments compared to the conventional positive ion MS2 and MS3 data. The availability of both positive and negative ion LC-UV-MS methodologies will allow future endophyte products to be more thoroughly screened for different classes of secondary metabolites. Extracts of mouldy walnuts were analysed for the presence of tremorgenic mycotoxins after a dog was found to exhibit symptoms characteristic of tremorgenic mycotoxicosis. LC-UV-MS analysis of the mouldy walnuts identified the tremorgenic mycotoxins penitrems A-F, thus confirming the veterinarian's tentative diagnosis of canine tremorgenic mycotoxicosis the first reported case in New Zealand.
Type of thesis
Babu, J. (2009). Bioactive Chemicals of Importance in Endophyte-Infected Grasses (Thesis, Doctor of Philosophy (PhD)). The University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/2608
The University of Waikato
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