Microbes Associated with Hylobius abietis: A Chemical and Behavioral Study Muhammad Azeem Doctoral Thesis Stockholm 2013 Akademisk avhandling som med tillstånd av Kungliga Tekniska Högskolan i Stockholm framlägges till offentlig granskning för avläggande av doktorsexamen i kemi med inriktning mot organisk kemi torsdagen den 30 maj kl 10.00 i sal F3, KTH, Lindstedtsvägen 26, Stockholm. Avhandlingen försvaras på engelska. Opponent är Dr. Irena Valterová, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague. Cover: Pine (Pinus sylvestris L.) seedling, Pine weevil (Hylobius abietis L.) feeding on pine seedling, Pine weevil frass, Penicillium expansum on pine bark broth, Pine bark, Co-culture of Heterobasidion parviporum and Bacillus subtilis A19 (Photos by Claes Hellqvist and Muhammad Azeem) ISBN 978-91-7501-727-3 ISSN 1654-1081 TRITA-CHE-Report 2013:23 © Muhammad Azeem, 2013 E-Print, Stockholm Muhammad Azeem, 2013: “Microbes Associated with Hylobius abietis: A Chemical and Behavioral Study” KTH Chemical Science and Engineering, Royal Institute of Technology, SE-100 44 Stockholm, Sweden. Abstract This thesis is based on three inter-related studies: the first part deals with the microbial consortium, the identification of microbes and their volatiles, the second part deals with the study of bio-chemical control methods of two conifer pests; the pine weevil Hylobius abietis (L.) and the root rot fungi Heterobasidion spp., and the third part describes the production of styrene by a fungus using forest waste. The large pine weevil (Hylobius abietis L.) is an economically important pest insect of conifers in reforestation areas of Europe and Asia. The female weevils protect their eggs from feeding conspecifics by adding frass (mixture of weevil feces and chewed bark) along with the eggs. In order to understand the mechanism behind frass deposition at the egg laying site and to find repellents/antifeedants for pine weevils, microbes were isolated from the aseptically collected pine weevil frass. Microbial produced volatile organic compounds (VOCs) were collected by solid phase micro extraction and analyzed by GC-MS after cultivating them on weevil frass broth. The major VOCs were tested against pine weevils using a multi-choice olfactometer. Ewingella sp., Mucor racemosus, Penicillium solitum, P. expansum, Ophiostoma piceae, O. pluriannulatum, Debaryomyces hansenii and Candida sequanensis were identified as abundant microbes. Styrene, 6-protoilludene, 1- octene-3-ol, 3-methylanisole, methyl salicylate, 2-methoxyphenol and 2- methoxy-4-vinylphenol were the VOCs of persistently isolated microbes. In behavioral bioassay, methyl salicylate, 3-methylanisole and styrene significantly reduced the attraction of pine weevils to their host plant volatiles. Heterobasidion spp. are severe pathogenic fungi of conifers that cause root and butt rot in plants. Bacterial isolates were tested for the antagonistic activity against fungi on potato dextrose agar. Bacillus subtilis strains significantly inhibited the growth of H. annosum and H. parviporum. Styrene is an industrial chemical used for making polymeric products, currently produced from fossil fuel. A strain of Penicillium expansum isolated from pine weevil frass was investigated for the production of styrene using forest waste. Grated pine stem bark and mature oak bark supplemented with yeast extract produced greater amounts of styrene compared to potato dextrose broth. Keywords: Hylobius, Ewingella, Penicillium, Heterobasidion, Bacteria, Fungi, Bark, Forest waste, Metabolites, Styrene, Methyl salicylate. Abbreviations ANOVA Analysis of variance CFU Colony forming unit DNA Deoxyribonucleic acid DVB Divinylbenzene FRASS Mixture of pine weevil feces and chewed bark GC Gas chromatography GPB Grated pine stem bark GPBYE Grated pine stem bark with yeast extract GYE Glucose yeast extract HS Headspace ITS Internal transcribed spacer MeS Methyl salicylate MS Mass spectrometry PCR Polymerase chain reaction PDA Potato dextrose agar PDB Potato dextrose broth PDMS Polydimethylsiloxane PN Pine needles PW Pine wood chips SE Standard error SPME Solid phase micro extraction VOCs Volatile organic compounds WF Weevil frass WFA Weevil frass agar WFB Weevil frass broth YE Yeast extract List of Publications This thesis is based on the following papers, referred to in the text by their Roman numerals I-V: I. Chemo- and biodiversity of microbes associated with pine weevil (Hylobius abietis) Muhammad Azeem, Gunaratna Kuttuva Rajarao, Kazuhiro Nagahama, Olle Terenius, Henrik Nordenhem, Göran Nordlander and Anna-Karin Borg-Karlson Manuscript II. Fungal metabolite mask the host plant odor of the pine weevil (Hylobius abietis) Muhammad Azeem, Emil Norin, Olle Terenius, Gunaratna Kuttuva Rajarao, Göran Nordlander, Henrik Nordenhem and Anna Karin Borg-Karlson Submitted III. Penicillium expansum volatiles reduce pine weevil attraction to host plants Muhammad Azeem, Gunaratna Kuttuva Rajarao, Henrik Nordenhem, Göran Nordlander and Anna Karin Borg-Karlson J. Chem. Ecol. 2013, 39, 120-128 IV. Antagonistic activity of Bacillus subtilis A18 – A19 against Heterobasidion species Muhammad Azeem, Anna Karin Borg-Karlson and Gunaratna Kuttuva Rajarao Submitted V. Sustainable bio-production of styrene from forest waste Muhammad Azeem, Anna Karin Borg-Karlson and Gunaratna Kuttuva Rajarao Submitted List of papers not included in this thesis: VI. Benzyl isothiocyanate, a major component from the roots of Salvadora persica is highly active against gram-negative bacteria Sofrata A, Santangelo E M, Azeem M, Borg-Karlson A K, Gustafsson A & Putsep K PlosOne. 2011, 6, 23045 VII. Anti-schistosomiasis triterpene glycoside from the Egyptian medicinal plant Asparagus stipularis Hesham R El-Seedi, Rehan El-Shabasy, Hanem Sakr, Mervat Zayed, Asmaa M A El-Said, Khalid M H Helmy, Ahmed H M Gaara, Zaki Turki, Muhammad Azeem, Ahmed M Ahmed, Loutfy Boulos, Anna- Karin Borg-Karlson and Ulf Göransson Braz. J. Pharmacog. 2012, 22, 314-318 VIII. Chemical composition and repellency of essential oils from four medicinal plants against Ixodes ricinus nymphs (Acari: Ixodidae) Hesham R El-Seedi, Nasr S Khalil, Muhammad Azeem, Eman A Taher, Ulf Göransson, Katinka Pålsson, and Anna Karin Borg-Karlson J. Med. Entomol. 2012, 49, 1067-1075 Dedication To my family, especially to my mother, and to the memories of my father (late) Table of Contents Abstract Abbreviations List of publications 1. Introduction ............................................................................................... 1 1.1. The aims of this thesis ........................................................................... 1 1.2. Conifers ................................................................................................. 3 1.3. Pine weevil ............................................................................................ 4 1.4. Reforestation problem of conifers ......................................................... 4 1.5. Seedling protection methods ................................................................. 5 1.5.1. Insecticides .................................................................................... 5 1.5.2. Physical protection ........................................................................ 7 1.5.3. Biocontrol ...................................................................................... 8 1.5.4. Silvicultural methods..................................................................... 8 1.5.5. Antifeedants .................................................................................. 9 1.6. Towards a new solution based on pine weevil egg protection strategy10 1.7. Microbes…. ........................................................................................ 11 1.7.1. Bacteria ....................................................................................... 11 1.7.2. Fungi ........................................................................................... 12 1.8. Metabolites of bacteria and fungi ........................................................ 13 2. Materials and Methods ............................................................................ 15 2.1. Collection of weevil frass and feces .................................................... 15 2.2. Microbial culturing media ................................................................... 16 2.3. Isolation of microbes ........................................................................... 17 2.4. Identification of microbes ................................................................... 18 2.5. Collection of volatiles ......................................................................... 19 2.5.1. Static or biodynamic collection of volatiles using SPME ………19 2.5.2. Dynamic collection of volatiles ................................................... 20 2.6. Identification of volatiles .................................................................... 21 2.7. Bioassay .............................................................................................. 22 2.7.1. Pine weevil behavior ................................................................... 22 2.7.2. Fungal inhibition ......................................................................... 22 2.8. Data analysis ....................................................................................... 24 3. Microbes associated with Hylobius abietis ............................................. 25 4. Fungal volatiles