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      Bioactive Chemicals of Importance in Endophyte-Infected Grasses

      Babu, Jacob
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      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
      Permanent Research Commons link: https://hdl.handle.net/10289/2608
      Abstract
      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.
      Date
      2009
      Type
      Thesis
      Degree Name
      Doctor of Philosophy (PhD)
      Publisher
      The University of Waikato
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