Heteronychus Arator) in Perennial Ryegrass (Lolium Perenne L.) Infected with AR1 Endophyte
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Resistance to African black beetle (Heteronychus arator) in perennial ryegrass (Lolium perenne L.) infected with AR1 endophyte A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Biological Sciences at The University of Waikato by Kathryn Mary Ross _________ The University of Waikato 2016 Abstract The insect pest, African black beetle (Heteronychus arator (Fabricus, 1775)) is of considerable economic cost to the New Zealand agricultural industry, in regions where the insect is established and regular outbreaks are now occurring. Selection for Epichloë festucae var. lolii (Latch, M.J. Chr. & Samuels) C. W. Bacon & Schardl, stat. nov. et comb. nov. endophyte-perennial ryegrass (Lolium perenne L.) associations which do not cause toxicity to livestock and with strong resistance to African black beetle (H. arator), would be of significant value to farmers in regions where this pest is a problem. Epichloë festucae var. lolii strain AR1 endophyte does not produce ergovaline, the alkaloid known to deter African black beetle (H. arator), yet pastures infected with this endophyte have moderate resistance to adult African black beetle (H. arator). Research into AR1 is of importance, as to date there have been no reports of toxicity in livestock brought about by consumption of from AR1-infected pastures and in the absence of African black beetle (H. arator) this ‘novel’ endophyte can provide highly productive pastures. AR1 endophyte produces secondary metabolites that are simple indole diterpenes, including paxilline and paxilline-like compounds. The numerous paxilline-like compounds produced by AR1 are detected by a paxilline ELISA (enzyme linked immunosorbant assay) and quantified collectively as paxilline immunoreactive equivalents. Earlier work suggested that these paxilline-like compounds could be the bioactive, or linked-marker associated with the bioactive compound(s), that provide some resistance to adult African black beetle (H. arator) feeding. The overall aim of this research was to determine if increased concentration of paxilline immunoreactivity was associated with a reduction in feeding damage using a series of adult African black beetle (H. arator) feeding trials on closely related AR1-infected perennial ryegrasses (L. perenne). A negative relationship was established between feeding damage from adult African black beetle (H. arator) and plant pseudostem paxilline immunoreactivity in AR1-infected perennial ryegrass (L. perenne) post exposure to beetles. However, it was not a simple relationship being complicated by: i) paxilline immunoreactivity iii not reflecting endophyte concentrations; ii) the influence of cultivar on the expression levels of paxilline immunoreactivity; iii) the effect of African black beetle (H. arator) presence on expression levels of paxilline immunoreactivity; and iv) the paxilline ELISA quantifying the complete complex of paxilline-like compounds and not simply those that are bioactive. The highest concentrations of paxilline immunoreactivity were found in plant undamaged pseudostem and were not strongly correlated with the lower concentrations found in herbage. Therefore undamaged pseudostem is recommended as the section sampled if the entire plant pseudostem was not available. Low levels of feeding from adult African black beetle (H. arator) accentuated plant tiller production. However, the negative effects of high feeding pressure still affected plants four weeks post-exposure to beetles, with reduced paxilline immunoreactivity production and plant tiller numbers. Adult African black beetle (H. arator) were able to compensate the deterrent effects of AR1 once an endophyte-free food source was available. Results from this research contribute towards further understanding the bioactivity of AR1 endophyte against adult African black beetle (H. arator) and will underpin further research into the chemical basis for this resistance. The paxilline ELISA could potentially be used for screening E. festucae endophyte ryegrass associations that produce unknown compounds which deter African black beetle (H. arator) and which do not cause toxicity to livestock. iv Acknowledgements When researching living and seasonal organisms, a part-time PhD does have its benefits. However, with a partner working out of town and a young child at home, it is a test of stamina and I wouldn’t have made it without the support of family, friends and work colleagues – thank you all. I wish to thank my supervisors; Alison Popay, Lyn Briggs, Peter Molan and Ian Hogg. Their guidance, encouragement and support were invaluable and much appreciated. I have learned a lot from each supervisor and appreciate the immense time and effort afforded to myself during my PhD. The research in this thesis would not have been possible without funding from the TR Ellett Trust, for which I am most appreciative. Many thanks to Errol Thom for the part he played in providing funding and for the encouragement he gave. I gratefully acknowledge the support of AgResearch Ltd throughout this project. In particular, my special thanks are extended to Heather Armour and my colleagues in the Toxinology group; Sarah Finch, Allan Hawkes, John Kerby, Jan Sprosen and Sangata Kaufononga for their wisdom and encouragement, and for showing a keen interest in my research. I’m very grateful to Colleen Podmore, Joan Fitzgerald and Derrick Wilson for helping with the experimental work. Many thanks to Joanne Jensen, Louise Hennessy and the rest of the Plant Protection crew for their kindness and making me part of their team. I thank David Hume, Wade Mace, Doug Ryan and Tom Lyons, of the Endophyte team, for the assistance and expertise they gave me. To the statistics team down the corridor, in particular Vanessa Cave, Catherine Cameron, Pip Maternaghan and Harold Henderson, thanks for the encouraging chats, support, de-stressing ten-pin bowling sessions, and allowing me to be an honorary statistician to come and eat cake. Special thanks to Vanessa Cave for statistical advice and for keeping me sane. Special thanks are due to PGGW Seeds and Grasslandz Technology for allowing the use of their seed lines. I’m grateful to the University of Waikato for allowing me the opportunity to study for a doctorate. I thank Cheryl Ward for her expertise and help formatting my thesis. v I would like to express my gratitude and thanks to the examiners who took the time to read and critically evaluate my thesis. Your time and effort is appreciated and your comments, corrections and suggestions were valuable, insightful and have improved the final version of my thesis. Thanks to my wonderful friends for their kindness and encouragement. Many thanks to both Kirstin Wurms and Tracy Wilde, for taking care of my son when I was busy with experimental work or in the school holidays, when dad was out of town. A big thank you to Karen Drummond for her great company, support, and allowing me to test her culinary and cheese making skills on many occasions. A big thank you to Clare Browne for her support in the final stages of this journey. Finally, many thanks to my wonderful family for all their love, patience and support they have provided me through this journey. Thank you Reimana, Kahurangi, my mum Carole, and my fur-babies; Marama, Te Po and Fatboy. vi Dedication This thesis and PhD is dedicated to: my late father, Gavin John Ross, who always believed in me to my mother Carole Shirley Ross, who gave her all to support me in this journey to my partner Reimana, we have struggled and battled through these tough times together, it has only made us stronger and closer to my beautiful son, Kahurangi for being so wise beyond your years, and for being so helpful, caring, thoughtful and understanding vii Table of Contents Abstract .............................................................................................................. iii Acknowledgements ............................................................................................. v Dedication ......................................................................................................... vii Table of Contents ............................................................................................... ix List of Figures ................................................................................................. xvii List of Tables.................................................................................................... xxi Abbreviations .................................................................................................. xxv Taxonomy ...................................................................................................... xxix Chapter 1: Introduction ......................................................................................