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.

Microbes Associated with Hylobius abietis: A Chemical and Behavioral Study

Muhammad Azeem

Doctoral Thesis

Stockholm 2013

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 affect pine weevil behavior ........................................... 32 5. Inhibition of Heterobasidion and Ophiostoma species by Bacillus subtilis A18 – A19 ................................................................... …………………40 6. Sustainable bio–production of styrene from forest biomass ................... 46 7. Conclusions ............................................................................................. 50 8. Proposed future study .............................................................................. 52 9. Acknowledgements ................................................................................. 53

10. References ............................................................................................... 55 Appendix A: author’s contributions .................................................................... 66

1

1. Introduction

Forests provide raw materials for many industries such as furnisher, chemicals, pulp and paper among many others and thus play an important role in the economy of a country. Since the beginning of the last century, in order to fulfill the increasing demands for wood based raw material, modern forest management practices of clear cutting and thinning were introduced. With clear cutting all the trees are harvested from a large area followed by re-plantation of small plants. This practice created a critical problem for the reforestation of conifers due to the extensive feeding of pine weevil (Hylobius abietis) on newly planted seedlings. In the thinning process selective trees are removed from a tree stand to improve the growth rate of remaining trees. Stumps in a tree stand after thinning are the main source of root rotting fungi (Heterobasidion spp.) infection to remaining healthy trees. These two problems of modern forestry cause large economical loss to conifer forestry every year.

1.1. The aims of this thesis The main objectives of this thesis were: To find environmentally friendly antifeedants and

repellents for pine weevils by understanding the chemical mechanism of the egg laying behavior of female pine weevil.

Understand the chemical signals from microorganisms in the weevil frass (mixture of weevil feces and chewed

2

bark) at oviposition site that prevent conspecifics from feeding.

The hypothesis of this thesis is that microorganisms present in pine weevil frass grow in the moist conditions around the oviposition site and continuously produce volatile/non-volatile compounds to deter conspecifics from feeding close to that particular site. To test this hypothesis the following studies were conducted:

• Investigation of the microbial community associated with pine weevil frass and feces. Isolation and identification of volatile organic compounds (VOCs) of isolated microbes and microbial consortium (Paper I).

• Investigation of the production of major VOCs from

selected microbial isolates over a time span and their effect on pine weevil behavior (Paper II & III).

Other objectives of the thesis: Study the antagonistic activity of selected bacterial strains

for the biocontrol of Heterobasidion spp., and Ophiostoma sp. Heterobasidion spp. cause severe root and butt rot in conifers whereas Ophiostoma sp. introduces blue staining in conifer sap wood. Infection of either fungal species results in significant economic loss to forestry every year.

• In vitro biocontrol study of Heterobasidion spp. and Ophiostoma sp. by Bacillus subtilis strains (Paper IV)

Study the potential of an isolated fungus from pine weevil

frass for the sustainable production of styrene using forest waste. Styrene is an industrial chemical used for the

3

manufacturing of polymer products; it is currently synthesized from petroleum products.

• Investigation of optimum culturing conditions and feeding

material from forest related waste for the bio-production of styrene (Paper V).

1.2. Conifers

Conifers are woody, mostly evergreen, cone-bearing seed plants widespread throughout the Northern Hemisphere. They are used as raw material for a number of products such as furnisher, pulp, paper, fuel, and chemicals. Because of this, their economic impact on our society is very significant. They are long-lived organisms and their success is in part due to their potent dynamic defense mechanisms against herbivores and pathogens (Franceschi et al. 2005). The defense depends on the production of an abundant structural variety of phenolics, tannins and terpenes (Klepzig et al. 1996). The biosynthesis of these defense chemicals can be induced during an insect or fungus attack allowing conifers to evade being fed upon by most insects and pathogens during their long life span. However, some insects and fungi have evolved the ability to overcome the toxicity of these chemical defenses and manage to utilize the conifer trees as a food source.

In Sweden, forestry based products make a substantial contribution to the economy. More than 80% of the forest in Sweden consists of conifers, mainly Norway spruce (Picea abies (L.) H. Karst) and Scots pine (Pinus sylvestris L.) (Wigrup 2012).

4

1.3. Pine weevil

The large pine weevil, Hylobius abietis (L.) (Coleoptera: Curculionidae), feeds on the soft bark of actively growing parts of conifers, e.g. crowns of conifers (Örlander et al. 2000). The adult weevil is 8–14 mm long and has yellow patches on its body (Eidmann 1974). The weevil can utilize the bark of living as well as recently dead trees and feeds above and below ground but a sheltered feeding position is preferred. The weevil feeding on mature tree crowns do not cause any significant damage to trees but the extensive feeding on the stem bark of small plants girdle the bark and destroy the transportation system of the plants that delivers sugars and other nutrients from leafs to other parts of the plant, resulting in mortality of the plant.

Pine weevils breed in stumps of freshly dead trees. During spring weevils of both sexes migrate in search of areas with freshly felled trees (Örlander et al. 2000). Compounds such as α-pinene and ethanol attract pine weevils to suitable hosts for oviposition (Nordlander 1991). During the summer season pine weevil’s main activity is to eat and mate (Tilles et al. 1988). On the average a female weevil lays 70–80 eggs per season (Bylund et al. 2004), laid either in the soil near the stump roots or in the root bark of stumps (Nordlander et al. 1997).

1.4. Reforestation problem of conifers

Because pine weevils lay eggs in freshly dead tree stumps and larvae develop under the root bark of stumps their population remained under control, due to the limited availability of breeding sites in self re-generated forests, until the start of the last century

5

(Leather et al. 1999). However, due to the use silvicultural practices of clear cutting, which provides plenty of reproduction sites, the population of pine weevil increased considerably (Örlander et al. 1997; Nordlander et al. 2011).

Weevils emerge from eggs after 12 to 36 months (Leather et al. 1999; Day et al. 2004) and feed on the stem bark of newly planted seedlings. Weevils can kill up to 80-90% of the newly planted seedlings every year, if no proper preventive measures are taken, and cause significant financial losses (Örlander et al. 1997; vonSydow & Birgersson 1997; Petersson & Örlander 2003). This damage to newly planted conifer seedlings in the reforestation areas make them economically the most important pest insect of conifers in reforestation areas in large parts of Europe and Asia (Långström & Day 2004).

1.5. Seedling protection methods

A number of methods have been studied or adopted to protect the newly planted seedlings from the feeding pine weevils in the managed forests.

1.5.1. Insecticides

The most commonly used method to protect newly planted seedlings in European forests is the use of insecticides. The seedlings are dipped in insecticide solution prior to planting in the field (Stoakley 1968) or alternatively they are sprayed after planting. Dichlorodiphenyltrichloroethane (DDT) has been used for this purpose; however, it was banned in 1972 for agriculture and forestry use due to its harmful effects on higher animals.

6

DDT was replaced by pyrethroid based synthetic permethrin. In contrast to DDT permethrin is bio degradable but due to its adverse effects on aquatic organisms (Hill 1989; Mian & Mulla 1992) it was later replaced with cypermethrin. Unfortunately cypermethrin has been found to be even more toxic to aquatic organisms than permethrin (McLeesc et al. 1980). At present, imidacloprid is being used to control pine weevils in reforestation areas but it has been shown to be highly toxic to bees (Suchail et al. 2001) and aquatic life (Federoff et al. 2008).

OO

O

Cl

Cl

OO

O

Cl

Cl

N

N

NN

HN NO2

Cl

Cl

Cl

Cl

ClCl

Permethrin

Cypermethrin

Imidacloprid

Dichlorodiphenyltrichloroethane (DDT)

Figure 1. Chemical structure of insecticides have been using for pine weevil control

7

The major problem of using insecticides is their adverse effect on non-targeted insects. The mortality of non-targeted organisms gives rise to ecological disturbances in ecosystems (Pimentel 2009). Moreover there is concern about health effects of the insecticides on forestry workers applying them (Kolmodin-Hedman et al. 1995), and their use has been questioned in many countries (Mian & Mulla 1992).

1.5.2. Physical protection

Instead of insecticidal application, there have been attempts to introduce physical barriers (fences) around the plants (Petersson & Örlander 2003; Petersson et al. 2004). The barriers are usually made of plastics or paper with or without a collar at the top. Barriers with a collar proved more effective in plant protection than those without (Petersson et al. 2004). In one study, plastic barriers with polytetrafluoroethylene coating the outer walls were used to hinder insects from climbing up, this treatment reduced the seedling mortality by 83% compared to untreated control plants (Eidmann et al. 1996). In other studies seedling stems were coated with wax that reduced weevil feeding (Watson 1999) or flexible polymer coatings with embedded sand particles that increased the seedling survival rate form 27% to 91% during a three year field trial (Nordlander et al. 2008). In spite of their effectiveness, which is comparable to the use of insecticides, there are technical issues which are related to plant growth and large scale application (Eidmann et al. 1996).

8

1.5.3. Biocontrol

In biocontrol methods the pests are managed by using other living organisms including predators or parasites. Biocontrol of pests is considered to be environmentally friendly compared to insecticide application. For some decades there has been an increased interest in biocontrol methods to cope the populations of weevils in clear cut areas. In some European countries field trials are undergoing to study the use of entomopathogenic nematodes to control pine weevil populations at larval and pupae stages (Dillon et al. 2007). Another option is the use of entomopathogenic fungi Metarhizium robertsii and Beauveria bassiana to kill pine weevils in different life stages (Ansari & Butt 2012). Recently, Harvey et al. (2012) showed that the risk of damage to non-target insects is high in using entomopathogenic nematodes and fungi for the management of pine weevils.

1.5.4. Silvicultural methods

In addition to chemical control, physical barriers and bio control methods, reduction of pine weevil damage to small seedlings can be achieved by employing a number of silvicultural measures. These include soil scarification or plantation under sheltered wood (Örlander & Nilsson 1999). Soil scarification is the removal of the humus layer to expose of the mineral soil. This helps the seedlings to establish in the ground (Nordborg & Nilsson 2003) and to some extent reduces damage by weevils to the planted seedlings (Petersson et al. 2005). In shelter wood treatment some trees are left behind as a food source for the weevils. When shelter wood practice was compared to clear cutting where all trees were cut down it was observed that weevil

9

population density was same, however more damage to seedlings was observed in the clear cut areas (von Sydow & Örlander 1994; Nordlander et al. 2003). A synergistic protection effect for the seedlings can be achieved if different silvicultural methods are combined with physical barrier methods (Petersson & Örlander 2003).

1.5.5. Antifeedants

An antifeedant is defined as a substance that deters or reduces feeding activity. Many plants and microorganisms produce such chemicals that protect them from being fed upon. Antifeedants don’t kill the insects directly as in the case of insecticides but the insects could die due to other reasons related to their digestive system. A number of studies have been carried out on plant derived antifeedants and the most vibrant example is azadirachtin which is extracted from the seeds of Indian neem tree (Azadirachta indica) (Isman et al. 1997). Azadirachtin is used to control many insects including aphids, beetles, weevils, and moths. This compound also exhibited antifeedant activity on pine weevils (Bryan 2003; Thacker et al. 2003). In other studies, carvone and nonanoic acid showed potent antifeedant activity against H. abietis (Schlyter et al. 2004; Månsson et al. 2006).

Methanol extract of pine weevil feces exhibited antifeedant activity towards pine weevils (Borg-Karlson et al. 2006). Moreover, structure activity studies of synthetic hydroxybenzoic acid derivatives revealed many potent antifeeding agents for pine weevils (Legrand et al. 2004; Unelius et al. 2006). Antifeedants are more environmentally friendly because they directly target pest insects.

10

1.6. Towards a new solution based on pine weevil egg protection strategy

Pine weevils use stumps of recently dead trees for

reproduction, they oviposit in the root bark of stumps or in the soil nearby roots with a preference for moist areas (Nordlander et al. 1997). Eggs are laid in soil or in special egg cavities made in the root bark of stumps. The female pine weevil excavates a cavity with her snout, places an egg, adds her feces along with the egg and closes the cavity with the chewed bark (Borg-Karlson et al. 2006), which may protect her egg from conspecific predation (Nordlander et al. 1997). Inserting eggs in cavities and adding feces along with the eggs is a common behavior in many other weevils (Trudel et al. 2001; Zhang & Langor 2004) and also other Hylobius species (Wilson & Schmiege 1975).

In a previous study, weevil feedings were not observed close to oviposition sites (Bylund et al. 2004; Borg-Karlson et al. 2006). Moreover, Egg protection habit is well known in many insects. Females add some chemical cues at oviposition site to protect their eggs from predation and also as an indication of oviposition site for other female to reduce competition between offspring (Blum & Hilker 2008).

In a previous study, methanol extract of pine weevil feces showed antifeedant activity against pine weevils lasting for some hours (Borg-Karlson et al. 2006). However, it takes 1–4 weeks for an egg to hatch (Eidmann 1974; Leather et al. 1999) hence, there should be deterrence for conspecifics and predators over this time period. Here we propose an hypothesis about the egg protection strategy of female pine weevils such that there might be microorganisms associated with pine weevil frass that grow in the moist conditions around egg laying sites and produce repellents and antifeedants for conspecifics (Azeem et al. 2013).

11

1.7. Microbes

Microorganisms are indispensable components of our ecosystem. They make possible the cycles of carbon, oxygen, nitrogen, and sulfur that take place in terrestrial and aquatic systems (Prescott et al. 2002). Insects, being the most diverse macroscopic organisms on the planet, are inevitably associated with microscopic organisms including bacteria, fungi, protozoa, nematodes, viruses and multicellular parasites (Sanchez-Contreras & Vlisidou 2008). These interactions may range from neutral to positive like commensalism, mutualism and endosymbiosis. Alternatively, these interactions can be negative for either of the partners, like antagonism or pathogenicity. In commensalism, one organism gets a benefit without being beneficial or harmful to the other organism. In a mutualistic interaction both organisms get benefit from each other. In antagonistic and pathogenic interactions, one organism benefits at the expense of the second organism. All these different types of interactions are known between insects and microorganisms. The association of bacteria and fungi and their interactions with Hylobius abietis is the focus of this thesis.

1.7.1. Bacteria

A number of interactions between bacteria and insects are known. Some bacteria like Bacillus thuringiensis have strong antagonistic relationships with insect so they can kill the insects (Aronson et al. 1986). On the other hand many bacteria have strong endo-symbiotic relationship with insects (Dillon & Dillon 2004; Gibson & Hunter 2009). Bacteria facilitate their hosts in digesting polymeric components of their food or providing necessary nutrients to the host (Hoyoux et al. 2009; Adams et al.

12

2011). In some cases bacteria can also help their host insects to cope with toxic chemicals in their food (Dobler et al. 2011) or produce antifungal compounds to protect their host from pathogenic fungi (Currie et al. 1999; Cardoza et al. 2006). Up to now, there is no report on the association of bacteria with pine weevil.

1.7.2. Fungi

Like bacteria, some fungi have antagonistic interactions with insects. Metarhizium robertsii and Beauveria bassiana can kill many insects including pine weevils (Abdul-Wahid & Elbanna 2012; Ansari & Butt 2012; Behie et al. 2012).

Fungi are also well known for their mutualistic relationship with arthropods (Yamaoka et al. 1997; Rivera et al. 2009; Roe et al. 2011). They benefit their associated organisms in a number of ways including food digestion (Suh et al. 2003; Suh et al. 2005; Rivera et al. 2009), providing nutrient rich food to their hosts (Currie et al. 1999; Long et al. 2010) and facilitating colonization of wood substrate (Krokene & Solheim 1998).

A special example is the leaf rolling weevil that inoculate egg laying site with their associated fungi (Kobayashi et al. 2008) to protect their larvae from pathogens and provide them readily digestible food (Li et al. 2012). Similar behavior is also found in stag beetles (Tanahashi et al. 2009).

Previously, some fungal species were reported to be associated with the pine weevil. These fungi were found on the body (Levieux & Cassier 1994; Levieux et al. 1994) or in the feces (Kadlec et al. 1992) of the weevils. Moreover, the contribution of pine weevils for the dispersal of Ophiostomatoid fungi to conifer seedlings has also been described (Piou 1993; Jankowiak & Bilanski 2013).

13

1.8. Metabolites of bacteria and fungi

Fungi and many bacteria are heterotrophic; unlike plants they are unable to produce their essential nutrients. Instead they utilize organic molecules from other organisms such as plants or animals as sources of carbon and energy. They obtain their food from living organisms as well as from the degradation of dead organisms. These microbes not only acquire enough energy and nutrients from other organisms essential for their growth, development and reproduction but also utilize it to produce a wide variety of volatile and non-volatile organic compounds (Demain 1999; Thakeow et al. 2006; Karlshoj et al. 2007; Citron et al. 2012).

The function of these organic compounds produced by bacteria and fungi is not fully understood, but it seems that the compounds have many different purposes owing to their remarkable variety and many different chemical structures. Toxins are well known to be produced by bacteria (Moita et al. 2005; Alfonzo et al. 2012) and fungi (Ezra et al. 2004; Karlshoj et al. 2007). These metabolites with toxic effects might be used by bacteria and fungi to compete or kill their competitors (Schnepf et al. 1998; Quesada-Moraga & Vey 2004; Quesada-Moraga et al. 2006). However, there are reports describing fungal species that use their volatile metabolites to attract insects (Fäldt et al. 1999; Becher et al. 2012) which may be a tool to disperse their spores, on the other hand some fungi produce insect repelling compounds (Daisy et al. 2002; Wang et al. 2007), perhaps to reduce competition on a food source.

14

OH

O

OCH3

OHO

OH

OCH3

Methyl salicylate 2-Methoxy-4-vinylphenol

Styrene

OH

Phenol

OH

OCH3

2-Methoxyphenol

H

H

OH

OCH3OCH3

Benzylalcohol3-Methylanisole Estragole

6-Protoilludene

3-Octanone

3-Octanol

OH1-Octene-3-ol

Figure 2. Structure of major compounds from microbes associated with pine weevil

15

2. Materials and Methods

2.1. Collection of weevil frass and feces

Field collected pine weevils of both sexes (Paper I & III) or only female weevils (Paper I) were starved for 24-48 hrs. Water was provided by placing an autoclaved wet filter paper in their storage container. Starved weevils were placed in a sterilized container with a sieve at the bottom, which fitted into another sterilized container (Figure 3).

Figure 3. Setup used for the aseptic collection of weevil frass and feces

The weevils were provided with fresh surface sterilized P. sylvestris twigs along with water. The pine weevil frass (mixture of their feces and pine bark particles chewed by weevils) fell through the sieve into the container below. Fully fed weevils (after 48-72 hrs) were placed in similar container with only water and their feces collected in the container below. Frass and feces collections were repeated at least seven times over a period of

16

three years. Each time fresh weevils and P. sylvestris twigs from different trees were used.

2.2. Microbial culturing media

A set of culturing media were developed and frequently used during the studies. Weevil frass agar (WFA)

WFA was used for the isolation of microbes from weevil frass and feces. It was also used to maintain pure microbial isolates (Paper I, II & III). The culturing medium was prepared by adding 10 g weevil frass (WF) and 20 g agar in 1000 ml demineralized water and the mixture was autoclaved for 40 min at 121 °C. Weevil frass broth (WFB)

WFB was used to cultivate microbial consortium and pure microbial isolates (Paper I, II, & III) for the analysis of volatiles organic compounds (VOCs). The medium was prepared by adding 20 g WF in 1000 ml demineralized water and autoclaving the mixture at 121 °C for 40 min. Potato dextrose agar (PDA)

This culturing medium was used to isolate bacteria and yeasts. Furthermore, it was also used to maintain microbial isolates (Paper I) and to conduct fungal inhibition bioassay (Paper IV). It was prepared by dissolving 20 g potato extract, 20 g

17

glucose and 15 g agar to 1000 ml demineralized water and autoclaving the mixture for 20 min at 121 °C.

2.3. Isolation of microbes

In order to maximize the number of different microbes isolated from weevil frass or feces, three different incubation methods were used. These methods were modified from Peterson et al. (2009).

A. Moist weevil frass or feces (1 g) were incubated in a beaker.

B. Weevil frass or feces particles (5 mg) were spread on WFA plates without adding water.

C. Serial dilutions of weevil frass or feces (5 mg) suspension in water were prepared and spread on WFA Petri plates.

Microbial colonies growing on frass or feces were isolated

according to the method described by Peterson et al. (2009) and Lam et al. (2010) with some modifications. For this purpose WFA and PDA culturing media in Petri plates were used. Method A was used for the isolation of fungi as described in Paper I whereas Methods B and C were used in Papers II & III.

Isolated microbes were grouped based on their morphological characteristics including color, texture, diffusible pigment, growth zone and growth rate after culturing on WFA and PDA plates. In addition, the comparison of produced VOCs when growing on WFB was used as an extra tool to confirm the grouping (Paper I, III).

18

There are many reports demonstrating that microbes can be differentiated by analyzing their VOCs (Cox & Parker 1979; Larsen & Frisvad 1995; Fischer et al. 1999; Fiedler et al. 2001; Karlshoj et al. 2007; Zhu et al. 2010). At least two isolates from each distinct group were identified by molecular methods (Paper I, III).

2.4. Identification of microbes Fungal DNA extraction and PCR (Paper I & III)

Pure fungal isolates were cultivated in GYE broth for two days and a pellet of the cells was obtained by centrifugation. DNA from the cell pellet was liberated by mechanical beating using glass beads in a phosphate buffer. DNA was isolated from the fungal isolates according to the method described by Adams & Frostick (2009). The ITS region of the DNA was amplified by PCR using universal primers ITS 1 and ITS 4 (Geib et al. 2008). Bacterial DNA extraction and PCR (Paper I)

Pure bacterial isolate was cultivated in nutrient broth (NB) overnight. The cell pellet was obtained by centrifugation and DNA was extracted from the cell pellet using illustra bacteria genomic Prep Mini Spin Kit (GE Healthcare) according to the protocol provided by the manufacturer. The 16SrRNA gene was amplified using 27f and 1492r primers (Frank et al. 2008).

19

Sequencing and identification

The PCR products of bacterial and fungal isolates were sequenced by the KTH Genome Center, Biotechnology School (Paper III) and at Macrogen (Korea) (Paper I). The sequences were analyzed by using a BLAST search in the NCBI database.

2.5. Collection of volatiles

Living organisms often produce small quantities of volatile organic compounds (VOCs). In order to detect the minute amounts of VOCs in the large volumes of their headspace (HS) an enrichment is often necessary prior to chemical analysis (Fäldt et al. 2000). HS is the gas space above a sample in an open or closed container. These VOC sample enrichment and collection techniques are normally categorized as static or dynamic.

2.5.1. Static or biodynamic collection of volatiles using SPME

Solid phase micro extraction (SPME) is often used for collecting VOCs under static conditions, where volatiles are adsorbed on polymer matrixes coated on silicon fibers (Zhang & Pawliszyn 1993). For the collection of volatiles by SPME an equilibrium of analytes between the SPME fiber, the releasing source and the HS is desirable. However, with living organisms like microbes, this equilibrium condition is never reached because they continuously release metabolites in their HS; therefore the term “biodynamic” collection can be used instead of “static”.

20

VOCs from the HS of un-inoculated (control) and

microbe inoculated WFB were collected by SPME. The SPME fiber used for the studies was 65 µm polydimethylsiloxane /divinylbenzene (PDMS/DVB) coated on a stable flex fiber (Supelco, USA). Microbes were cultivated in 50 ml WFB placed in 250 ml Erlenmeyer flasks and VOCs were collected by inserting the SPME fiber in their HS for 15 min (Paper III) or 30 min (Paper I, II) or 60 min (Paper I) through a pin hole in the aluminum foil cover (Figure 4a).

2.5.2. Dynamic collection of volatiles

In the dynamic VOCs collection method, an air stream is passed over a sample and through a glass tube packed with an adsorbent (for instance, Tenax TA, Porapak Q or charcoal). Volatiles trapped on the adsorbent are eluted thermally or using an appropriate solvent (Fäldt et al. 2000). This method has advantages over SPME that it is more reliable for quantification than SPME and the sample can be stored for a longer period if desorbed in a solvent.

In this study, dynamic collection of volatiles was used for the analysis of styrene produced by Penicillium expansum from forest waste (Paper V). Tenax TA (60/80 mesh, Supelco, Sweden) was used as an adsorbent packed in glass tubes. The HS air of 1 L flasks was pumped through the Tenax TA using a pump (NMP 830 KNDC B, KNF Germany) and flow meter at the rate of 200 ml/min for 60 min (Figure 4b). The adsorbed volatiles were desorbed in (330 µl or 550 µl) hexane and 1 µl of the hexane extract obtained was used for the GC-MS analysis.

21

Figure 4. Collection of volatiles (a) headspace volatiles collection using SPME (static or

biodynamic), (b) setup used for the collection of volatiles under dynamic condition.

2.6. Identification of volatiles

A Varian 3400 GC was used to separate the VOCs produced by the microbes. The GC was equipped with either a DB-WAX (Agilent USA) or a SPB-1 (Supelco, USA) column with 30 m length, 0.25 mm internal diameter and 0.25 µm film thickness. Isothermal temperature was used for GC injector and interphase between GC and MS. High purity helium at a constant pressure of 10 psi was used as the carrier gas. The temperature program was adjusted to obtain highest possible separation of the volatiles (for more details se Paper I, II and III).

The GC was connected to a Finnigan SSQ 7000 mass spectrometer (MS) equipped with electron ionization (EI). Ion source temperature was 150 °C and electron energy used for ionization was 70 eV. The MS scan range was 30-600 m/z. The electron ionization is a hard ionization technique, which causes the molecular ion to fragment with characteristic patterns for each compound. These patterns are useful for compound identification.

22

In addition to commercially available reference compounds, retention indices and MS libraries (NIST-2008 and Chem Finder 3) were used for compound identification.

2.7. Bioassay

2.7.1. Pine weevil behavior

The response of both sexes of H. abietis towards selected microbial VOCs was examined in a multi-choice arena (Paper II & III). Compounds were presented as pure reference and in combination with conifer host odor (fresh pine twigs). Each of the following four treatments was used randomly in four of the 16 traps: (1) fresh pine twig, (2) fresh pine twig + dispenser with test compound, (3) dispenser with test compound, and (4) empty dispenser (Figure 5). The behavior of 50 male or female weevils was observed on two parallel arenas for 18 hrs; the test was replicated 5 times for each sex.

2.7.2. Fungal inhibition To test the antagonistic activity of selected bacterial

isolates the PDA plate was prepared as shown in Figure 6. An aliquot of fifty µl bacterial isolate suspension in water (1E8 CFU/ml concentration) was evenly applied in the small compartment of the PDA plate and a plug of fully grown fungus was placed at the center of remaining area of the plate (Figure 6). The length of mycelia growth on test and control plates was marked and measured from the center of fungus plug to the tip of the mycelium.

23

Figure 5. Multi-choice arena set up for testing pine weevil behavior (a) arena from above (b) close-up of one of 16 traps on arena (c) weevil collection setup. The number of weevils in each trap was counted after the test occasion (d) inside of a trap, pine twig and test compound dispenser hanging far from the entering pine weevils

Figure 6. Bioassay set up for testing antagonistic activity of bacterial isolates against fungi

24

2.8. Data analysis

Analysis of variance (ANOVA) with Duncan’s post-hoc tests were used to determine the significant difference (p < 0.05) between fungal growth on control and test Petri plates, moreover the difference in fungal inhibition activity of different bacterial isolates on a fungus species (Paper IV) by using program package Statistica 10 (Statsoft Inc. USA).

The response of female and male pine weevils to different treatments in the multi-choice bioassay was analyzed separately using a logistic regression (Paper II & III). This was performed by fitting a generalized model to each data set using the procedure PROC GENMOD in SAS® (SAS Institute, version 9.2).

25

3.

Microbes associated with Hylobius abietis

(Paper I)

Female pine weevils deposit their feces and chewed bark at the oviposition sites to protect eggs from conspecifics. To test our hypothesis that microbes in the frass produce antifeedants and repellents against conspecifics, pine weevil feces and frass were collected in the lab under controlled conditions. In order to mimic the conditions at the weevil egg laying site, collected frass was incubated under moist conditions at room temperature and the HS volatiles were analyzed. In this study we focused on the following points:

1. Which microbes are persistently isolated from feces and frass replicates?

2. Which VOCs are emitted by the microbial consortium of weevil frass (to mimic the oviposition site conditions)?

3. Identification of VOCs produced by pure microbial isolates after cultivating them on WFB.

A total of sixty eight microbial isolates isolated from five

replicates of weevil frass and feces. The number of isolates was calculated in such a way that each distinct type of isolate was counted once from each frass and feces sample irrespective of its abundance on the culture plate. The same numbers of microbial isolates were isolated from frass and feces replicates; moreover, there was no difference in microbes isolated from male and

26

female feces/frass. More isolates of filamentous fungi were isolated from frass and feces compared to bacteria and yeast.

Bacterial isolates

One bacterium, Ewingella sp., was isolated from 80% of frass and feces replicates. It was found in three replicates of frass and five replicates of feces. The occurrence of Ewingella sp. in greater numbers in feces replicates compared to frass suggests that it might exist in the weevil’s gut. Previously, Ewingella americana has been reported in the intestines of mollusks (Muller et al. 1995), and the malaria mosquito Anopheles stephensis (Pumpuni et al. 1993). Recently, E. americana has been reported to be associated with the pine wood nematode Bursaphelenchus xylophilus (Wu et al. 2013). Yeast isolates

Two yeast species, Candida sequanensis and Debaryomyces hansenii, were frequently found in weevil frass and feces replicates. D. hansenii and C. sequanensis were isolated from four out of five replicates of feces. C. sequanensis was isolated from two frass samples, and D. hansenii was isolated from three frass replicates.

C. sequanensis has been isolated from oat grains (Saez & Demiranda 1984). However, there is no previous report describing its association with arthropods. D. hansenii has been reported to be associated with insects gut, for example in the gut of click beetle Melanotus villosus (Ravella et al. 2011) and in the gut and excrements of cockroach, Blattella germanica (Zheltikova et al. 2011).

27

Filamentous fungi isolates

Overall four species of Penicillium were isolated from five replicates of frass and feces. Some species were frequently found from frass/feces samples whereas others were isolated from one or two replicates of frass and feces. P. solitum and P. expansum were the dominate species isolated from 70% and 60% of frass/feces samples respectively. On the other hand, P. spinulosum, P. lividum and P. expansum-2 were only found in 30% and 20% of frass/feces samples respectively. Penicillium spp. were more frequently found in frass compared to feces. Mucor racemosus was equally isolated from frass and feces samples i.e. isolated three times from both frass/feces samples.

Penicillium spp. are common soil fungi that can be found on degrading matter in nature (Fischer et al. 1999). Kobayashi et al. (2008) and Li et al. (2012) reported the association of Penicillium spp. and Mucor sp. with leaf rolling weevils. The leaf rolling weevils inoculate their oviposition sites with fungi to provide easy digestible food to their offspring in the form of degraded leaf polymers. M. racemosus has also been found in Thrypticus truncates and T. sagittatus larval feeding mines (Hernandez et al. 2007), and in the dung of various animals (Santiago et al. 2011).

Ophiostoma piceae was the most abundant fungal species, isolated from 80% of the feces/frass samples. It was found in equal numbers of frass and feces replicates i.e. 4 out of 5. A closely related species, O. pluriannulatum was isolated from three replicates of frass and two replicates of feces.

Ophiostoma spp. are plant pathogenic fungi that cause sap staining in many tree species. Their association with bark beetles is well known (Yamaoka et al. 1997; Krokene & Solheim 1998). Piou (1993) reported the presence of Ophiostoma spp. in the

28

galleries of new emerging H. abietis. Many related fungi were also identified from the feeding scars of weevils on small seedlings (Levieux & Cassier 1994; Jankowiak & Bilanski 2013).

VOCs of microbial consortium

Seventeen compounds were found in the HS above a culture of microbial consortium cultivated in WFB that were not present in the HS of un-inoculated WFB. The identified compounds were: isopentylalcohol, styrene, 3-octanone, anisole, 3-methylanisole, 6-protoilludene, α-cedrene, phenol, estragole, β-sesquiphellandrene, 3-pinanone, methyl salicylate and 2-methoxyphenol. There were some un-identified sesquiterpenes. VOCs of pure microbial isolates

VOCs identified from the HS of pure microbial isolates after cultivating them in WFB are listed in Table 1. Major VOCs were styrene, octatriene isomers, 6-protoidullene, 3-octanol, 3-octanone, 1-octene-3-ol, methyl salicylate, 2-methoxyphenol, and 2-methoxy-4-vinylphenol.

The isolated microbial species produced several VOCs but there were some characteristic volatiles that could be used to differentiate the species from each other when using the same medium. 6-Protoilludene was only produced by two Ophiostoma species. The amount of this compound from O. piceae was far greater that from O. pluriannulatum. The unknown sesquiterpene was the characteristic constituent of the A4-25D2 fungal isolates.

Octatriene isomers and aliphatic alcohols were produced by some Penicillium spp. but those could be differentiated from each other due to the presence of one or two other metabolites (Table 1). Two strains of P. expansum were identified based on

29

their VOC profile, the major VOCs of P. expansum-1 were styrene and 3-methylanisole whereas P. expansum-2 produced four octatriene isomers and 3-methylanisole.

Phenolic compounds such as 2-methoxyphenol, 2-methoxy-4-vinylphenol and phenol were the characteristic metabolites of bacterial and some yeast isolates. Ewingella sp., Candida sp., and Hormonema sp. can be differentiated from each other on the basis of their most abundant VOCs (Table 1). From this study we can conclude that if controlled conditions and “special culturing media” are used the microbial VOCs can be used for initial screening, parallel to morphological characteristics.

30

Table 1. VOCs produced by pure microbial isolates after cultivating them in WFB

Ewi† Cse Hde Pe1 Pso Pe2 Pli Psp Opi Opl Dha Eca A4 Mre Octatriene isomers ‡‡‡ ‡‡‡‡ Ethanol ‡‡ ‡‡ Isopentylalcohol ‡‡ ‡‡ ‡ ‡‡ ‡

Hexanol tr ‡‡

Heptanol ‡‡

3-Octanol* ‡‡ ‡‡‡‡ ‡‡‡‡ ‡‡

3-Octanone ‡‡ ‡‡‡‡ ‡‡‡‡ ‡‡ ‡‡ 1-Octen-3-ol* ‡‡‡ ‡‡ ‡‡‡ ‡ ‡‡ ‡‡

p-Menthan-7-ol* ‡‡ 3-Caren-10-al* ‡‡ Unknown sesquiterpene* M204ꜜ ‡‡‡‡ γ-Muurolene* ‡‡‡ 6-Protoilludene* ‡‡‡‡‡ ‡‡ β-Sesquiphellandrene* ‡‡ ‡‡

Styrene ‡‡‡‡ ‡‡‡ ‡‡‡‡ ‡‡‡‡ Benzylalcohol ‡‡

2-Phenylethanol ‡ tr Benzylmethylether ‡‡ 1,4-Dimethoxy benzene ‡‡‡

31

4-Ethyl-1,2-dimethoxybenzene ‡‡ Methyl 4-isopropylbenzoate ‡‡ Methyl salicylate ‡ ‡ ‡‡‡ ‡‡‡ ‡‡‡ ‡‡ ‡

Phenol ‡‡ ‡‡ ‡‡ ‡‡ Anisole ‡ ‡‡‡ 3-Methylanisole ‡‡‡ ‡‡‡‡ ‡‡ 4-Ethylanisole ‡‡ ‡‡‡ tr Estragole (4-allylanisole) ‡‡‡ ‡ ‡‡‡ 4-Vinylanisole ‡‡ ‡‡ 2-Methoxyphenol ‡‡‡ ‡‡ 2-Methoxy-4-vinylphenol ‡‡‡ ‡‡‡ ‡‡‡

In the table above ‡‡‡‡‡ denotes for > 1E9 TIC counts, ‡‡‡‡ 1E9 - 1E8, ‡‡‡ 1E8 - 1E7, ‡‡ 1E7 - 1E6, ‡ 1E6 - 5E5 and tr for less than 5E5 Ewi (Ewingella sp), Cse (Candida sequanensis), Hde (Hormonema dematioides), Pe1 (Penicillium expansum-1), Pso (P. solitum), Pe2 (P. expansum-2), Pli (P. lividum), Psp (P. spinulosum), Opi (Ophiostoma piceae), Opl (O. pluriannulatum), Dha (Debaryomyces hansenii), Eca (Eucasphaeria capensis), A4 (A4-25D2), and Mre (Mucor racemosus) ꜜ MS of unknown sesquiterpene: 204, 189, 164, 151,149, 135, 123, 109, 95, 43 *Enantiomers of chiral compounds were not separated † Species in bold were frequently isolated (> 50%) from pine weevil frass and feces replicates

32

4.

Fungal volatiles affect pine weevil behavior

(Paper II & III)

In these studies we focused on the following questions:

1. What is the effect of incubation time on the production of VOCs from different microorganisms?

2. How do the pine weevils behave in the presence of microbial produced VOCs? Methylsalicylate (MeS) was produced by a number of

microorganisms isolated from H. abietis frass and feces. Three of those microbes, D. hansenii, O. piceae and O. pluriannulatum, produced MeS in large amounts when cultured in WFB. Similarly, styrene was also produced by a number of microorganisms. Three fungal species, P. expansum, P. solitum and C. sequanensis, produced styrene in higher amounts compared to others microbes (Paper I). Effect of incubation time on the production of volatiles

(Paper II, III)

The yeast Debaryomyces hansenii started MeS production within 24 hrs of incubation whereas the other two microbes started MeS production at the 5th day of incubation. D. hansenii and O. piceae continued to produce increasing amounts of MeS over 30 days of incubation, in contrast to O. pluriannulatum

33

where MeS amounts reached the highest level at day 10 (Figure 7).

Figure 7. Mean amount of methyl salicylate detected in the headspace of D. han (D. hansenii), O.plu (O. pluriannulatum) and O. pic (O. piceae) microbial species cultivated on weevil frass broth. Volatiles were collected after different time span. Error bars = SE

Interestingly, MeS was only produced by the microbes when cultivated on WFB or wood related media and not when cultivated on synthetic or semi synthetic media. This suggests that MeS is a degradation product of lignin. Besides MeS, two Ophiostoma spp. produced 6-protoilludene (Table 1). Previous studies have reported the production of 6-protoilludene by wood decaying fungi. For instance, Hanssen et al. (1986) identified this compound in the culture broth of Ceratocystis piceae. Furthermore, Thakeow et al. (2006) detected this compound in the HS above beech wood infected with Gloeophyllum trabeum. Engels et al. (2011) used Armillaria gallica to produce 6-protoilludene from farnesyl diphosphate. Unfortunately, it was not possible to collect enough of this compound for NMR analysis or behavioral test on weevils.

The yeast Candida sequanensis started styrene production within 24 hrs of inoculation and continued until the 8th day of

34

incubation. Both Penicillium species started styrene production on the 4th day of inoculation and continued producing it for more than 20 days (Figure 8). In addition to styrene, 3-octanone, 1-octene-3-ol and 3-octanol were also detected in the headspace above P. solitum culture. 3-Methylanisole was produced by P. expansum and 2-methoxy-4-vinylphenol produced by C. sequanensis along with styrene (Table 1). 3-Methylanisole was detected from the cultures a few days after the styrene production started.

Figure 8. Amount of styrene produced by C. seq (Candida sequanensis), P. exp (Penicillium expansum) and P. sol (Penicillium solitum) cultivated on weevil frass broth after different days of incubation. Error bars = SE Pine weevil response to fungal volatiles Effect of MeS (Paper II)

Pine weevil response to MeS was tested in a multi-choice arena (Figure 5) using dispensers with two different release rates of MeS (0.4 mg and 1.4 mg per 24 hrs). A greater number of weevils were caught in traps loaded with host odor (freshly cut

35

pine twig) compared to traps with pine twig plus MeS and the other two treatments (Figure 9).

Figure 9. Mean number of pine weevils captured in traps with different baits in a multi choice bioassay, examining the effect of MeS (methyl salicylate) at two different release rates (0.4mg and 1.4 mg per 24 hrs). Columns with the same letter are not significantly different (p < 0.05) to each other when comparisons were made between treatments for male and female weevils independently. Error bars = SE

The difference in the number of weevils caught in traps

with pine twigs and traps with pine twigs plus MeS was greater for the higher release rate of MeS compared to those with a lower

36

release rate of MeS (Figure 9). At the low release rate, MeS attracted weevils, compared to empty traps, but the number of weevils caught was far lower than in pine twig plus MeS and pine twig traps. At the high release rate of MeS, there was no significant difference in the number of weevils caught in empty or MeS traps (Figure 9). Taken together, the data indicates that MeS has a masking effect, i.e. in the presence of MeS weevils cannot detect their host plant odor. Interestingly, Kännaste et al. (2009) reported similar behavior of pine weevils towards Norway spruce volatiles infested with spider mite which release MeS along with linalool and β-farnesene. Effect of styrene (Paper III)

In these tests host plant traps caught significantly more weevils than empty traps. Furthermore, significantly fewer weevils were caught in the traps containing styrene plus host plant compared to host plant alone. Pure styrene without the host odor did not have any effect on pine weevil behavior; similar numbers of weevils were caught in traps with styrene as in empty traps (Figure 10). Effect of 3-methylanisole (Paper III)

Male pine weevil response was greater towards 3-methylanisole compared to styrene. The difference between the number of male weevils caught in pine twig traps and pine twig plus 3-methylanisole was greater compared to those with styrene, but it was comparable to those traps with the higher release rate of MeS (Figure 9, 10 & 11). Female weevils did not differentiate between the odor of pine twig and pine twig plus 3-methylanisole.

37

Figure 10. Mean number of pine weevils captured in traps with different baits in a multi choice bioassay, examining the effect of styrene (5 mg/ 24 hrs). Columns with different letters are significantly different from each other when comparisons were made between treatments for male and female weevils independently. Error bars = SE Effect of fungal volatiles on the attraction of the pine weevil to host odors (Papers II and III)

It has been shown previously that pine weevils are highly attracted to α-pinene (Nordlander 1990) which is present in the phloem and bark of P. sylvestris (Sjödin et al. 1996). Similar compounds were present in the freshly cut pine twigs used in this behavioral study as host odor. However, the addition of microbial produced compounds (i.e. MeS, styrene and 3-methylanisole) to the attractive host volatiles reduced their attraction for pine weevils. We hypothesized that “microbes in weevil frass grow in the moist conditions at the egg laying site and produce conspecific repelling compounds”. The avoidance of pine weevils to their favorite host plant volatiles in the presence of microbial produced compounds favors our hypothesis.

38

Figure 11. Mean number of pine weevils captured in traps with different baits in a multi choice bioassay, examining the effect of 3-methylanisole (1 mg per 24 hrs). Columns with different letters are significantly different from each other when comparisons were made between treatments for male and female weevils independently. Error bars = SE

The tested compounds did not show any repellency against weevils when presented in pure form but in combination with the weevils host plant they deterred the weevils significantly. The presence of these compounds in host plant odor might be a signal of microbial infection in food. In a previous study, infection of Phlebia gigantea and Trichoderma harzianum to pine twigs significantly reduced the attraction of pine weevils towards infected twigs compared to uninfected twigs (Skrzecz & Moore 1997). Avoidance from fungal infected food is known in many other phytophagous insects (Rayamajhi et al. 2006; Röder et al. 2007; Menjivar et al. 2011).

Moreover, the time span of host masking VOC production from weevil frass/feces microbes is also related to egg protection. It takes 1-4 weeks for the weevil egg to hatch (Eidmann 1974; Olenici & Olenici 2007). It seems that volatiles from different

39

microbes protect the egg from conspecifics at different time periods. The yeasts start producing odor masking compounds (styrene and MeS) during early days of incubation whereas filamentous fungi produced the same compounds during the later period.

40

5.

Inhibition of Heterobasidion and Ophiostoma species by Bacillus subtilis A18 – A19

(Paper IV)

Introduction

Heterobasidion species are important pathogens of conifers throughout the Northern Hemisphere. These fungi cause root and butt rot disease to several species of conifers and the result is a significant economic loss to the forest industry in Northern Europe (Woodward et al. 1998). The economic loss related to Heterobasidion infection of conifers in Europe is estimated about 800 million euros annually (Asiegbu et al. 2005). There are three major species of Heterobasidion found in Europe, H. annosum, H. parviporum and H. abietinum (Niemelä & Korhonen 1998), of them H. annosum and H. parviporum are the most important.

Heterobasidion spp. produce basidiospores that reach stumps via air or from insects colonizing the stump roots which in turn infect healthy tree roots via root connections (Asiegbu et al. 2005). In managed forests the stumps are frequently treated with chemicals (urea or borates) or Phlebiopsis gigantea to prevent Heterobasidion infection after thinning. Chemical treatments create ecological problems and P. gigantean is not very effective because it does not show any antagonistic activity against Heterobasidion. However, it does prevent Heterobasidion infection by competing for nutrient resources and is now widely used for the protection of stumps.

41

Ophiostoma piceae is known to be associated with bark

beetle (Krokene & Solheim 1998) and pine weevil. This fungus does not degrade wood but creates blue staining in sap wood (Fleet et al. 2001) and as a result decreases the economic value of timer to be used for furniture or pulp and paper making.

Antagonistic activity of B. subtilis A18 – A19

B. subtilis A19 started inhibiting H. parviporum and O. piceae from day 6 after inoculation whereas on H. annosum the inhibition activity was observed on the 8th day of incubation. B. subtilis A18 started inhibiting all test fungi from day 8 (Figure 12, 13, 14). A19 exhibited significantly higher activity against H. parviporum and O. piceae (p < 0.05) than A18 throughout the incubation period (Figure 12, 14). Both B. subtilis strains showed similar inhibition activity against H. annosum.

The bacterial strains were inoculated in the form of water suspension (5E5 CFU/plate) at a 30 mm distance from the fungal inoculum so bacterium took some days to grow on plates and produce antifungal compounds. These compounds diffused through the medium and inhibited the fungal growth after 6 or 8 days of inoculation. The inhibitory effect was only found on the side of fungal growth facing bacterial growth directly whereas on the opposite side the fungal growth was comparable to that on control plates (Figure15). This indicates that these bacterial isolates inhibited fungi growth by producing non-volatile compounds. Moreover, butanol extract of bacterial cultures grown for 5 days in potato dextrose broth showed inhibition for both Heterobasidion spp. (Paper IV).

42

Figure 12. Growth of H. parviporum in the absence and presence of B. subtilis A18 and A19. Columns with different letters are significantly different (p < 0.05) to each other. Comparisons were made between treatments within a day of observation. Error bars = SE

Figure 13. Growth of H. annosum in the absence and presence of B. subtilis A18 and A19. Columns with different letters are significantly different (p < 0.05) to each other. Comparisons were made between treatments within a day of observation. Error bars = SE

43

Figure 14. Growth of O. piceae in the presence and absence of B. subtilis A18 and A19 strains. Columns with different letters are significantly different (p < 0.05). Comparisons were made between treatments within a day of observation. Error bars = SE Other tests

Cell free supernatant of culture broth of B. subtilis A 19 reduced the germination of O. piceae spores (Figure 16 b, c). Even fewer spores were germinated when culture broth with bacterial cells was tested (Figure 16d).

In another test, when the bacterium suspension with a higher cell count was spread over the whole surface of a PDA plate and followed by fungal inoculation, no fungal growth was observed at all.

During a preliminary study, both B. subtilis strains were able to grow on WFA and inhibited the growth of all the tested fungi for two weeks, however, the inhibition activity was lower than that on PDA medium.

44

Figure 15. Effect of B. subtilis A19 on the growth of fungi (a) Ophiostoma piceae, (b) Heterobasidion annosum and (c) H. parviporum. In upper plates bacteria and each fungus were co-cultured whereas in lower plates only respective fungi are inoculated (control)

Figure 16. Effect of B. subtilis and culture extracts on spore germination of Ophiostoma piceae. (a) control culture, (b) water extracts of culture supernatant (10 days), (c) and (d) 3 day old bacteria culture supernatant alone and with bacteria grown in nutrient broth respectively.

The most commonly used Heterobasidion infection controlling fungus (P. gigantea) only possesses preventive activity due to its fast growth rates on stumps compared to

45

Heterobasidion. In some previous studies, Heterobasidion infections have been observed in stumps that were not fully covered with P. gigantea suspension (Berglund & Rönnberg 2004; Rönnberg & Cleary 2012).

The B. subtilis strains A18 – A19 exhibited the potential to inhibit root rot fungi and could act as future candidates for controlling Heterobasidion infection of stumps. Furthermore, they are potential candidates to be tested for curative measures of infected plants by inoculating them on plant roots. From a chemical prospective, further studies are needed to isolate and identify antifungal compounds from these isolates.

46

6.

Sustainable bio–production of styrene from forest biomass

(Paper V)

Introduction

Everyday life is highly dependent on polymeric products. Styrene, which is the principal monomer of ubiquitous polystyrene plastics and resins, is synthesized from petroleum products (James & Castor 2005). Due to dwindling world petroleum resources alternative ways of producing energy and important industrial compounds are needed, preferably using environmentally friendly ways and utilizing biomass as starting material (Cherubini 2010; McKenna & Nielsen 2011).

Over the last few decades interest in using renewable resources for energy production and raw materials for large consumer industrial products has increased. Agriculture and forestry waste are major sources of biomass that could be used for this purpose. Wood bark, saw dust and leaves are an abundant and cheap biomass source for making energy and industrial raw materials. Previously, the possibility of using glucose as a starting material for the bio-production of styrene has been investigated (Beck et al. 2008; McKenna & Nielsen 2011).

Here, we investigated the potential of Penicillium expansum isolated from pine weevil frass for the production of styrene using different kinds of forest waste biomass abundantly available in Sweden. The fungus was cultivated on forest biomass

47

supplemented with yeast extract and the production rate was compared with PDB medium. Styrene production using pine tree related waste biomass

Styrene production from P. expansum cultivated on pine tree related biomass started after 5 days of incubation whereas from PDB it started after 9 days (Figure 17). The highest production rate was observed from grated pine bark (GPB) with 52.5µg/h after 9 days of incubation. GPB consisted of bark from actively growing stems of pine tree, thus rich in nutrients like nitrogen and minerals. Fungal hyphae could easily penetrate the soft bark to degrade and extract nutrients. The lowest production rate of styrene from fungus was observed when cultivated on pine wood (PW) medium (Figure 17). This low production might be explained by the fact that wood is more compact and rigid compared to the other feeding sources tested here. In a previous study, P. expansum was not able to produce styrene when cultivated on conifer wood medium (Fiedler et al. 2001). Styrene production using bark of different trees

When mature bark of Scots pine, Norway spruce, oak (Quercus robur L.) and birch (Betula pubescens Ehrh.) were used as culturing media, styrene production from all media started after 5 days of fungal inoculation. Fungus grown on pine and birch bark produced the highest amount of styrene on day 5 whereas from oak and spruce bark media the highest production rate was observed after 9 days of inoculation. The highest styrene production among all mature bark media was observed from oak bark medium with 41µg/h and 22µg/h from spruce bark (Figure 18).

48

Figure 17. Styrene production rate from P. expansum cultivated on PDB (potato dextrose broth), GPB (grated pine stem bark), PN (pine needles) and PW (pine wood chips) monitored on four occasions. Error bars = SE

Figure 18. Styrene production rate form P. expansum cultured on mature bark of pine, spruce, oak and birch monitored on four occasions. Error bars =SE

From this screening study, oak bark proved best for the production of styrene with fungus producing pure styrene comparable to PDB medium (Paper V). Fungus grown on GPB

49

medium produced the highest amount of styrene compared to all tested media but the collected VOCs consisted of terpene volatiles from bark itself that decreased the purity of collected styrene. A similar problem was found when using pine, spruce and pine needle media (Paper V).

In this study, a dynamic volatile collection method was used to collect fungal produced VOCs from tested media. The major fungus VOC was styrene in all cases, in contrast to Paper III where 3-methylanisole was the major compound adsorbed on SPME fiber in an older culture. During this study (Paper V), a small amount of 3-methylanisole was observed compared to styrene in collected samples. It could be that fungal culture flasks were well aerated during the experiment described in Paper V compared to culture flasks used in Paper III.

Bark of hardwood and softwood consist of more lignin compared to cellulose and hemicellulose (Harkin & Rowe 1971). Even though the fiber length of bark is similar to wood fiber (Miranda et al. 2012) the high lignin content and lipophilic extractive (Sakai 2001; Miranda et al. 2012) make it unsuitable for making pulp. The major use of tree bark today is for heat and power generation. It might be possible to first use forestry waste for the production of styrene, and the residue can be fed to bio refineries for power generation. Moreover, fungal biomass can be used as a cheap source of protein.

50

7. Conclusions

Microbes were isolated from adult pine weevil frass/feces

that female weevils add to their oviposition sites for the protection of eggs from feeding conspecifics. Both sexes of weevil were deterred from their preferred food source in the presence of microbial volatiles (styrene and methyl salicylate). Both compounds were found to be produced from frass associated microbes for the period needed for an egg to hatch.

Sixty eight microbial isolates representing nine distinct genera were isolated from aseptically collected pine weevil frass and feces. Eight species were isolated from more than 50% of frass/feces replicates. More frequently isolated species were Ewingella sp., Ophiostoma piceae, O. pluriannulatum, Penicillium solitum, P. expansum, Mucor racemosus, Candida sequanensis and Debaryomyces hansenii. Bacteria and yeasts were more frequently found in feces than in frass samples.

The major volatile compounds identified in the HS above pure microbes associated with pine weevils were: styrene, octatriene isomers, 1-octene-3-ol, 3-methylanisole, 6-protoilludene, methyl salicylate, phenol, 2-methoxy-4-vinylphenol, and 2-methoxyphenol. Most of microbes produced phenolic compounds only when cultivated on wood related media (WFB). Moreover, VOCs of pure microbial isolates were also detected in the HS of

51

microbial consortium of weevil frass. If “special culturing medium” is used, it is possible to differentiate microbial isolates based on their VOC chemical profile.

Two isolates of Bacillus subtilis (A18, A19) inhibited the growth of two root rot fungi Heterobasidion annosum and H. parviporum and a blue staining fungi Ophiostoma piceae. Chemicals involved in fungal inhibition are non-volatile water soluble compounds.

Penicillium expansum produced styrene when cultivated on forest waste biomass medium supplemented with yeast extract. When using mature oak bark and potato dextrose broth pure styrene was produced at 41µg/h and 26µg/h respectively.

52

8.

Proposed future study

1. In this study, we found that presence of microbial VOCs in weevil food deter both sexes. It would be interesting to test egg laying behavior of female weevils in the presence of combined odor of host plant and microbial VOCs.

2. Preliminary studies indicated the potential of non-volatile compound of microbes. Thus, it would be interesting to test antifeedant activity of non-volatile metabolites of bacteria and fungi against pine weevils or other insects.

3. Some isolated microbes have the potential to degrade wood polymers e.g. lignin. The polymer degrading activity should be tested by using wood polymers in combination with carbon free minimal medium.

4. Bacillus subtilis strains have the potential to exhibit their antagonistic activity in vivo. They should be studied on roots of small plants to test whether the plants could withstand them and whether these bacteria could inhibit Heterobasidion fungus infection to small plants and trees.

5. Large scale production of styrene from biomass should be developed. Moreover chemical analysis of feeding stock should be done to see which type of structural or non-structural chemicals are consumed from the bark when grown with fungus for the production of styrene. Moreover, the fungus medium should be extracted with solvents and tested for antifeedant activity against pine weevil.

53

9. Acknowledgements

Thank to Almighty Allah ‘the most beneficent, the most merciful,’ for giving me the strength, steadfastness and patience to complete this challenging task.

I am extremely thankful to my supervisor Prof. Anna

Karin Borg-Karlson for accepting me as a PhD student under her kind supervision, giving me the opportunity to work on such an interesting and interdisciplinary project, for sharing her knowledge in such a nice way, and for being so encouraging, kind and patient during the years.

Prof. em. Tobjörn Norin, Dr. Jenny Lindh, and Dr. Akbar Ali are gratefully acknowledged for their valuable comments and suggestions on this thesis and Dr. Douglas Jones for linguistic revision and nice comments.

The projects provided me an opportunity to work with Dr. Gunaratna Kuttuva Rajarao, Prof. Göran Nordlander and Henrik Nordenhem, Dr. Kazuhiro Nagahama and Karolin Axelsson. Thank you very much all of you for sharing your knowledge and for your contributions to this thesis and publications.

I would like to express my sincere gratitude to Dr. Kazuhiro Nagahama and Dr. Gunaratna for teaching practical microbiology; Prof. Peter Baeckström for introducing and explaining MPLC technique and for being so nice and friendly; Dr. Raimondas Mozuraitis for help in statistics and giving technical training of analytical instruments; Dr. Hesham El-seedi for being an excellent teacher and friend during the years–you are the person who always gave me an inspiration, motivation and encouragement to work hard.

54

I thank Dr. Tao Zhao for being so kind with me since the start of my PhD, helping in learning lab techniques and all the nice conversations on scientific and common topics; All my collaborators on projects not mentioned in this thesis; All my students for providing me the opportunity to work and learn to improve my teaching abilities; Lena Skowron, Ulla Jacobsson and Henry Challis for all your help with practical matters.

I also would like to thank all the former and present members in ecological chemistry group for being nice and kind during my stay, for your cooperation in scientific and social matters; all the present and former colleagues at Organic Chemistry and at other divisions and departments at KTH, who participated in creating the good working environment; all the Pakistani friends for their nice company after study hours, making it possible to live happily far from family.

Higher Education Commission of Pakistan and Formas, Sweden are gratefully acknowledged for financial support that allowed me to pursue my PhD study. The Aulin-Erdtman foundation-KTH, Linder’s foundation-KTH, Hugo and Emma Björkmans foundation-KSLA and International Society of Chemical Ecology, for proving travel grants and making it possible to participate in interesting and inspiring conferences.

I would like to thank my family and friends for their continuous support and care. Specially, my mother for her love, encouragements, support and prayers; my wife for care, motivations and support during the last three years; and my brother, Dr. Akabr Ali, for his limitless support and motivation.

55

10.

References Abdul-Wahid OA & Elbanna SM, 2012. Evaluation of the insecticidal activity

of Fusarium solani and Trichoderma harzianum against cockroaches; Periplaneta Americana. African Journal of Microbiology Research 6: 1024-1032.

Adams AS, Jordan MS, Adams SM, Suen G, Goodwin LA, Davenport KW, Currie CR & Raffa KF, 2011. Cellulose-degrading bacteria associated with the invasive woodwasp Sirex noctilio. The ISME Journal

Adams JDW & Frostick LE, 2009. Analysis of bacterial activity, biomass and diversity during windrow composting. Waste Management 29: 598-605.

Alfonzo A, Lo PS, Conigliaro G, Ventorino V, Burruano S & Moschetti G, 2012. Antifungal peptides produced by Bacillus amyloliquefaciens AG1 active against grapevine fungal pathogens. Annals of Microbiology 62: 1593-1599.

Ansari MA & Butt TM, 2012. Susceptibility of different developmental stages of large pine weevil Hylobius abietis (Coleoptera: Curculionidae) to entomopathogenic fungi and effect of fungal infection to adult weevils by formulation and application methods. Journal of Invertebrate Pathology 111: 33-40.

Aronson AI, Beckman W & Dunn P, 1986. Bacillus thuringiensis and related insect pathogens. Microbiological reviews 50: 1-24.

Asiegbu FO, Adomas A & Stenlid J, 2005. Conifer root and butt rot caused by Heterobasidion annosum (Fr.) Bref. s.l. Molecular Plant Pathology 6: 395-409.

Azeem M, Rajarao G, Nordenhem H, Nordlander Gr & Borg-Karlson A, 2013. Penicillium expansum Volatiles Reduce Pine Weevil Attraction to Host Plants. Journal of Chemical Ecology 39: 120-128.

Becher PG, Flick G, Rozpędowska E, Schmidt A, Hagman A, Lebreton S, Larsson MC, Hansson BS, Piškur J, Witzgall P et al., 2012. Yeast, not fruit volatiles mediate Drosophila melanogaster attraction, oviposition and development. Functional Ecology 26: 822-828.

Beck JJ, Merrill GB, Palumbo JD & O'Keeffe TL, 2008. Strain of Fusarium oxysporum isolated from almond hulls produces styrene and 7-methyl-1,3,5-cyclooctatriene as the principal volatile components. J. Agric. Food Chem. 56: 11392-11398.

Behie SW, Zelisko PM & Bidochka MJ, 2012. Endophytic Insect-Parasitic Fungi Translocate Nitrogen Directly from Insects to Plants. Science 336: 1576-1577.

56

Berglund M & Rönnberg J, 2004. Effectiveness of treatment of Norway spruce stumps with Phlebiopsis gigantea at different rates of coverage for the control of Heterobasidion. Forest Pathology 34: 233-243.

Blum MS & Hilker M, 2008. Chemical Protection of Insect Eggs. In: (eds), Chemoecology of Insect Eggs and Egg Deposition, Blackwell Publishing Ltd, pp. 61-90.

Borg-Karlson AK, Nordlander G, Mudalige A, Nordenhem H & Unelius CR, 2006. Antifeedants in the feces of the pine weevil Hylobius abietis: Identification and biological activity. Journal of Chemical Ecology 32: 943-957.

Bryan W, 2003. Efficacy of plant protection products containing the botanical antifeedant, azadirachtin, against the large pine weevil (Hylobius abietis). In: (eds), BCPC International Congress Crop Science & Technology 2003, Vol 1 and 2, Congress Proceedings, pp. 623-626.

Bylund H, Nordlander G & Nordenhem H, 2004. Feeding and oviposition rates in the pine weevil Hylobius abietis (Coleoptera: Curculionidae). Bulletin of Entomological Research 94: 307-317.

Cardoza YJ, Klepzig KD & Raffa KF, 2006. Bacteria in oral secretions of an endophytic insect inhibit antagonistic fungi. Ecological Entomolgy 31: 636-645.

Cherubini F, 2010. The biorefinery concept: Using biomass instead of oil for producing energy and chemicals. Energy Conversion and Management 51: 1412-1421.

Citron CA, Rabe P & Dickschat JS, 2012. The scent of bacteria: headspace analysis for the discovery of natural products. Journal of Natural Products 75: 1765-1776.

Cox CD & Parker J, 1979. Use of 2-aminoacetophenone production in identification of Pseudomonas aeruginosa. Journal of Clinical Microbiology 9: 479-484.

Currie CR, Scott JA, Summerbell RC & Malloch D, 1999. Fungus-growing ants use antibiotic-producing bacteria to control garden parasites. Nature 398: 701-704.

Daisy BH, Strobel GA, Castillo U, Ezra D, Sears J, Weaver DK & Runyon JB, 2002. Naphthalene, an insect repellent, is produced by Muscodor vitigenus, a novel endophytic fungus. Microbiology 148: 3737-3741.

Day KR, Nordlander G, Kenis M & Halldorson G, 2004. General biology and life cycles of bark weevils. In: Lieutier F, Day KR, Battisti A, Gregoire J-C & Evans HF (eds), Bark and Wood Boring Insects in Living Trees in Europe, a Synthesis., Kluwer Academic Publishers, Dordrecht, pp. 331-349.

Demain AL, 1999. Pharmaceutically active secondary metabolites of microorganisms. Applied Microbiology and Biotechnology 52: 455-463.

57

Dillon AB, Downes MJ, Ward D & Griffin CT, 2007. Optimizing application of entomopathogenic nematodes to manage large pine weevil, Hylobius abietis L. (Coleoptera: Curculionidae) populations developing in pine stumps, Pinus sylvestris. Biological Control 40: 253-263.

Dillon RJ & Dillon VM, 2004. The gut bacteria of insects: nonpathogenic interactions. Annual Review of Entomology 49: 71-92.

Dobler S, Petschenka G & Pankoke H, 2011. Coping with toxic plant compounds - The insect's perspective on iridoid glycosides and cardenolides. Phytochemistry 2011: 25.

Eidmann H, 1974. Hylobius Schönh. In: Schwenke W (eds), Die Forstschadlingc Europas, , Vol, 2 Verlag Paul Parey Hamburg, Berlin, pp. 272-293.

Eidmann HH, Nordenhem H & Weslien J, 1996. Physical protection of conifer seedlings against pine weevil feeding. Scandinavian Journal of Forest Research 11: 68-75.

Engels B, Heinig U, Grothe T, Stadler M & Jennewein S, 2011. Cloning and characterization of an Armillaria gallica cDNA encoding protoilludene synthase, which catalyzes the first committed step in the synthesis of antimicrobial melleolides. Journal of Biological Chemistry 286: 6871-6878.

Ezra D, Hess WM & Strobel GA, 2004. New endophytic isolates of Muscodor albus, a volatile-antibiotic-producing fungus. Microbiology-Sgm 150: 4023-4031.

Fäldt J, Jonsell M, Nordlander G & Borg-Karlson AK, 1999. Volatiles of bracket fungi Fomitopsis pinicola and Fomes fomentarius and their functions as insect attractants. Journal of Chemical Ecology 25: 567-590.

Fäldt J, Eriksson M, Valterova I & Borg-Karlson AK, 2000. Comparison of headspace techniques for sampling volatile natural products in a dynamic system. Zeitschrift Fur Naturforschung C-a Journal of Biosciences 55: 180-188.

Federoff NE, Barrett MR & Vaughan A, 2008. Problem formulation for the imidacloprid environmental fate and ecological risk assessment. eds), US EPA, pp. 1-36.

Fiedler K, Schutz E & Geh S, 2001. Detection of microbial volatile organic compounds (MVOCs) produced by moulds on various materials. International Journal of Hygiene and Environmental Health 204: 111-121.

Fischer G, Schwalbe R, Möller M, Ostrowski R & Dott W, 1999. Species-specific production of microbial volatile organic compounds (MVOC) by airborne fungi from a compost facility. Chemosphere 39: 795-810.

58

Fleet C, Breuil C & Uzunovic A, 2001. Nutrient consumption and pigmentation of deep and surface colonizing sapstaining fungi in Pinus contorta. Holzforschung 55: 340-346.

Franceschi VR, Krokene P, Christiansen E & Krekling T, 2005. Anatomical and chemical defenses of conifer bark against bark beetles and other pests. New Phytologist 167: 353-375.

Frank JA, Reich CI, Sharma S, Weisbaum JS, Wilson BA & Olsen GJ, 2008. Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Appllied Environmental Microbiology 74: 2461-2470.

Geib SM, Filley TR, Hatcher PG, Hoover K, Carlson JE, Jimenez-Gasco MD, Nakagawa-Izumi A, Sleighter RL & Tien M, 2008. Lignin degradation in wood-feeding insects. Proceedings of the National Academy of Sciences of the United States of America 105: 12932-12937.

Gibson CM & Hunter MS, 2009. Extraordinarily widespread and fantastically complex: comparative biology of endosymbiotic bacterial and fungal mutualists of insects. Ecology Letters 13: 223-234.

Hanssen HP, Sprecher E & Abraham WR, 1986. 6-Protoilludene, The Major Volatile Metabolite from Ceratocystis-Piceae Liquid Cultures Phytochemistry 25: 1979-1980.

Harkin JM & Rowe JW, 1971. Bark and its possible uses. Forest Products Laboratory, US Forest Service.

Harvey CD, Alameen KM & Griffin CT, 2012. The impact of entomopathogenic nematodes on a non-target, service-providing longhorn beetle is limited by targeted application when controlling forestry pest Hylobius abietis. Biological Control 62: 173-182.

Hernandez MC, Pildain MB, Novas MV, Sacco J & Lopez SE, 2007. Mycobiota associated with larval mines of Thrypticus truncatus and T. sagittatus (Diptera, Dolichopodidae) on waterhyacinth, Eichhornia crassipes, in Argentina. Biological Control 41: 321-326.

Hill IR, 1989. Aquatic organisms and pyrethroids. Pesticide Science 27: 429-457.

Hoyoux C, Zbinden M, Samadi S, Gaill F & Compère P, 2009. Wood-based diet and gut microflora of a galatheid crab associated with Pacific deep-sea wood falls. Marine Biology 156: 2421-2439.

Isman MB, Gunning PJ & Spollen KM, 1997. Tropical timber species as sources of botanical insecticides. In: Hedin PA, Hollingworth RM, Masler EP, Miyamoto J & Thompson DG (eds), Phytochemicals for Pest Control, Vol, 658 American Chemical Society, pp. 27-37.

James DH & Castor WM, 2005. Styrene. (eds), Wiley-VCH, Weinheim. Jankowiak R & Bilanski P, 2013. Diversity of ophiostomatoid fungi associated

with the large pine weevil, Hylobius abietis, and infested Scots pine seedlings in Poland. Annals of Forest Science 1-12.

59

Kadlec Z, Starý P & Zumr V, 1992. Field evidence for the large pine weevil, Hylobius abietis as a vector of Heterobasidion annosum. European journal of forest pathology 22: 316-318.

Kännaste A, Nordenhem H, Nordlander G & Borg-Karlson AK, 2009. Volatiles from a mite-infested spruce clone and their effects on pine weevil behavior. Journal of Chemical Ecology 35: 1262-1271.

Karlshoj K, Nielsen PV & Larsen TO, 2007. Differentiation of closely related fungi by electronic nose analysis. Journal of Food Science 72: M187-M192.

Karlshoj K, Nielsen PV & Larsen TO, 2007. Prediction of Penicillium expansum spoilage and patulin concentration in apples used for apple Juice production by electronic nose analysis. Journal of Agricultural and Food Chemistry 55: 4289-4298.

Klepzig K, Smalley E & Raffa K, 1996. Combined chemical defenses against an insect-fungal complex. Journal of Chemical Ecology 22: 1367-1388.

Kobayashi C, Fukasawa Y, Hirose D & Kato M, 2008. Contribution of symbiotic mycangial fungi to larval nutrition of a leaf-rolling weevil. Evolutionary Ecology 22: 711-722.

Kolmodin-Hedman B, Akerblom M, Flato S & Alex G, 1995. Symptoms in forestry workers handling conifer plants treated with permethrin. Bulletin of Environmental Contamination and Toxicology 55: 487-493.

Krokene P & Solheim H, 1998. Pathogenicity of four blue-stain fungi associated with aggressive and nonaggressive bark beetles. Phytopathology 88: 39-44.

Lam K, Tsang M, Labrie A, Gries R & Gries G, 2010. Semiochemical-mediated oviposition avoidance by female house flies, Musca domestica on animal Feces colonized with harmful fungi. Journal of Chemical Ecology 36: 141-147.

Långström B & Day KR, 2004. Damage, control and management of weevil pests, especially Hylobius Abietis. In: Lieutier F, Day KR, Battisti A, Grégoire J-C, Evans HF, Långström B & Day KR (eds), Bark and Wood Boring Insects in Living Trees in Europe, a Synthesis, Springer Netherlands, pp. 415-444.

Larsen TO & Frisvad JC, 1995. Characterization of volatile metabolites from 47 Penicillium-taxa. Mycological Research 99: 1153-1166.

Leather SR, Day KR & Salisbury AN, 1999. The biology and ecology of the large pine weevil, Hylobius abietis (Coleoptera: Curculionidae): a problem of dispersal? Bulletin of Entomological Research 89: 3-16.

Legrand S, Nordlander G, Nordenhem H, Borg-Karlson A-K & Unelius CR, 2004. Hydroxy-methoxybenzoic methyl ester: Synthesis and antifeedant activity on the pine weevil, Hylobius abietis. Z. Naturforsch., B: Chemical Science 59: 829-835.

60

Levieux J & Cassier P, 1994. The transportation of conifers pathogenic fungi by the Europian xylophagous beetler. Annee Biologique 33: 19-37.

Levieux J, Piou D, Cassier P, Andre M & Guillaumin D, 1994. Association of phytopathogenic fungi for the Scot pine (Pinus Sylvestris L) with the European weevil Hylobius abietis (L) (Col curculionidae). Canadian Entomologist 126: 929-936.

Li X, Wheeler G & Ding J, 2012. A leaf-rolling weevil benefits from general saprophytic fungi in polysaccharide degradation. Arthropod-Plant Interactions 6: 417-424.

Long Y-H, Xie L, Liu N, Yan X, Li M-H, Fan M-Z & Wang Q, 2010. Comparison of gut-associated and nest-associated microbial communities of a fungus-growing termite (Odontotermes yunnanensis). Insect Science 17: 265-276.

Månsson PE, Schlyter F, Eriksson C & Sjoedin K, 2006. Nonanoic acid, other alkanoic acids, and related compounds as antifeedants in Hylobius abietis pine weevils. Entomologia Experimentalis Et Applicata 121: 191-201.

McKenna R & Nielsen DR, 2011. Styrene biosynthesis from glucose by engineered E. coli. Metabolic Engineering 13: 544-554.

McLeesc DW, Metcalfe CD & Zitko V, 1980. Lethality of permethrin, cypermethrin and fenvalerate to salmon, lobster and shrimp. Bulletin of Environmental Contamination and Toxicology 25: 950-955.

Menjivar RD, Cabrera JA, Kranz J & Sikora RA, 2011. Induction of metabolite organic compounds by mutualistic endophytic fungi to reduce the greenhouse whitefly Trialeurodes vaporariorum (Westwood) infection on tomato. Plant and Soil 352: 233-241.

Mian LS & Mulla MS, 1992. Effects of pyrethroid insecticides on nontarget invertebrates in aquatic ecosystems. Journal of Agricultural Entomology 9: 73-98.

Miranda I, Gominho J & Pereira H, 2012. Incorporation of bark and tops in Eucalyptus Globulus wood pulping. BioResources 7.

Moita C, Feio SS, Nunes L, Curto MJM & Roseiro JC, 2005. Optimisation of physical factors on the production of active metabolites by Bacillus subtilis 355 against wood surface contaminant fungi. International Biodeterioration & Biodegradation 55: 261-269.

Muller HE, Fanning GR & Brenner DJ, 1995. Isolation of Ewingella americana from Mollusks. Current Microbiology 31: 287-290.

Niemelä T & Korhonen K, 1998. Taxonomy of the genus Heterobasidion. In: Woodward S, Stenlid J, Karjalainen R & Hüttermann A (eds), Heterobasidion annosum: Biology, Ecology, Impact and Control, CAB International, London, pp. 27-33.

Nordborg F & Nilsson U, 2003. Growth, damage and net nitrogen uptake in Picea abies (L.) Karst. seedlings, effects of site preparation and fertilisation. Annals of Forest Science 60: 657-666.

61

Nordlander G, 1990. Limonene inhibits attraction to α-pinene in the pine weevils Hylobius abietis and H. pinastri. Journal of Chemical Ecology 16: 1307-1320.

Nordlander G, 1991. Host finding in the pine weevil Hylobius abietis: effects of conifer volatiles and added limonene. Entomologia Experimentalis Et Applicata 59: 229-237.

Nordlander G, Nordenhem H & Bylund H, 1997. Oviposition patterns of the pine weevil Hylobius abietis. Entomologia Experimentalis Et Applicata 85: 1-9.

Nordlander G, Nordenhem H & Hellqvist C, 2008. A flexible sand coating (Conniflex) for the protection of conifer seedlings against damage by the pine weevil Hylobius abietis. Agricultural and Forest Entomology 11: 91-100.

Nordlander G, Bylund H, Örlander G & Wallertz K, 2003. Pine weevil population density and damage to coniferous seedlings in a regeneration area with and without shelterwood. Scandinavian Journal of Forest Research 18: 438-448.

Nordlander G, Hellqvist C, Johansson K & Nordenhem H, 2011. Regeneration of European boreal forests: Effectiveness of measures against seedling mortality caused by the pine weevil Hylobius abietis. Forest Ecology and Management 262: 2354-2363.

Olenici N & Olenici V, 2007. Oviposition preferences of the large pine weevil, Hylobius abietis (L.) for different coniferous species. Analele ICAS 50: 213-222.

Örlander G & Nilsson U, 1999. Effect of reforestation methods on pine weevil (Hylobius abietis) damage and seedling survival. Scandinavian Journal of Forest Research 14: 341-354.

Örlander G, Nilsson U & Nordlander G, 1997. Pine weevil abundance on clear-cuttings of different ages: A 6-year study using pitfall traps. Scandinavian Journal of Forest Research 12: 225-240.

Örlander G, Nordlander G, Wallertz K & Nordenhem H, 2000. Feeding in the crowns of Scots pine trees by the pine weevil Hylobius abietis. Scandinavian Journal of Forest Research 15: 194-201.

Peterson RA, Bradner JR, Roberts TH & Nevalainen KMH, 2009. Fungi from koala (Phascolarctos cinereus) faeces exhibit a broad range of enzyme activities against recalcitrant substrates. Letters in Applied Microbiology 48: 218-225.

Petersson M & Örlander G, 2003. Effectiveness of combinations of shelterwood, scarification, and feeding barriers to reduce pine weevil damage. Canadian Journal of Forest Research 33: 64-73.

Petersson M, Orlander G & Nilsson U, 2004. Feeding barriers to reduce damage by pine weevil (Hylobius abietis). Scandinavian Journal of Forest Research 19: 48-59.

62

Petersson M, Örlander G & Nordlander G, 2005. Soil features affecting damage to conifer seedlings by the pine weevil Hylobius abietis. Forestry 78: 83-92.

Pimentel D, 2009. Environmental and economic costs of the application of pesticides primarily in the United States. In: Peshin R & Dhawan AK (eds), Integrated Pest Management: Innovation-Development Process, Springer Netherlands, pp. 89-111.

Piou D, 1993. Role d' Hylobius abietis (L)(Col, Curculionidae) dans le transport de Leptographium procerum (Kendr) Wingf et son inoculation au pin sylvestre. Annales Des Sciences Forestieres 50: 297-308.

Prescott LM, Harley JP & Klein DA, 2002. The History and scope of microbiology. In: (eds), Microbiology, McGraw – Hill companies Inc., New York, pp. 1-16.

Pumpuni CB, Beier MS, Nataro JP, Guers LD & Davis JR, 1993. Plasmodium falciparum: Inhibition of sporogonic development in Anopheles stephensi by gram-negative bacteria. Experimental Parasitology 77: 195-199.

Quesada-Moraga E & Vey A, 2004. Bassiacridin, a protein toxic for locusts secreted by the entomopathogenic fungus Beauveria bassiana. Mycological Research 108: 441-452.

Quesada-Moraga E, Carrasco-Diaz JA & Santiago-Alvarez C, 2006. Insecticidal and antifeedant activities of proteins secreted by entomopathogenic fungi against Spodoptera littoralis (Lep., Noctuidae). Journal of Applied Entomology 130: 442-452.

Ravella SR, Donovan N, James SA, Shivaji S, Arunasri K, Bond CJ, Roberts IN & Hobbs PJ, 2011. Candida northwykensis sp nov., a novel yeast isolated from the gut of the click beetle Melanotus villosus. Current Microbiology 63: 115-120.

Rayamajhi MB, Van TK, Pratt PD & Center TD, 2006. Interactive association between Puccinia psidii and Oxyops vitiosa, two introduced natural enemies of Melaleuca quinquenervia in Florida. Biological Control 37: 56-67.

Rivera FN, Gonzalez E, Gomez Z, Lopez N, Hernandez-Rodriguez C, Berkov AMY & Zuniga G, 2009. Gut-associated yeast in bark beetles of the genus Dendroctonus Erichson (Coleoptera: Curculionidae: Scolytinae). Biological Journal of the Linnean Society 98: 325-342.

Röder G, Rahier M & Naisbit RE, 2007. Coping with an antagonist: the impact of a phytopathogenic fungus on the development and behaviour of two species of alpine leaf beetle. Oikos 116: 1514-1523.

Roe AD, James PMA, Rice AV, Cooke JEK & Sperling FAH, 2011. Spatial community structure of mountain pine beetle fungal symbionts across a latitudinal gradient. Microbial Ecology 62: 347-360.

63

Rönnberg J & Cleary MR, 2012. Presence of Heterobasidion infections in Norway spruce stumps 6 years after treatment with Phlebiopsis gigantea. Forest Pathology 42: 144-149.

Saez H & Demiranda LR, 1984. The Yeast Candida sequnanesis Sp-Nov. Antonie Van Leeuwenhoek Journal of Microbiology 50: 379-381.

Sakai K, 2001. Chemistry of bark. In: Hon DN-S & Shiraishi N (eds), Wood and Cellulosic Chemistry, Marcel Dekker inc, New York, pp. 243-274.

Sanchez-Contreras M & Vlisidou I, 2008. The diversity of insect-bacteria interactions and its applications for disease control. Biotechnology and Genetic Engineering Reviews 25: 203-243.

Santiago ALCMdA, Trufem SFB, Malosso E, Santos PJPd & Cavalcanti MAdQ, 2011. Zygomycetes from herbivore dung in the ecological reserve of Dois Irmaos, Northeast Brazil. Brazilian Journal of Microbiology 42: 89-95.

Schlyter F, Smitt O, Sjödin K, Högberg HE & Löfqvist J, 2004. Carvone and less volatile analogues as repellent and deterrent antifeedants against the pine weevil, Hylobius abietis. Journal of Applied Entomology. 128: 610-619.

Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR & Dean DH, 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiology and molecular biology reviews 62: 775-806.

Sjödin K, Persson M, Borg-Karlson A-K & Norin T, 1996. Enantiomeric compositions of monoterpene hydrocarbons in different tissues of four individuals of Pinus sylvestris. Phytochemistry 41: 439-445.

Skrzecz I & Moore R, 1997. The attractiveness of pine branches infected with selected wood colonizing fungi to the large pine weevil (Hylobius abietis). (Gregoire JC, Liebhold, A.M., Stephen, F.M., Day, K.R., Salom, S.M., eds), pp. 146-152.

Stoakley JT, 1968. Control of the pine weevil, Hylobius abietis L., and of Hylastes species. Forestry 41: 182-188.

Suchail S, Guez D & Belzunces LP, 2001. Discrepancy between acute and chronic toxicity induced by imidacloprid and its metabolites in Apis mellifera. Environmental Toxicology and Chemistry 20: 2482-2486.

Suh S-O, Marshall CJ, McHugh JV & Blackwell M, 2003. Wood ingestion by passalid beetles in the presence of xylose-fermenting gut yeasts. Molecular Ecology 12: 3137-3145.

Suh SO, McHugh JV, Pollock DD & Blackwell M, 2005. The beetle gut: a hyperdiverse source of novel yeasts. Mycological Research 109: 261-265.

Tanahashi M, Matsushita N & Togashi K, 2009. Are stag beetles fungivorous? Journal of Insect Physiology 55: 983-988.

Thacker JRM, Bryan WJ, McGinley C, Heritage S & Strang RHC, 2003. Field and laboratory studies on the effects of neem (Azadirachta indica) oil

64

on the feeding activity of the large pine weevil (Hylobius abietis L.) and implications for pest control in commercial conifer plantations. Crop protection 22: 753-760.

Thakeow P, Weißbecker B & Schütz S, 2006. Volatile organic compounds emitted from fungal-rotting beech (Fagus sylvatica). Mitt. Dtsch. Ges. Allg. Angew. Ent. 15: 157-160.

Tilles DA, Eidmann HH & Solbreck B, 1988. Mating stimulant of the pine weevil Hylobius abietis (L.). Journal of Chemical Ecology 14: 1495-1503.

Trudel R, Lavallée R & Bauce É, 2001. Oviposition biology of Pissodes strobi (Coleoptera: Curculionidae) on white pine (Pinaceae) under laboratory conditions. The Canadian Entomologist 133: 333-341.

Unelius CR, Nordlander G, Nordenhem H, Hellqvist C, Legrand S & Borg-Karlson A-K, 2006. Structure-activity relationships of benzoic acid derivatives as antifeedants for the pine weevil, Hylobius abietis. Journal of Chemical Ecology 32: 2191-2203.

von Sydow F & Örlander G, 1994. The influence of shelterwood density on Hylobius abietis (L.) occurrence and feeding on planted conifers. Scandinavian Journal of Forest Research 9: 367-375.

vonSydow F & Birgersson G, 1997. Conifer stump condition and pine weevil (Hylobius abietis) reproduction. Canadian Journal of Forest Research-Revue Canadienne De Recherche Forestiere 27: 1254-1262.

Wang L, Huang J, You M, Guan X & Liu B, 2007. Toxicity and feeding deterrence of crude toxin extracts of Lecanicillium (Verticillium) lecanii (Hyphomycetes) against sweet potato whitefly, Bemisia tabaci (Homoptera: Aleyrodidae). Pest Management Science 63: 381-387.

Watson PG, 1999. Influence of insecticide, wax and biofungicide treatments, applied to Pinus sylvestris and Picea abies, on the olfactory orientation of the large pine weevil, Hylobius abietis L. (Coleoptera: Curculionidae). Agricultural and Forest Entomology 1: 171-177.

Wigrup I, 2012. Swedish statistical yearbook of forestry. Swedish Forestry Agency, Jönköping.

Wilson LF & Schmiege DC, 1975. Pine root collar weevil. Vol. Forest Pest Laeflet 39 (Agriculture USDo, eds), U.S. Government Printing Office, pp. 1-8.

Woodward S, Stenlid J, Karjalainen R & Hüttermann A, 1998. Preface. In: Woodward S, Stenlid J, Karjalainen R & Hüttermann A (eds), Heterobasidion annosum: Biology, Ecology, Impact and Control, CAB International, London, pp. xi-xii.

Wu XQ, Yuan WM, Tian XJ, Fan B, Fang X, Ye JR & Ding XL, 2013. Specific and functional diversity of endophytic bacteria from pine wood nematode Bursaphelenchus Xylophilus with different virulence. International Journal of Biological Sciences 9: 34-44.

65

Yamaoka Y, Wingfield MJ, Takahashi I & Solheim H, 1997. Ophiostomatoid fungi associated with the spruce bark beetle Ips typographus f. aponicus in Japan. Mycological Research 101: 1215-1227.

Zhang H, YE, H., Haack, R.A., & Langor DW, 2004. Biology of Pissodes yunnanensis (Coleoptera: Curculionidae), a pest of Yunnan pine in southwestern China. The Canadian Entomologist 136: 719-726.

Zhang ZY & Pawliszyn J, 1993. Head space solid phase microextraction. Analytical Chemistry 65: 1843-1852.

Zheltikova TM, Glushakova AM & Alesho NA, 2011. The yeast community associated with the digestive tract of the German cockroach Blattella germanica L. Med Parazitol (Mosk) 18-22.

Zhu J, Bean HD, Kuo Y-M & Hill JE, 2010. Fast detection of volatile organic compounds from bacterial cultures by secondary electrospray ionization-mass spectrometry. Journal of Clinical Microbiolgy 48: 4426-4431.

66

Appendix A: author’s contributions

The following is a description of my contributions to the Publications I to V, as requested by KTH. Paper I: I participated in the planning of experiments, except the identification of microbes, isolated the microorganisms, analyzed their produced volatiles and wrote the manuscript. Paper II: I participated in the planning of the experiments, performed the chemical analyses and wrote the manuscript. Paper III: I planned the chemical part of experiments, performed the chemical analysis and wrote the manuscript. Paper IV: I formulated the research problem, performed part of the study and wrote the manuscript. Paper V: I formulated the research problem, performed the experimental work and wrote the manuscript.