Ca r d if f UNIVERSITY PR! FYSGOL C a e RD y g > BINDING SERVICES Tel +44 (0)29 2087 4949 Fax +44 (0)29 20371921 e-mail [email protected] Repellent, Antifeedant & Molluscicidal Effects of Commiphora spp. Oleoresins, and their extracts, on Deroceras reticulatum and Helix aspersa A thesis presented for the degree of Doctor of Philosophy Ahmed Ali March 2005 School of Biosciences Cardiff University UMI Number: U584779 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U584779 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 DECLARATION This work has not previously been accepted in substance for any degree and is not being concurrently submitted in candidature for any degree. Signed ..............z.Vxv r. .....................................................(Candidate) Date.................................................................................... STATEMENT 1 This thesis is the result of my own investigations, except where otherwise stated. Other sources are acknowledged by footnotes giving explicit references. A bibliography is appended. Signed.. .................................................. (Candidate) Date ............................................... STATEMENT 2 I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed... ...........M..: ........................................ (Candidate) Date ................................................... i Acknowledgments I would like to thank my supervisors: Professor Ifor Delme Bowen: for his technical advice, support and continuous enthusiasm throughout the duration of this research study. Dr. Carsten Muller: for his support in analytical chemistry as well as his general advice in chemical ecology. I would like to acknowledge and thank the Compton Group, and in particular the managing director Peter Ballard, for their financial support throughout the study. I would also like to thank Dr Peter Randerson for his valuable help in all things statistical. I would like to thank Mr Lyndon Tuck for his horticultural advice during field trials and for his never ending supply of slugs. I would also like to acknowledge Mr Mike O’Reilly for his technical help with the smoothing running of the gas chromatography- mass spectrometry (GC-MS) analytical equipment. I would like to acknowledge Professor Santaya Kela, from Bauchi University (Nigeria) for his part in this research project, which originally started with his sourcing of Nigerian molluscicidal plants. I would like to thank my colleagues Ahmed Bahashwan and Nargis Gani for their help and support during this research period. Finally I would like to thank my wife Fadumo Samsam and my son Robleh for their patience and support during this research period. Abstract The oleoresin exudates from two species of Commiphora trees, and their extracts, were evaluated as novel methods of controlling terrestrial molluscs. Various test methods were employed including terraria trials, leaf disc assays, caged field trials and spray trials. Laboratory terraria trials with C. molmol (myrrh) and C. guidotti (opoponax) oleoresins, showed them to be effective repellent barriers against the terrestrial molluscs Deroceras reticulatum, Arion hortensis and Helix aspersa. Solid repellent barriers comprised of reduced amounts of myrrh oleoresin, mixed with inert materials (sawdust, corncob and sharp sand) and sawdust coated with extracts of myrrh and opoponax, were also very effective in repelling terrestrial molluscs. The botanical origin of myrrh and opoponax oleoresins were confirmed using gas chromatography-mass spectrometry (GC-MS) analytical techniques. The chemical compounds identified for myrrh were consistent with those reported for C. molmol, comprising mainly of sesquiterpenes and furano-sesquiterpenes, whilst the chemical compounds identified for opoponax were consistent with those reported for C. guidotti, comprising mainly of monoterpenes and sesquiterpenes. Comparison of the chemicals identified for commercial myrrh (Yemeni) and Somali myrrh (Guban) showed them both to contain the same bouquet of chemical compounds. Differences were observed in the gas chromatographic profile of Somali and Yemeni myrrh. Somali myrrh contained high levels of P-elemene, whilst Yemeni myrrh was dominated by the furano-sequiterpenes, curzerene and Furanoeudesma -1,3- diene. a-Santalene was the major sesquiterpene identified for the liquid extracts of opoponax, whilst Jra«s-p-ocimene was the dominant chemical identified for the volatile odour associated with the opoponax oleoresin and its extracts. Leaf disc assays, with D. reticulatum slugs, confirmed the extracts of myrrh and opoponax, to be strong antifeedants at concentrations of 0.5% and 1% respectively. Both extracts significantly reduced the feeding behaviour of the slugs. A number of terpenoid chemicals were also evaluated, using the leaf disc assay, and showed significant antifeedant properties. The most potent of these chemicals was found to be toms-p-ocimene, a major component of opoponax, and was found to possess both antifeedant and molluscicidal properties towards slugs. The molluscicidal nature of this monoterpene depended upon the polarity of the medium used to prepare it. Leaf discs assays with H. apersa snails, showed that higher concentrations of myrrh and opoponax extracts (3%) was required to deter the snails from feeding on the lettuce leaf discs. In addition higher concentration levels of frww-p-ocimene (5%) was required to cause a similar antifeedant effect. In contrast to the slugs, no snail mortality was observed with these strong antifeedant extracts, however 100% snail mortality was observed after treating lettuce leaf discs with pure /ra«s-P-ocimene oil. Emulsion stability was found to be dependent upon nature of the non-ionic surfactant incorporated into the formulation. Oil in water emulsions based on myrrh, opoponax and trans- p-ocimene oils, containing 3 to 5% surfactant, were stable for time periods ranging from two weeks to more than 10 months. Emulsions based on Synperonic 91/8 were stable for two weeks, whilst those containing Tween 80 and Tween 20 were stable for approximately 10 months to one year. Caged field trials with repellent physical barriers comprised of 100% myrrh and opoponax oleoresins, reduced myrrh oleoresin mixed with inert substrates, and sawdust treated with ethanol and essential oil extracts of myrrh, all showed significant repellency properties towards D. reticulatum slugs for 14 days. Spray trials with myrrh, opoponax and trans- p-ocimene, under controlled temperature conditions, showed them to be very effective in deterring slugs and snails from consuming lettuce plants. Myrrh essential oil and /ra«s-P-ocimene were also molluscicidal against the small field slug. Little slug mortality was observed when ethanol extracts of myrrh were employed, whilst still maintaining its strong repellent properties. No incidences of snail mortalities were observed throughout the spray trials. Myrrh and opoponax oleoresins were found to have no toxic effects on earthworms and their 3% extracts showed very little phytotoxic effects against lettuce plants. 7><ms-p-ocimene (5%) extracts were well tolerated but marginally affected one variety of curly lettuce. This study has shown the novel application of myrrh and opoponax oleoresins, their extracts, and their chemical components in affecting the feeding activity of terrestrial molluscs. IV Publications Peer Reviewed 1. Ali, A. Y., Muller, C. T., Randerson, P. and Bowen, I. D. (2003) Screening African plants for mollusc repellency. In: Slugs & Snails Agricultural, Vetinary & Enviromental Perspectives, BCPC Symposium Proceedings, 80, 319-324. 2. Ali, A. Y., Muller, C. T., Randerson, P. and Bowen, I. D. (2003) Molluscicidal and repellent properties of African plants. In: Slugs & Snails Agricultural, Vetinary & Enviromental Perspectives, BCPC Symposium Proceedings, 80, 135-141. 3. Ali, A. Y. and Bowen, I. D. (2005) Antifeedant and molluscicidal activity of scented myrrh applied as a spray. In: OILB/IOBC Working Meeting On Integrated Control o f Soil Pests, Stuttgart, Germany. In Press. Patents 1. Bowen, I. D. and Ali, A. (2003) The use of plant materials as a terrestrial molluscicidal and/or mollusc repellent agent. International Patent WO 03/092385 Al. Table of Contents Page No. Declaration i Acknowledgments ii Abstract iii Publications v Table of contents vi 1. CHAPTER 1: GENERAL INTRODUCTION 1 1.1 Deroceras reticulatum 2 1.1.1 Description, Classification And Life History 2 1.1.2 Feeding Behaviour 4 1.2 Helix aspersa 6 1.2.1 Description, Classification And Life History 6 1.2.2 Feeding Behaviour 8 1.3 Terrestrial Molluscs As Pests 9 1.4 Methods of Controlling Terrestrial Molluscs 10 1.4.1 Chemical Control 10 1.4.1.1 Metaldehyde 11 1.4.1.2 Methiocarb 12 1.5 Biological Control 13 1.5.1 Carabid Beetles 13 1.5.2 Nematodes 15 1.6 Botanical