Imperial College London Department of Biology Effectiveness of prosulfocarb-based treatments for the control of sensitive and herbicide resistant Lolium spp. populations Geraldine Charlotte Bailly 26th September 2011 Submitted in part fulfilment of the requirements for the degree of Doctor of Philosophy in Weed Science of Imperial College London and the Diploma of Imperial College London 1 Declaration I herewith certify that all material in this dissertation which is not my own work has been properly acknowledged. Geraldine Charlotte Bailly 3 Abstract The rapid evolution of resistance to post-emergence herbicides in Lolium spp. (ryegrass species) has complicated weed management in cereals. En- suring good pre-emergence control is therefore increasingly important to protect yields. Prosulfocarb is a broad spectrum thiocarbamate herbicide that kills plants by disrupting the biosynthesis of very long chain fatty acids. This is the first report of resistance to prosulfocarb in Lolium spe- cies from farm sites and glasshouse selection. The recurrent selection pro- cess with prosulfocarb applied pre-emergence at its maximum labelled rate (4,000 gai/ha) on a susceptible Lolium multiflorum Lam. population showed that the evolution of resistance due to repetitive use was possible but slow. Over three generations, it resulted in modicum levels of resistance which were not significant at practical field rates. For the last progeny obtained, there was no evidence of either cross-resistance or increased sensitivity to an herbicide under-development with a mode of action similar to prosulfo- carb (RF50 = 0.99, 95% CI = 0.77 - 1.27). Likewise, high and increasing levels of non-target-site based resistance to clodinafop-propargyl did not result in cross-resistance to prosulfocarb. Prosulfocarb exerted low levels of negative cross-resistance on LL1781, SS1999, GG2078 and RR2088, four different ACCase mutant L. multiflorum sublines (average RF50 = 0.67). In order to control susceptible and resistant populations in winter cereals, prosulfocarb was mixed with diflufenican, metribuzin, pyroxasulfone and the formulated mixture fiodosulfuron:mesosulfurong. Over the 86 mixtures tested, 11 showed a high potential. Prosulfocarb + diflufenican at 2,400 + 32 gai/ha presented interesting levels of synergism (+20 points on av- erage). A non-random survey in 34 farm sites from England showed that most Lolium samples (80%) were sensitive or only partially resistant to pro- sulfocarb. However, prosulfocarb efficacy was lower where other herbicides had been used intensively. Future research may now concentrate on the determination of prosulfocarb resistance pathways in Lolium spp. 5 A mouse is an animal that, when killed in sufficient quantities, under controlled conditions, produces a doctoral thesis. Woody Allen Also applicable to ryegrass species, plants. 7 Acknowledgments From the newly graduated Master student to the Dr. to be, this PhD has really been quite a journey! Some would say, a roller coaster, I would rather say an initiatory journey. I would not be there now without Deepak who chose me as his first PhD student, believed I could make it as a researcher and put together a Research Proposal that was in competition at Syngenta level for funding (BBSRC In- dustrial Case Award). I am really grateful to all the persons who supported the project from its drafting stage to the end, especially Jason Tatnell and Gael Le Goupil who were always here to advise on technical questions and to review publications. Warm thanks also go to Luc Flamant, from whom I learnt a lot, although he failed to honor his field trials visit promise and who should really consider quitting smoking. Finally, I do really appreciate the great opportunity I had to have been able to conduct all my experi- mental work at Jealott's Hill. This would not have been possible without Simon Archer and Denis Wright's approvals and without Mark Spinney's flexibility. Giving me Bay 2 for three years was a big sacrifice. I really enjoyed the glasshouse work and I can now state that I master the way of growing grass. Quite a technical skill, isn't it? My glasshouse time was that successful as I knew I could always count on Adam Stacey to cheer me up when I felt blue and to give me a hand when I needed strong arms. I'm particularly thinking of the excessively heavy green wheelie bins, the moist silver sand bags and the metal pieces that covered the glasshouse gully. Adam, we need to schedule another pub lunch! I also knew I could abandon my plants, as difficult as it may be, for some time and enjoy the (too few) holidays and conferences without any worries as Barry Elsdon was there to take care of my babies and trim them as needed. Thanks Barry. Thanks also to Sarah-Jane Hutchings. General thanks to Plant Production folks, Phil - keep your funky T-shirt collection, there are some I really liked; Peter 9 - try to keep the Resistance Seed Store tidy and for God's sake, please stop stealing or hiding resistant seed batches; Diane - thanks for the rhubarb and please keep an eye on Peter; Lesley - thanks for the sugar cane courses and the transplanting help. Glasshouse is good, lab is better! Growing plants was one thing, but I also had to figure out what was really going on at a lower scale, yes man, at the enzyme scale and even worst, at the gene scale. Damn! How to do that? Calling 118 118 could have been a solution, but I opted for Richard Dale's teaching skills. Poor Rich! He had no choice really and saying he was terrified when I refused to hold the multichannel pipette to load a gel is an understatement. Soon after this silver pipette became my best friend and I wouldn't have lent it for anything. So, Rich thank you for your patience, your help and your knowledge. I'm glad it was you who taught me some tricks of the trade. I had an amazing, awesome (!) time in Lab 136 and I wish I had more molecular work to do. More thanks go to Rachael Blain who was my centrifuge buddy and the best labmate. Pauline may have already said it, but I say it again. A wink to John Ray and our Tour de France conversations. Quite a good one this year, and guess what, I saw the last stage! In this busy schedule, there was still some time left for enzymatic studies. It was the era of ACCase extraction, purification, fraction testing, western blotting. Mama Mia! Hopefully, Steve Elvidge and his magic fingers were there when the Akta misbehaved. Thanks Steve. Thanks to Samantha Hall for the radiochem training and to Sheila Atten- borough for the guidance on the protein identification work. All - your help was invaluable. A thought to Nathalie Dupen who ordered everything I needed and chased up the orders when required. Knowing your science consumables will arrive on time is a significant advantage. The icing on the cake was the statistical analyses. One cannot imagine a biology PhD thesis without them. For that, I am strongly indebted to Eddie McIndoe. The theoretical courses you get at the Uni are one thing, the reality of the experiments another. So, thank you Eddie for having taken time whenever needed. A final thought for the Bible that accompanied me during these years, I mean the R book, and huge congratulations to Michael J Crawley for having written it and introduced this black magic to the Silwoodians! As promised, a line to Linda Romain who managed to make Denis and Simon sign the paperwork and send it one month after the official deadline but two months before the informal one. That was a relief!. Last warm thanks 10 to David Brocklehurst for having scrutinised my thesis before the viva day, to Colin Turnbull and Stephen Moss for their helpful comments during the viva voce examination. Exhausting research must work in tandem with relaxing time. Now, I wish to thank all the persons who accompanied and supported me during this long journey. First and foremost, thanks to my local family who pampered me and my little car for three years. Leaving with you was really enjoyable. Joe - you were right, I brought back my bike to France, but I still haven't used it and the tyres are still flat; Saskia - keep going, you will do well at school and beyond. Les filles, Karine, Olivia, ¸cam'a fait un bien fou de vous savoir `ames c^ot´es.On aurait quand m^emepu organiser plus de soir´eesWii, on a mal g´er´el`a; d'autant plus que la promise n'a toujours pas apparu `aBry ! Iain, take care of The Management and stay as you are, always happy and relax on the surface. Pauline, on ne se sera malheureusement pas c^otoyer longtemps, j’esp`oreque tu trouveras ton bonheur, en Suisse ou ailleurs ! Martina, Yael and Gabriel, thanks for your support, for the lovely dinners we had together, for all the postcards and letters I received from you. Rekha, I'm glad you moved on, hugs to Anjali and thanks for having initiated me to the Indian cuisine. Enfin, merci `aMaman et `aMarc David, inutile d'en dire plus, vous savez tr`esbien ce que vous avez fait, faites et ^etespour moi. 11 Contents Abstract7 Acknowledgments9 List of Figures 17 List of Tables 21 List of Abbreviations 25 1 General Introduction 29 1.1 Ryegrass.............................. 29 1.1.1 General characteristics.................. 29 1.1.2 Ryegrass species as a weed............... 30 1.2 Herbicides and ryegrass control................. 32 1.2.1 ACCase-inhibiting herbicides, HRAC A........ 35 1.2.2 ALS-inhibiting herbicides, HRAC B.........