Investigations into the potential of classical biological control of the invasive aquatic weed, Crassula helmsii Thesis submitted for the degree of Doctor of Philosophy School of Biological Sciences Royal Holloway, University of London By Sonal Varia October 2020 Declaration of Authorship I, Sonal Varia, hereby declare that this thesis and the work presented in it is entirely my own with the exception of the following: • Marion Seier assisted in the design and assessment of the fungal natural enemies of Crassula helmsii as outlined in chapter 3 • Suzy Wood assisted in the host-range testing assessments of Hydrellia perplexa as outlined in chapter 3 and the host-range testing assessments of Aculus crassulae in chapter 4, under my supervision • Robert Allen assisted in the host-range testing of Aculus crassulae and assessment of the impact study assessments and data analysis as outlined in chapter 4 under my supervision • Tim Beale developed the maps of the potential distribution of Aculus crassulae in chapter 4 • Corin Pratt assisted in the assessment of the application of glyphosate and Aculus crassulae as outlined in chapter 5 under my supervision • Aylin Kerim assisted in the data collection of the mite phenology experiment as outlined in chapter 6 under my supervision 2 Acknowledgements First and foremost, I would like to thank my supervisors, Dr Sean Murphy and Professor Alan Gange for their valued guidance and support throughout this PhD, particularly during the writing of this thesis. I am grateful to CABI for allowing me the funding, time and facilities to carry out this PhD. Thank you to my CABI colleagues in the Invasives team who have not only supported me in the laboratory and in the field over the past seven years but whose help, advice and words of encouragement have been invaluable. In particular I would like to thank Dr Marion Seier, Suzy Wood, Dr Carol Ellison, Dr Norbert Maczey, Kate Pollard, Corin Pratt, Nikolai Thom, Dr Dick Shaw and Djami Djeddour. Thanks must also go to the interns and students who have provided assistance in data collection over the years; Robert Allen, Aylin Kerim and Jacob Horner. Thank you also to my colleagues based in Australia for facilitating surveys and arthropod shipments; Dr Robin Adair, Dr John Ireson and Richard Holloway. I am also grateful to Defra for funding this project and to members of the steering committee for providing guidance throughout the biological control project. A special thank you to my family for always supporting me, particularly during the final stages of this PhD. Finally, I would like to thank my husband George for always being there to support me in every way. 3 Abstract Crassula helmsii, a semi-aquatic plant native to Australia and New Zealand, was first introduced to the UK over a hundred years ago. Since then, it has spread across much of the UK and is present in parts of Western Europe. Crassula helmsii can dominate aquatic habitats and there are limited options for its management, particularly in protected habitats. Consequently, alternative methods of control have been encouraged by the UK government. The potential for using classical biological control for C. helmsii was investigated and the results are presented in this thesis. Assessments of the plant and its associated natural enemy complex in south-eastern Australia were made and several natural enemies with high potential as biological control agents were selected and underwent further investigation to assess their suitability. The gall-forming mite, Aculus crassulae (Eriophyidae) was found to be the most promising natural enemy based on its high host specificity and the damage inflicted on C. helmsii. Further laboratory-based assessments predicted its ability to tolerate the low temperatures experienced during winters in the UK confirming that there was potential for the mites to survive in the UK. Field experimentation with A. crassulae was also undertaken and mite populations and C. helmsii growth were monitored, verifying predictions that the mites were able to survive natural conditions in the winter and develop robust populations in the spring and summer. Furthermore, the effect of shade was assessed and it was found that winter survival of mites increased under these conditions. With herbicide application being the main control measure employed against C. helmsii, the impact of the use of a low dose of glyphosate in combination with the mites was also investigated. Findings were varied although the trend for increased plant decay when both were applied together warrants further study into the interactions between the mite and the herbicide. 4 Table of Contents Declaration of Authorship ............................................................................................ 2 Acknowledgements ...................................................................................................... 3 Abstract ........................................................................................................................ 4 Table of Contents ......................................................................................................... 5 List of Figures ............................................................................................................ 10 List of Tables.............................................................................................................. 14 1 General Introduction .......................................................................................... 15 2 Literature Review ............................................................................................... 17 2.1 Invasive non-native species ......................................................................... 17 2.2 Biological control of weeds ......................................................................... 18 2.3 Crassula helmsii .......................................................................................... 23 2.3.1 Taxonomy and nomenclature ............................................................... 23 2.3.2 Introduction and distribution ................................................................ 23 2.3.3 Biology of Crassula helmsii ................................................................ 24 2.3.4 Impact of Crassula helmsii .................................................................. 26 2.3.5 Current control methods ....................................................................... 27 2.3.6 Research on the biological control of Crassula helmsii....................... 28 2.4 Aims and objectives .................................................................................... 29 3 An assessment of the Australian natural enemies of Crassula helmsii as potential biological control agents ............................................................................. 30 3.1 Introduction ................................................................................................. 30 3.1.1 Native range surveys ............................................................................ 30 3.1.2 Agent prioritisation .............................................................................. 31 3.1.3 Host-range testing ................................................................................ 32 3.1.4 Natural enemies of Crassula helmsii ................................................... 33 3.2 Objectives .................................................................................................... 34 3.3 Native range surveys ................................................................................... 34 5 3.3.1 Materials and methods ......................................................................... 34 3.3.1.1 Surveys for invertebrate natural enemies ...................................... 36 3.3.1.2 Surveys for fungal natural enemies .............................................. 36 3.3.2 Results .................................................................................................. 36 3.3.2.1 Crassula helmsii sites surveyed .................................................... 36 3.3.2.2 Surveys for invertebrate natural enemies ...................................... 37 3.3.2.3 Surveys for fungal natural enemies .............................................. 39 3.3.2.4 Conclusion of native range survey................................................ 43 3.4 Investigations into the biology of the natural enemies of Crassula helmsii 43 3.4.1 Materials and methods ......................................................................... 43 3.4.1.1 Assessment of Hydrellia perplexa as a biological control agent .. 43 Host-range testing ........................................................................................ 43 3.4.1.2 Assessment of fungal pathogens as biological control agents of Crassula helmsii ............................................................................................. 46 Isolation from plant material ....................................................................... 46 Colletotrichum spp. studies ......................................................................... 46 Alternaria sp. studies (W2631A) ................................................................ 48 3.4.2 Results .................................................................................................. 50 3.4.2.1 Assessment of Hydrellia perplexa as a biological control agent .. 50 3.4.2.2 Assessment of fungal pathogens as biological control agents