An investigation on the interactions between entomopathogenic nematodes and plant growth promoting bacteria By Mary Jo Hurley BSc. A Thesis presented for the Degree of Doctor of Philosophy Submitted to the Higher Education and Training Awards Council (HETAC) Supervisors: Dr. Dina Brazil and Dr. Thomais Kakouli-Duarte External Examiner: Prof. Dr. Stephen Sturzenbaum Internal Examiner: Dr. Kieran Germaine Submitted to the Institute of Technology Carlow July 2018 There are so many people that contributed to getting to this stage and I am grateful to all of them. Firstly, I would like to wholeheartedly thank my supervisors, Dr Thomaé Kakouli-Duarte and Dr Dina Brazil. Thank you for your insight, advice and continued encouragement and for supporting choices I made for my career, even though they made this process significantly longer and more challenging! I would like to acknowledge the staff of IT Carlow: the technicians, caretakers, IT, administration and porters, thanks for everything over the years. A special thanks to Sarah Clarke, Aisling Fitzgerald, Dr Guiomar Garcia-Cabellos, Dick Farrell, Bob Stacey and Ray Dermody. To Dr Xuemei Germaine and all of the MicroGen team, thank you for giving me such an amazing opportunity to learn and develop as a researcher and a scientist. You were all incredibly encouraging, inspiring, knowledgeable and patient. I will never forget my time with MicroGen and I wish you all the best. Thank you to all the postgraduate students in the Dargan Centre and a special thank you to the ‘originals’ for the nights in and the nights out. I am so grateful to have made life- long friends in John C and Eilis, Richie and Rachel, Eileen, Emma, Sean, Ridhdhi, Debbie, Nikki and John B. This experience would have been unbearable without you all, although it may have been much shorter and with fewer hangovers’. The train and plane journey home from Rhizosphere4 is something I never want to repeat and will never forget. A special thank you to Aoife Egan, I could not have gotten through this ‘writing phase’ or the ‘Portugal experience’ without you. Graduation caps at the ready for November, we’ve got this! To my all friends, we’ve been together as far back as I can remember, through communions, and confirmations, graduations, weddings and births. To, Andrea, Aisling, Kylie, Lisa, Maria and Paula thanks for being a great support system. You may not have always been the best influence, but you were always fun. I would like to thank my mam and dad for their endless support, I always appreciated you but never realised how much you did for us. Can I move back in? Thanks to my brothers and sisters for your encouragement and for never asking me when I will be finished. Finally (and most importantly), a special thanks to Jamie and Cathal, who probably know as much about nematodes and PGP bacteria as I do. It’s finally done and I’m looking forward to the adventures we have in store with our little man. Cathal, you didn’t make this process any easier but you definitely made my days brighter and always kept me on my toes. I’m not sure what is next but I can’t wait to find out, together. ii Increasing demands on global food production have resulted in the overuse of chemical fertilisers and pesticides, leading to an increase in environmental pollution and pest resistance. Traditional fertiliser applications are carried out to supplement soil with a bioavailable form of nutrients essential for plant growth, and pesticides are applied to prevent crop damage from economically detrimental pests. The adverse impacts of these conventional agricultural practices include water, air and soil pollution in addition to direct negative effects on human health. There is growing public and political concern on these issues and if current intensive practices are continued, natural resources including clean water, fertile soil, and biodiversity, in flora and fauna, are under severe threat for future generations. In order to facilitate the transition away from the use of conventional agri-chemicals and prevent further environmental pollution, research, and resulting application, must focus on the use of naturally occurring biofertilisers and the biological control of economically important pests. This study was the first of its kind and examined the basic fundamental interactions between entomopathogenic nematodes (EPN; Rhabditida: Heterorhabditis and Steinernema) and plant growth promoting (PGP) bacteria in order to provide a framework for the development of an environmentally sustainable, reliable, cost-effective, multi-function product for biocontrol and PGP. A comprehensive examination on the effects of PGP bacteria on EPN showed that these naturally occurring soil organisms did not significantly affect EPN survival, infectivity, virulence, attraction to their host or reproduction. Major findings of this work include Greenhouse results which revealed that a combination of Heterorhabditis bacteriophora and the PGP bacteria Pseudomonas fluorescens F113 gfp and P. fluorescens L321 gfp individually contributed significantly to an increase in oilseed rape (Brassica napus) fresh weight. Laboratory-based bioassay results showed that P. fluorescens F113 gfp successfully colonised infective juveniles (IJ) of Steinernema feltiae SB 12(1) and H. bacteriophora. Moreover, there appears to be some level of interaction between P. fluorescens F113 gfp and EPN and their associated endosymbionts (Xenorhabdus bovienii and Photorhabdus luminescens) as P. fluorescens F113 gfp survived inside Galleria mellonella following nematode infection and insect mortality. Interactions between EPN and bacterial species and strains varied. However, the overall results are positive and indicate that the EPN and PGP bacteria examined here are compatible and show promising potential to be developed and formulated as a combined agricultural product, for insect control and increased soil and plant health. iii Results from this study have been disseminated at the following national and international conferences: Hurley. M J., Brazil. D. and Kakouli-Duarte, T. (2012). Effects of bacterial endophytes on the survival and virulence of entomopathogenic nematodes, unpublished paper presented at: Association of Applied Biologists annual conference in ‘Advances in Nematology. The Linnean Society, London, 11-13 December 2012. Hurley. M J., Brazil. D. and Kakouli-Duarte, T. (2014). The effects of endophytic bacteria isolated from bioenergy crops on the biology and behaviour of entomopathogenic nematodes, unpublished paper presented at: Environmental Sciences Association of Ireland, Environ. Trinity College, Dublin, 26-28 February, 2014. Hurley. M J., Brazil. D. and Kakouli-Duarte, T. (2014). Entomopathogenic nematode biology and behaviour: An investigation on nematode infectivity and fecundity following endophytic bacterial exposure, unpublished poster presented at: Institute of Technology Carlow Research symposium. Institute of Technology, Carlow, 2 May 2014. Hurley. M J., Brazil. D. and Kakouli-Duarte, T. (2015). An investigation on the colonisation potential of endophytic bacteria in oilseed rape, grown in the presence and absence of the entomopathogenic nematodes, unpublished paper presented at: Rhizosphere4. Maastricht, the Netherlands, 21-25 June, 2015. Hurley. M J., Brazil. D. and Kakouli-Duarte, T. (2015). Endophytic bacterial effects on entomopathogenic nematode infectivity and virulence, unpublished paper presented at: the 32nd symposium of the European Society of Nematologists. Braga, Portugal, 28 August 28 - September 1, 2016. iv Figure 1.2.1 Classification of the Phylum Nematoda based on molecular phylogenetic analyses of the small subunit ribosomal RNA gene from Blaxter (2011). ..................... 29 Figure 1.3.1 Generalised life cycle of EPN from Campos-Herrera, 2015 ...................... 32 Figure 1.3.2 Variation in the location of EPN symbiotic bacteria. a) Photorhabdus sp. bacterium from Heterorhabditis sp. occupying a large portion of the lumen of the nematode gut and b) Xenorhabdus bacterium from Steinernema sp. localised within a vesicle in the posterior of the pharynx. Image from Goodrich-Blair & Clarke (2007). 34 Figure 1.5.1 Both EPN and bacterial endophytes play an integral role in integrated pest management. This pyramid shows the most important IPM processes (from Stenberg [2017]). ............................................................................................................................ 57 Figure 2.2.1 Bioassay plate setup to investigate the antimicrobial activity of endophytic bacteria F113 and L321 against P. luminescens and X. bovienii .................................... 82 Figure 2.3.1 S. feltiae SB 12(1) infective juvenile mortality following exposure to endophytic bacteria (108CFU/ml) for 24 and 48 hr. The black dotted line indicates 50% nematode mortality. Asterisk(*) indicates significant differences between control and bacterial treatment at the respective time points (24 and 48 hr). .................................... 83 Figure 2.3.2 S. feltiae (e-nema) infective juvenile mortality following exposure to endophytic bacteria (108CFU/ml) for 24 and 48 hr. The black dotted line indicates 50% nematode mortality. Asterisk(*) indicates significant differences between control and bacterial treatment. .......................................................................................................... 84 Figure 2.3.3 S. carpocapsae infective juvenile mortality following exposure to endophytic bacteria (108CFU/ml) for 24 and