Mimosa pigra dieback in the Northern Territory, Australia: Investigation into possible causes Aniline D. Sacdalan Master of Science in Biology A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2015 School of Agriculture and Food Sciences Abstract In this study, Mimosa pigra dieback is described as plants or population of plants which appeared to suffer from ill health, death of their growing points and the emergence of green reshoots coming from the base. Dieback is an important phenomenon, not only in many crops and natural forests worldwide, but also in many invasive trees and shrubs in Australia. The dieback phenomenon is thought to be caused by complex and interacting factors and causes are often difficult to disentangle. In some cases it is a result of an interaction between both biotic and abiotic factors. Since 1988, dieback has been widely observed in Mimosa pigra L. (Fabaceae), a woody shrub that is one of the most serious exotic invaders of floodplains and riparian areas in the Top End of the Northern Territory. However, there has been no conclusive evidence of links between M. pigra dieback and specific causal factors. In addition, the dieback phenomenon has not been previously quantified. This thesis aimed to describe M. pigra dieback phenomenon at plant and population levels and its effect on plant growth, survival and demography. Specifically, the aim of this thesis was to evaluate the role of biotic factors (insect borers and fungal pathogens), and abiotic factors (soil pH and salinity), in relation to the development of M. pigra dieback. In addition, the thesis examines the pathogenicity of the fungal endophytes/pathogens associated with dieback- affected and healthy M. pigra. M. pigra has been a focus of biological control programs for more than 30 years, but it still remains a serious threat in the wetlands and conservation areas in the Top End of the Northern Territory. While extensive studies have been conducted to evaluate the impact of biological control programs, little is actually known about dieback and its causal factors. To evaluate the causal factors of M. pigra dieback, repeated ground surveys, plant health assessment and specimen collection of stems and soils, were conducted in the period 2011 to 2013 in established M. pigra sites on Melaleuca Station, Mary River, Northern Territory. Insect borers were quantified and putative fungal pathogen abundance were surveyed. A combined morphological and phylogenetic approach was used for fungal pathogens/endophyte identification. Using a subset of isolates, trials were performed to examine differences in the pathogenicity of the recovered fungal pathogens/endophytes. Results of field research showed that dieback in M. pigra is highly dynamic and fluctuates in its severity. Different sites showed increases and/or decreases in plant health over time. ii Dieback affects plants in all age classes, from seedlings, juveniles to adult plants, resulting in partial mortality of plants in all age categories over the trial period. Contrary to expectations, none of the biotic and abiotic factors examined showed a direct relationship to the changing population dynamics of M. pigra dieback over time. Nevertheless, fungal isolations demonstrated that M. pigra is a host to a diverse range of endophytes, with the most frequently isolated taxa belonging to the Botryosphaeriales, Xylariales and Diaporthales. Results of the fungal identification studies showed that M. pigra harbours diverse endophyte communities. The use of morphological characters was crude and unreliable. However, species identification using the ITS sequence data was also insufficiently discriminatory while inclusion of sequence data based on EF1-α allowed the discrimination between sibling species, especially among the confusing taxa within the Botryosphaeriaceae. The pathogenicity trials undertaken revealed the potential of fungal species from Botryosphaeriaceae to kill seedlings and cause lesion development in juvenile and adult plants in both controlled and field conditions. Overall, the research findings highlight the challenges in attributing a single factor as being responsible for the dieback phenomenon. This is the first study to investigate M. pigra dieback and the results reveal that M. pigra dieback has potential dramatic plant population-level effects. The results also provide evidence of the ubiquity of the phytophagous insects and fungal endophytes across all sites and seasons. Furthermore, among the 1,352 fungal isolations, it is likely that new fungal species will be identified. However, a number of factors were able to be ruled out in relation to potential causes of M. pigra dieback. Continued efforts are needed to focus on interaction studies of biotic and abiotic factors in relation to M. pigra dieback. The fact that population (stand) dieback in M. pigra occurs in multiple catchments in the Northern Territory, and over large areas, makes the dieback phenomenon to be of considerable ecological importance. iii Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. iv Publications during candidature Sacdalan, A., Galea, V., Goulter, K., Elliott, L., and van Klinken R.D. (2012).Preliminary investigations of the Mimosa pigra dieback phenomenon. In: Valerie Eldershaw, Proceedings of the 18th Australasian Weeds Conference. 18th Australasian Weeds Conference 2012, Melbourne Australia, (356-358). 8-11 October 2012. Sacdalan, A., Galea, V., Goulter, K., Elliott, L., and van Klinken R.D (2013). Dieback- affected and healthy Mimosa pigra stands have similar endophyte communities in the Northern Territory, Australia. In: Abstract and Programme Book: 19th Australasian Plant Pathology Conference, Auckland, New Zealand. Publications included in this thesis No publications included. Contributions by others to the thesis Galea, V. J (Thesis Principal Supervisor) for the experimental design, guidance and critical revision of the whole thesis. Goulter, K.C (Thesis Advisor) for the guidance, training and support in my laboratory, glasshouse and sequencing work and critical revision of the whole thesis. van Klinken, R. D (Thesis Advisor) for the guidance in the logic, set-up, data entry, data analysis and writing of my field ecology chapter and for the critical revision of the whole thesis. Lukitsch, B. (NT-DLRM) our dieback manager, for the organisation of fieldwork logistics from permit acquisition to field data collection. Elliott, L. (NT-DLRM) for the assistance in the logic of the thesis, suggestions for the field design and data collection. Bissett, A. (CSIRO) for the molecular sequencing identification of the first batch of mimosa isolates. v Statement of parts of the thesis submitted to qualify for the award of another degree None. vi Acknowledgements First and foremost, I thank my supervisors: Associate Professor Vic Galea, Dr Ken Goulter and Dr Rieks van Klinken. This work would have not been realised without their guidance and constant support. I truly appreciate that they had encouraged me to do my best and for being very understanding when it mattered. I would like to acknowledge the Australia Leadership Awards (AusAID) for the very generous 4 years of financial support and scholarship at the University of Queensland- Gatton Campus. Special thanks to Catherine Fitzgerald, Sue Quarton and Cheryl Brugman of UQ International. To the School of Agriculture and Food Science (SAFS) of the Faculty of Science staff: Allan Lisle, Victor Robertson, Mel, Lochie, John, Brigitte, Ross, Heidi, Katherine Raymont, Ross Bourne, Kylie and Kaye for the support given. I am indebted to the Department of Land and Resource Management (DLRM), Northern Territory Government. To: Bert, Lou, Tash, Susanne, and Phil. Thank you for all the time and assistance given during my fieldwork. To Tony Searle, for allowing us to do fieldwork on his property and for sharing his knowledge and expertise on mimosa in the area. Life as a graduate student can be very challenging, but I am glad to have amazing friends who cheered me up when I felt low. To Susanne Casanova and Michele Foley, thanks for all the wonderful times we spent together. I will surely miss you both. I am forever grateful to my beloved hubby, Alwen Jess, who has been very understanding and supportive of my research endeavour all these years. Life has been so much more enjoyable since you came along. To my parents and three sisters for the encouragement. Above all, I am thankful to the Lord Almighty for giving me the strength, courage and knowledge that I needed to finish my PhD journey.