Penicillium expansum Volatiles Reduce Pine Weevil Attraction to Host Plants

Muhammad Azeem 0 2 Gunaratna Kuttuva Rajarao 0 2 Henrik Nordenhem 0 2 Gran Nordlander 0 2 Anna Karin Borg-Karlson 0 2 0 G. K. Rajarao KTH Royal Institute of Technology, School of Biotechnology, Division of Environmental Microbiology , SE-100 44, Stockholm, Sweden 1 ) KTH Royal Institute of Technology, School of Chemical Science and Engineering, Department of Chemistry, Organic Chemistry , SE-100 44, Stockholm, Sweden 2 A. K. Borg-Karlson Tartu University, Institute of Technology, Division of Organic Chemistry , Tartu 50411, Estonia The pine weevil Hylobius abietis (L.) is a severe pest of conifer seedlings in reforested areas of Europe and Asia. To identify minimally toxic and ecologically sustainable compounds for protecting newly planted seedlings, we evaluated the volatile metabolites produced by microbes isolated from H. abietis feces and frass. Female weevils deposit feces and chew bark at oviposition sites, presumably thus protecting eggs from feeding conspecifics. We hypothesize that microbes present in feces/frass are responsible for producing compounds that deter weevils. Here, we describe the isolation of a fungus from feces and frass of H. abietis and the biological activity of its volatile metabolites. The fungus was identified by morphological and molecular methods as Penicillium expansum Link ex. Thom. It was cultured on sterilized H. abietis frass medium in glass flasks, and volatiles were collected by SPME and analyzed by GCMS. The major volatiles of the fungus were styrene and 3-methylanisole. The nutrient conditions for maximum production of styrene and 3-methylanisole were examined. Large quantities of styrene were produced when the fungus was cultured on grated pine bark with yeast extract. In a multi-choice arena test, styrene significantly reduced male and female pine weevils' attraction to cut pieces of Scots pine twigs, whereas 3-methylanisole only reduced male weevil attraction to pine twigs. These studies suggest that metabolites produced by microbes may be useful as compounds for controlling insects, and could serve as sustainable alternatives to synthetic insecticides. - Adult pine weevils, Hylobius abietis (L.), feed on the tender bark of a wide range of conifers. Small plants are the preferred host in reforested areas of managed forests (Day et al., 2004), and weevils can kill up to 80 % of planted pine and spruce seedlings every year if no preventive measures are taken (Petersson and rlander, 2003). Seedlings are protected routinely by insecticide treatment before planting, but insecticides may contaminate soil and water and may thus have significant adverse effects on the environment (Peshin et al., 2009). Non-targeted organisms also may be affected, leading to ecological disturbance. In addition, there is a concern that insecticide-treated seedlings may affect the health of forestry workers (Kolmodin-Hedman et al., 1995). It is necessary to develop alternative methods of controlling pest insects using naturally occurring and sustainable insect repellents and anti-feedants. At present, plants are the major source of such chemicals, but microbes including fungi and bacteria also produce a variety of insect repellents (Daisy et al., 2002; Dong et al., 2007; Sun et al., 2008; Lam et al., 2010) such as avermectin, a macrocyclic lactone isolated from a bacterial fermentation broth that has been successfully used for pest control (Hotson, 1982). We are interested in finding sustainable and ecologically non-disruptive methods to limit pine weevil damage on newly planted seedlings. Observations of oviposition and egg-protecting behavior of pine weevil females reveal that they lay their eggs in the roots of fresh conifer stumps. A cavity is gnawed in the bark and an egg is deposited in the cavity. After this, the female adds her feces and closes the cavity using fresh pieces of chewed bark. It has been suggested that the deposition of feces protects the eggs from conspecific feeding (Borg-Karlson et al., 2006). Although the function of added feces is not fully understood, it has been shown that compounds present in feces (e.g., methylanisole, p-cresol, and dihydroconiferylalcohol) have an antifeedant effect on pine weevils (Borg-Karlson et al., 2006). Structureactivity studies have identified a large number of potent antifeedants, mainly derivatives of benzoic acid methyl esters, and various phenylpropanoids and phenylpropenyl esters (Legrand et al., 2004; Unelius et al., 2006; Sunnerheim et al., 2007; Bohman et al., 2008). Our hypothesis is that compounds responsible for antifeedant and repellent effects on pine weevils originate from microbes present in frass and feces of ovipositing females. These microbes grow in the humid environment around the eggs and produce compounds that repel weevils continuously. A similar observation also has been made by Lam et al. in 2010: the volatiles produced by a fungus growing on chicken feces were found to repel domestic house files and decrease their oviposition rate. Identification of insectcontrolling chemicals produced by microbes and a clear understanding of how these natural products function may lead to the development of alternatives to insecticides. We describe the isolation and identification of the fungus Penicillium expansum Link ex. Thom associated with pine weevil frass and feces. The fungus was cultured on its natural medium (i.e., pine weevil frass) and volatiles released by the fungus were collected by SPME and analyzed using GC-MS. Various culture media were used to compare the production of the major fungus volatiles, which were identified as styrene and 3-methylanisole. Styrene and 3-methylanisole were tested for biological activity against pine weevil using a multi-choice laboratory bioassay. Methods and Materials Chemicals (+)--pinene, (+)-3-carene, (+)--pinene, (+)-limonene, octanal, styrene, and 3-methylanisole (all 99 % GCpurity), dimethyl sulfoxide, potato dextrose agar, glucose and yeast extract were purchased from Sigma-Aldrich, Sweden; agar was purchased from Fisher Scientific, Sweden. Collection of Pine Weevil Frass and Feces Pine weevils (H. abietis) of both sexes were collected in the field and starved for 24 h; water was provided by placing a sterilized wet filter paper in the container. Two hundred starved weevils, including both sexes, were placed for 2 d in a sterilized container with a sieve at the bottom, which fit into another sterilized container. The weevils were provided with fresh Pinus sylvestris twigs obtained from a single healthy tree. The1530 mm diam, 100 mm long twigs were surface sterilized in 70 % ethanol, and excessive ethanol was evaporated aseptically before feeding them to weevils. The pine weevil frass (mixture of feces and pine bark particles chewed by weevils) fell through the sieve into the container below for aseptic collection and storage at 5 C in a sterilized glass vial. Fully fed weevils were placed in sterilized and dried glass Pet (...truncated)