Volatiles of Conifer Seedlings

Composition and Resistance Markers

Astrid Kännaste

Doctoral Thesis

Stockholm 2008

KTH Chemical Science and Engineering

Akademisk avhandling som med tillstånd av Kungliga Tekniska Högskolan i Stockholm framlägges till offentlig granskning för avläggande av teknologie doktorsexamen i kemi med inriktning mot ekologisk kemi måndagen den 19 maj kl. 10.00 i sal F3, KTH, Lindstedtsvägen 26, Stockholm. Avhandlingen försvaras på engelska. Opponent är Doc. Anders Backlund, Institutionen för läkemedelskemi, Avdelningen för Farmakognosi, BMC, Box 574, 75123 Uppsala.

ISBN 978-91-7178-962-4 ISSN 1654-1081 TRITA-CHE Report 2008:34 © Astrid Kännaste Universitetsservice US AB, Stockholm

«Niisugust rohtukasvanud rada pead astuma kaugemale enda ette vaadates, siis juhib ta iseenesest õiges suunas, umbes nii nagu teeb jõesäng veevooluga. Aga kui püüda märke leida siitsamast jalge eest, siis kaob tee käest ja eksitab eesmärgist kõrvale.» (A walker on a path covered with weeds should look further on to find the right way; in a similar way a water-stream is guided along the river-bed. Watching the way right at your feet will not help you to see the track – the path will be lost and it will be misleading.) Fred Jüssi

Astrid Kännaste, 2008. Volatiles of Conifer Seedlings, Composition and Resistance Markers. ISSN 1654-1081 Abstract

Pine weevils cause major damage to newly planted conifer seedlings in reforestation areas. However, recent findings indicate that small (“mini”) seedlings, planted at the age of 7-10 weeks, are gnawed less by pine weevils than the larger, conventionally planted seedlings. Thus, it has been proposed that planting young conifer seedlings in clear-cut areas may reduce the damage caused by pine weevils. In attempts to determine why mini seedlings appear to be less damaged by pine weevils than “conventional” seedlings, the volatiles released by Norway spruce and Scots pine mini seedlings were investigated, since such chemicals are of great importance in herbivore-plant communication, inter alia acting as repellents, attractants or antifeedants. Volatiles from the seedlings were collected, separated and identified by solid phase microextraction (SPME) followed by gas chromatography-mass spectrometry.

The results show that there are high levels of chemodiversity among both spruce and pine seedlings. Between-tissue and age-related variations in their emissions were also found. Norway spruce clones infested by mites were also examined to assess genotype- and pest-specific stress reactions of Norway spruce. Finally, the effects of certain spruce defense compounds on the behavior of the large pine weevil Hylobius abietis were examined.

Keywords: Conifers, Norway spruce, Scots pine, Nalepella, Oligonychus ununguis, green leaf volatiles, terpenes, aromatics, benzenoids, SPME, induced defense reaction.

Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid ADH alcohol dehydrogenase BAMT benzoic acid carboxyl methyltransferase BEAT benzylalcohol acetyltransferase IEMT (iso)eugenol O-methyltransferase DMAPP dimethylallyl diphosphate DNA deoxyribonucleic acid F seedlings from the freezer FPP farnesyl diphosphate GLVs green leaf volatiles GC gas chromatography GGPP geranylgeranyl diphosphate GPP geranyl diphosphate HPLS hydroperoxide lyases IPP isopentenyl diphosphate MeJA methyl jasmonate MEP-pathway methylerythritol pathway MeSA methyl salicylate MS mass spectrometry MVA-pathway mevalonate pathway MW molecular weight OA seedlings from an outdoor area Other MT other monoterpenes PAL phenylalanine ammonia lyase PCA principal component analysis PDMS polydimethylsiloxane PDMA/DVB polydimethylsiloxane/divinylbenzene PDMS/CAR polydimethylsiloxane/carboxen RDA redundancy analysis RhAAT rose alcohol acetyltransferase RI retention index SAMT salicylic acid carboxyl methyltransferase SD standard deviation SE standard error SPME solid-phase microextraction SqT sesquiterpenes

List of publications This thesis is based on the following papers, which are cited in the text by the corresponding Roman numerals. I Pettersson, M., Kännaste, A., Lindström, A., Hellqvist, C., Stattin, E., Långström, B.,

Borg-Karlson, A.-K. 2008. Why are mini seedlings less attacked by Hylobius abietis than conventional ones – is plant chemistry the answer? Scandinavian Journal of Forest Research. In press.

II Kännaste, A., Tao, Z., Lindström, A., Stattin, E., Långström, B., Borg-Karlson, A.-K.

Odors of Norway spruce (Picea abies) seedlings: differences due to age and chemotype and their relevance for weevil resistance. Manuscript.

III Kännaste, A., Tao, Z., Lindström, A., Stattin, E., Långström, B., Borg-Karlson, A.-K.

Volatile emissions from pine seedlings - age-related changes and chemotypes. Manuscript.

IV Kännaste, A. and Borg-Karlson, A.-K. Correlations between terpenes emitted by mini-

seedlings of Norway spruce and Scots pine. Preliminary manuscript. V Kännaste, A., Vongvanich, N., Borg-Karlson, A.-K. 2008. Infestation by a Nalepella

species induces emissions of α- and β-farnesenes, (-)-linalool and aromatic compounds in Norway spruce clones of different susceptibility to the large pine weevil. Arthropod-Plant Interactions 2(1): 31-41.

VI Kännaste, A., Nordenhem, H., Nordlander, G., Borg-Karlson, A.-K. Volatiles from a

mite-infested spruce clone and their effects on pine weevil behavior. Submitted to Journal of Chemical Ecology.

Papers not included in the thesis. VII Lindh, J.M., Kännaste, A., Knols, B.G.J., Faye, I., Borg-Karlson, A.-K. 2008. Oviposition

responses of Anopheles gambiae s.s. (Diptera: Culicidae) and identification of volatiles from bacteria containing solutions. Journal of Medical Entomology. In press.

VIII Liblikas, I., Mõttus, E., Borg-Karlson, A.-K., Kuusik, S., Ojarand, A., Kännaste, A.,

Tanilsoo, J. 2003. Flea beetle (Coleoptera: Chrysomelidae) response to alkyl thiocyanates and alkyl isothiocyanates. Agronomy Research 1(2): 175-184.

IX Mõttus, E., Kahu, K., Kännaste, A., Liblikas, I., Ojarand, A., Mozuraitis, R.,

Ovsjannikova, J., Nikolajeva, Z. 2001. Experiments and results of a currant pest, Lampronia capitella. Transactions of the Estonian Agricultural University 213: 121-125.

X Liblikas, I., Mõttus, E., Kännaste, A., Kuusik, S., Ojarand, A., Mozuraitis, R., Santangelo,

E., Mõttus, M., Unelius, R.C., Borg-Karlson, A.-K. Activity of sex pheromone components on currant shoot borer males (Lampronia capitella (Cl.) (Lepidoptera: prodoxidae). Manuscript.

Table of contents 1. Introduction ............................................................................................................................ 1

1.1. Biosynthesis of secondary metabolites ......................................................................... 2 1.1.1. Terpenes ............................................................................................................... 2 1.1.2. GLVs .................................................................................................................... 4 1.1.3. Aromatic volatiles ................................................................................................ 5

1.2. Conifer Defenses ........................................................................................................... 5 1.3. Aims .............................................................................................................................. 6

2. Experimental investigations ................................................................................................... 7 2.1. Plant material................................................................................................................. 7 2.2. Arthropods..................................................................................................................... 7 2.3. Sampling and analyses of volatiles ............................................................................... 8

2.3.1. Collection of volatiles .......................................................................................... 8 2.3.2. Handling of plant material.................................................................................... 9 2.3.3. Separation and identification of volatiles............................................................. 9

2.4. Statistical analysis ......................................................................................................... 9 2.4.1. Multivariate data analysis..................................................................................... 9 2.4.2. Correlation Analysis........................................................................................... 10

3. Volatile profiles of conifers.................................................................................................. 11 3.1. Chemotypes of spruces and pines ............................................................................... 11

3.1.1. Spruces ............................................................................................................... 11 3.1.2. Pines ................................................................................................................... 13 3.1.2. Resistance of different chemotypes of spruce and pine seedlings to Hylobius

abietis ............................................................................................................... 14 3.2. Relative proportions of conifer volatiles..................................................................... 15

3.2.1. Spruce seedlings................................................................................................. 15 3.2.2. Pine seedlings..................................................................................................... 18

3.3. Enantiomeric composition of α-pinene and limonene ................................................ 19 3.3.1. Spruce seedlings................................................................................................. 19 3.3.2. Pine seedlings..................................................................................................... 20

3.4. Biosynthesis of terpenes of young seedlings............................................................... 22 3.4.1. Monoterpenes ..................................................................................................... 22 3.4.2. Sesquiterpenes.................................................................................................... 22

4. Volatile profiles of infested spruces..................................................................................... 24 4.1. Volatiles released by spruces attacked by mites ......................................................... 24

4.1.1. Enantiomeric composition of chiral monoterpenes in the whole plant emissions .......................................................................................................... 25

4.1.2. Enantiomeric composition of chiral monoterpenes stored in the phloem tissues of spruces attacked by mites ................................................................. 26

4.2. Correlations amongst induced terpenes....................................................................... 27 4.3. Biosynthetic relations of induced aromatic compounds ............................................. 28

4.4. Impact of induced spruce volatiles on pine weevils, Hylobius abietis........................ 29 5. Conclusions .......................................................................................................................... 33 References ................................................................................................................................ 35 Acknowledgements .................................................................................................................. 47 Appendixes............................................................................................................................... 49

Appendix 1 ................................................................................................................... 49 Appendix 2A ................................................................................................................ 50 Appendix 2B ................................................................................................................ 51

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1. Introduction Intensive forest management is one reason why the large pine weevil (Hylobius abietis L.)

is becoming a serious pest in reforestation areas. The most damage is caused by the adult weevils, which feed on the bark of young, newly planted conifer seedlings after being attracted to their area by the odor of fresh stumps. Efforts are being made to find effective, environmentally-friendly control methods, which could help to reduce weevils’ attacks on young spruce and pine seedlings (Wallertz, 2005). Options based on the effects of semiochemicals on the olfactory and/or gustatory receptors of weevils could be considered (Nordlander, 1990; Nordlander, 1991; Kleipzic and Schlyter, 1999; Månsson, 2005; Sibul, 2005; Borg-Karlson et al., 2006; Unelius et al., 2006). For instance, extracts of the tropical tree species Neem have been shown to be deterrent to weevils (Luik et al., 2000; Weathersbee and McKenzie, 2005).

Common plant volatiles include GLVs, terpenes, phenylpropanoids and benzenoids (Figure 1) (Dudareva et al., 2006). In most cases the odors of plants are complex mixtures of compounds (Borg-Karlson et al., 1985; Xugen and Luqin, 2006), implying that their biosynthetic pathways are active simultaneously.

In the bouquet of conifers there are large numbers of compounds that activate olfactory receptors on the antenna of forest pests (Wibe et al., 1997; Huber et al., 2000; Asaro et al., 2004). Bouquets of plant volatiles also carry information about the status of the plants (e.g., their species, developmental state, stress, and whether or not they are flowering – odors of flowers attract pollinators, for example). Various species-specific combinations of volatiles – including (3Z)-hexenal, (2E)-hexenal, (3Z)-hexenyl acetate, 4,8-dimethyl-1,3,7-nonatriene, α- and β-farnesenes and methyl salicylate (Figure 1) – are released by angiosperms and gymnosperms that are attacked by herbivores (V; VI; Paré and Tumlinson, 1999; Arimura et al., 2001; Martin et al., 2003; Himanen et al., 2005). The chirality, presence/occurrence and amounts of the compounds are all factors that influence the orientation of the herbivores (Nordlander, 1990; Klepzic and Schlyter, 1999; Wibe et al., 1998; Byers et al., 2000; Grant and Langevin, 2002; Fraser et al., 2003; De Boer and Dicke, 2004; Bichão et al., 2005; Røstelien et al., 2005; Xugen and Luqin, 2006; Chen and Fadamiro, 2007).

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(+) (-)limonene

(-) (+)α-pinene

(-) (+)β-pinene

(-) (+)

3-carene

OCH3

estragole

(E)-β-farnesene (E,E)-α-farnesene

H H

(E)-β-caryophyllene

OH

OCH3O

methyl salicylate(MeSA)

OCH3O

methyl benzoate

(3R)-(-)-linalool

OH

CHO(3Z)-hexenal

CH2OH

(3Z)-hexen-1-ol

CHO(2E)-hexenal

(3S)-(+)-linalool

OH

(3Z)-hexenyl acetate

Monoterpenes Sesquiterpenes

Green leaf volatiles (GLVs) Phenylpropanoids/benzenoids

OCH3

(E)-anethole

O

O

longifolene α-longipinene

Figure 1. Molecular structures of plant volatiles

1.1. Biosynthesis of secondary metabolites 1.1.1. Terpenes

Terpenoids, which comprise the largest class of plant secondary metabolites, originate from IPP (Scheme 1). Two different pathways are known to be involved in the biosynthesis of IPP: the MVA pathway, which starts with the formation of acetoacetyl-CoA and is located in the cytosol and endoplasmatic reticulum (Dewick, 1999); and the MEP-pathway which is initialized from pyruvate and is located in plastids (Lichtenthaler et al., 1997; Rohmer, 1999). The route in

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plastids provides IPP for the biosynthesis of isoprene, mono-, and diterpenes, while the cytosol-localized pathway provides IPP for sesqui- and triterpenes.

Scheme 1. Biosynthetic pathways of terpenes. DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; GPP, geranyl diphosphate; IPP, isopentenyl diphosphate.

Head-to-tail condensations of two or three IPP-units form the precursors of terpenes (GPP, FPP and GGPP), which in subsequent steps are converted into terpenes.

acetyl-CoA

mevalonic acid

glyceraldehyde-3-phosphate

pyruvate

IPP

deoxy-D-xylulose-5-phosphate

OPPDMAPPDMAPP

monoterpenes C10

sesquiterpenesC15

triterpenesC20

IPPOPP

GPP2 IPP

OPP

GGPP

diterpenesC20

2 IPP

OPP

FPP

isopreneC5

methylerythritol-4-phosphate

OPP

OPP

pyruvate

IPPOPP

HOOC OHOH

OPO

OH

OH

MVA pathway MEP pathway PLASTIDS CYTOSOL

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Genes encoding enzymes that catalyze the final biosynthetic steps of one or more terpenes have been discovered in several conifers to date, including Norway spruce (Picea abies), loblolly pine (Pinus taeda), grand fir (Abies grandis), sitka spruce (Picea sitchensis) and Douglas fir (Pseudotsuga menziesii) (Bohlmann et al., 1997; Bohlmann et al., 1998; Phillips et al., 1999; Byun McKay et al., 2003; Fäldt et al., 2003; Phillips et al., 2003; Martin et al., 2004; Huber et al., 2005).

Precursors of terpenes have been experimentally demonstrated to be transported from plastids to the cytosol, as shown in Scheme 1 (Dudareva et al., 2005; Bartram et al., 2006). Therefore, the production of terpenes is sometimes referred to as the result of “cross-talk” between the MEP- and MVA-pathways.

1.1.2. GLVs Due to their increasing use in perfumes and the food industry, intensive research efforts

have been focused on the biocatalysts responsible for the selective production of GLVs (Gargouri et al., 2004).

GLVs are formed via the lipoxygenase pathway, as shown in Scheme 2.

COOH

linolenic acid

lipoxygenase

COOHOOH

13-hydroperoxylinolenic acid

HPLS

CHO

(3Z)-hexenal

IE

CHO

CH2OH

(3E)-hexenal

(3E)-hexenol

ADH

ADH CH2OH

(3Z)-hexen-1-ol

COOHHOC

12-oxo-(9Z)-dodecenoic acid

CHO(2E)-hexenal

IE

IE

CH2OH(2E)-hexenol

ADH

acetyltransferase

O

(3Z)-hexenyl acetateO

Scheme 2. Biosynthetic pathways of GVLs from linolenic acid. HPLS, hydroperoxide lyase; ADH, alcohol dehydrogenase; IE, isomerization enzyme.

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Firstly, dehydrogenation of C18 acids released from the cell membrane at C9 or C13 position by lipoxygenases yields 9-hydroperoxy and 13-hydroperoxy derivates of polyenic acidsv which are further cleaved by HPLSs into oxo-acids and C6-aldehydes. With the help of ADHs aldehydes are converted into the corresponding alcohols. Unsaturated and saturated GLVs are produced from linolenic and linoleic acid, respectively (Hatanaka et al., 1978; Hatanaka et al., 1993; Dudareva et al., 2006).

1.1.3. Aromatic volatiles In recent decades substantial amounts of information have been obtained on the metabolic

pathways involved in the synthesis of secondary metabolites in plants. The increased activity of PAL by fungi and pathogens in conifers is investigated (Campbell and Ellis, 1992; Lange et al., 1994; Cvikrová et al., 2006). Although there is little or no information about the synthases of volatile phenylpropanoids and benzenoids in conifers, the activities of various enzymes (e.g. BAMT, BEAT, IEMT and SAMT) involved in the production of compounds such as benzyl acetate, methyl salicylate, methyl eugenol and methyl benzoate in angiosperms have been described (Wang et al., 1997; Dudareva et al., 1998; Dudareva et al., 2000a; Dudareva et al., 2000b; Negre et al., 2003; Guterman et al., 2006; Koeduka et al., 2006).

Aromatic volatiles are biosynthesized via the shikimic acid pathway, initialized by a condensation of erythrose 4-phosphate and phosphoenolpyruvate, an intermediate in the production of pyruvate. After numerous steps via 3-dehydroshikimic acid and chorismate, phenylalanine is produced. With the catalytic assistance of an enzyme (PAL) phenylalanine is converted into trans-cinnamic acid, which is a starting point for the synthesis of phenylpropanoids (phenylethanol, chavicol etc.) and benzenoids (benzaldehyde, methyl benzoate, methyl salicylate etc.) (Boatright et al., 2004; Dudareva et al., 2006). Via the transformation of trans-cinnamic acid into phenylpropenol various compounds, including volatiles, are formed (Dudareva et al., 2006; Koeduka et al., 2006; Pichersly and Dudareva, 2007). Benzenoids are derived by the shortening of the cinnamic side chain by two carbons (Dudareva et al., 2006).

1.2. Conifer Defenses The defenses of plants, including conifers, can be divided into two types: direct and indirect

(Dicke, 1999). In addition, direct defenses can be subdivided into constitutive and induced components. Furthermore, direct constitutive defenses include both physical (e.g. natural polymers and calcium oxalate crystals) and chemical (e.g. oleoresin, phenolic compounds) barriers (Franceschi et al., 2005). The effects of α-pinene, β-pinene, 3-carene and limonene on mites, GVLs on beetles, and limonene on pine weevils are good examples of the repellency effects that defense chemicals can have on herbivores (Cook, 1992; Dickens et al., 1992; Nordlander, 1990; Poland et al., 1998; Poland and Haack, 2000; Huber and Borden, 2001).

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Induced direct defense systems include the production of terpenes in pre-existing resin-producing structures and/or newly formed traumatic resin cell ducts (TRDs) caused by mechanical wounding, insect or pathogen attack, abiotic factors or MeJA (Rosemann et al., 1991; Kainulainen et al., 1993; Kainulainen et al., 1995; Nagy et al., 2000; Franceschi et al., 2002; Martin et al., 2002; Byun McKay et al., 2003; Miller et al., 2005; Turtola et al., 2003). The development of TRDs in conifer xylem or phloem tissues is species-specific (Hudgins et al., 2004), and the resin stored is chemically different from the resin found in the cells of non-stressed conifers of the same species (Steele et al., 1998; Martin et al., 2002; Byun McKay et al., 2003; Miller et al., 2005; Fäldt et al., 2006). In addition, there are significant genetically determined within-species differences in plants’ tolerance of stresses, as indicated by the effects of heavy metal contaminants on allelic frequencies in conifer seedling populations reported by Bergmann and Hosius (1995).

Chemical and biotic stressors increase the activity of various enzymes in needle tissues of spruces; resulting in dramatic changes in the composition of the volatiles they release (V; VI; Martin et al., 2003; Miller et al., 2005). Some of these de novo-synthesized chemicals are “SOS-signals”; mediators of indirect plant defenses that may attract carnivorous parasitoids or predatory mites, or trigger the defense reactions of neighboring plants (Dicke et al., 1990; Janssen, 1999; Arimura et al., 2001; De Boer and Dicke, 2004; De Boer et al., 2004; Mumm and Hilker, 2005; Chen and Fadamiro, 2007).

1.3. Aims The main aims of the studies underlying this thesis were to characterize the volatiles

emitted by spruce and pine seedlings, and to examine their effects on the major pest threatening them – the large pine weevil Hylobius abietis – focusing especially on volatiles that might influence the survival of mini-seedlings in reforestation areas.

Papers I, II, III and IV describe the chemical composition of the seedlings’ volatile profiles, including the enantiomeric composition of chiral monoterpenes, and variations related to differences in chemotypes and ages of the seedlings. Emissions from both unwounded seedlings and volatiles in phloem samples were investigated, and correlations between the volatiles produced and the seedlings’ performance in the field were examined.

In a second series of studies the chemical responses of Norway spruces induced by biotic stressors like “mites” were investigated (V, VI). These studies included analyses of between-clone and between-mite species variations in the composition of released volatiles, and the enantiomeric composition of chiral α-pinene, β-pinene, limonene and linalool released by both foliage and wounded phloem tissues of mite-infested clones (V, VI). Monoterpenes produced by conifers have been previously shown to affect the behavior of pine weevils (Nordlander, 1990; Nordlander, 1991; Wibe et al., 1998). Therefore, the impact of selected spruce stress volatiles on weevils was also evaluated (VI).

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2. Experimental investigations 2.1. Plant material

Results presented in Papers I, II, III and IV are based on chemical analyses of unwounded seedlings and pieces of phloem obtained from Norway spruces (Picea abies) and Scots pines (Pinus sylvestris). Firstly, the composition of volatiles released by ‘mini’ seedlings (young seedlings, 8-12 weeks old and 6-8 cm tall) and ‘conventional’ seedlings (13-15 cm, traditionally used in reforestation after clear cutting) was compared (I), and then emissions of volatiles from seedlings of different developmental stages were compared (Table 1 in II; Table 1 in III). Similarly the emissions from pine mini-seedlings (III) were compared to conventional pines.

The stress reactions of spruces infested by mites were examined by analyzing 4-year-old Norway spruce clones with varying degrees of resistance to the large pine weevil described by Weslien (2004): clone 1091 – low (36% not eaten), clone 72 - medium (72, 45 % not eaten) and clone 1090 - high (1090, 90% not eaten) (V). Using Norway spruce clone 1321 differences in the bouquet of spruce stress volatiles triggered by different species of mites were also characterized (VI).

2.2. Arthropods Mites: In nature, mites rarely cause severe stress reactions in spruces, but they are serious

pests in nurseries (Ehnstrom et al., 1974; Lehman, 1982; Lehman, 2002; Poteri et al., 2005). Furthermore, due to their rapid development from larval to adult stages, certain mites may produce five or more generations per year (Löyttyniemi, 1973; Lehman, 1982). Therefore in order to monitor the spruce defenses throughout a full period in which they could be attacked, naturally occurring mite species Nalepella Boczek var. Picea abietis (Acarina, Ereophyidae) (Phytoptidae) (Löyttyniemi, 1973) and Oligonychus ununguis Jacobi (Acarina, Tetranychidae) (Jeppson et al., 1975) were separately fed on the plants for an entire year (V, VI).

Insects: As mentioned in the introduction, H. abietis is a serious forest pest. The investigations included behavior studies in which female and male weevils were stored in darkness at 10 ºC and provided with fresh conifer bark as food, then placed in experimental conditions (18 h light/6 h dark cycles, 20 oC) with continuous access to food until 24 h before the tests, in which their preferences for various types of samples were evaluated (VI). Correlations between their preferences and the plants’ volatile profiles were then explored, as described below.

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2.3. Sampling and analyses of volatiles 2.3.1. Collection of volatiles

Several methods have been developed for collecting volatile compounds prior to their analysis (Agelopoulos and Pickett, 1998; Cavalli et al., 2003; Povolo and Contarini, 2003). One, developed in the early 1990s by a group of scientists headed by Prof. Janus Pawliszyn, is SPME (Zhang and Pawliszyn, 1993; Borg-Karlson and Mozuraitis 1996, Bartelt, 1997; Pawliszyn, 1997), which can be used for both qualitative and quantitative analyses (Matich et al., 1996; Zabaras and Wyllie, 2001; Demyttenaere et al., 2003; Custódio et al., 2006). An SPME device consists of a glass fiber coated with a suitable organic polymer attached to a stainless steel plunger mounted in a needle. After inserting the SPME to the headspace to be sampled, the fiber is extruded from the metal needle and analytes start to adsorb to the fiber. When the sampling period has finished, the fiber is retracted into the needle and subsequently extruded again in the injector of a gas chromatograph, in which the adsorbed compounds are thermally desorbed and transferred to the analytical column.

Due to their simplicity, sensitivity and wide range of fiber coatings (e.g. PDMS, PDMS/DVB, PDMS/CAR), SPME devices are used for analyzing various volatiles released by insects (Borg-Karlson and Mozuraitis, 1996; Andersson et al., 2000), fungi (Demyttenaere et al., 2003), plants (Vercammen and Sandra, 2000; An et al., 2001; Zabaras and Wyllie, 2001; Silvestrini et al., 2004; Custódio et al., 2006; Li et al., 2006), fruits (Matich et al., 1996; Song et al., 1997; Agelopoulos and Pickett, 1998), food products (Roberts et al., 2000; Cavalli et al., 2003; Povolo and Contarini, 2003), essential oils (Field et al., 1996) and drugs (Musshoff et al., 2002). In the investigations this thesis is based upon (Papers I – VI), volatiles were trapped using SPMEs with a 65 µm film of PDMS/DVB coating the fiber (Supelco, Bellefonte, PA, USA).

Characterizing constituents in a gas-phase associated with plant material is a complex task, because the emissions consist of large numbers of compounds with widely differing volatilities and partition coefficients between the gas phase and SMPE fiber coating ((Matich et al., 1996; Martos et al., 1997). In order to describe the composition of a plant headspace, the concentrations of substances in the gas-phase and SPME fiber coating have to be in equilibrium. Molecules of high MW, especially if released in small absolute amounts, need more time to reach equilibrium than small molecules (Matich et al., 1996; Martos and Pawliszyn, 1997). If volatiles are to be trapped above the extract, in some cases the long collection times sometimes required can be shortened by reducing the size of the headspace, heating or agitation (e.g. by allowing air to flow through the collection jar, thus creating a “dynamic” headspace) (Bartelt, 1997; Pawliszyn, 1997; Vercammen et al., 2000). However, due to practical limitations, and because living organisms were investigated (conifers and mites), these options could not be used. Therefore, volatiles in whole plant emissions were trapped at room temperature over 24-hour sampling periods (Papers I - VI).

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2.3.2. Handling of plant material Volatiles of unwounded spruce and pine seedlings were collected by placing the seedlings

into either glass jars (I, IV) or bags made of an oven-proof plastic material (II, III). Piercing the stem of a seedling was determined to release similar composition of volatiles as were trapped from a piece of phloem. Hence depending on the size of seedlings, the bouquets of phloem tissues were analyzed by either piercing the stem (I) or by placing pieces of phloem into a glass vial, then immediately sealing them with aluminum foil (II, III, IV).

Emissions from mite-infested spruce clones (V, VI) were collected by inserting the foliar parts of the spruces into plastic bags. Phloem volatiles of spruces attacked by Nalepella sp. (V) were trapped by piercing their stems and inserting the plants immediately into a bag up to the wounded stem.

Each spruce and pine was analyzed separately.

2.3.3. Separation and identification of volatiles Emissions of conifers were analyzed by GC-MS using a Varian 3400 chromatography,

equipped with a non-polar CB1 capillary column to separate the compounds, coupled to a Finnigan SSQ 7000 mass spectrometer. Reference libraries of NIST (National Institute of Standards and Technology) and MassFinder 3.06 together with mass spectra and other relevant data regarding reference samples (Davies, 1990; Orav et al., 1996; Ruther, 2000; Gargouri et al., 2004), were used for identifying the volatiles.

Since several authors have found that the relative proportions of enantiomers of some terpenes can influence the behavior of pine weevils and beetles (Nordlander, 1990; Nordlander, 1991; Wibe et al., 1998; Almquist et al., 2006), the enantiomeric composition of α-pinene and limonene was also examined when analyzing the emissions of healthy and stressed plants. In order to separate the chiral enantiomers a 2D-GC system (Borg-Karlson et al., 1993) was used, including two Varian 3400 instruments, one equipped with a polar and the other with a chiral column, thus allowing separate temperature programs to be applied, providing baseline separation of the peaks (with appropriate “cutting”) and helping to avoid the co-elution of substances.

2.4. Statistical analysis 2.4.1. Multivariate data analysis

Species, clone, age and stress related patterns in variations in the composition of the collected volatiles released by conifers were explored by multivariate data analysis using Canoco 4.5 software (developed by Braak and Smilauer, Biometris Plant Research International, The Netherlands). Data on the peak areas and relative amounts of compounds were subjected to PCA (Wold et al., 1987). After mean-centering, square-root or logarithmic data transformation was

10

used. On the score plots equal weighting was given to each compound. The significance of differences between the emissions was tested by Monte-Carlo permutation tests using RDA. If the differences between compared bouquets were significant, i.e. the comparisons yielded P value lowers than 0.05, F- and T-tests were used to evaluate the differences in more detail.

2.4.2. Correlation Analysis By measuring the correlations between selected constituents in the emissions we were able

to tentatively explore the biosynthetic routes of selected plant volatiles. For this purpose, the peak areas of selected volatiles derived from chromatograms were plotted against each other, and the correlations between them were calculated in terms of Pearson correlation coefficients using ExcelTM (Microsoft). High correlation was found between 3-carene and terpinolene (data not shown), which is known to be a by-product of the reaction catalyzed by the 3-carene synthase isolated from P. abies by Fäldt et al. (2003).

11

3. Volatile profiles of conifers The variation in chemical composition of secondary metabolites indicates certain

chemotypes (Hiltunen et al., 1975a; Esteban et al., 1976; Thoss et al., 2007), and since the occurrence of some polyphenols and terpenes is determined genetically, the compounds are used, in addition to isoenzymes and DNA, in studies of genetic variations in trees (Hiltunen et al., 1975b; Yazdani and Lebreton, 1991).

The genetic composition, variations and structure of various European conifers have been intensively investigated (Müller-Starck et al., 1992). Studies on the geographical distribution of conifer populations have shown (inter alia) that the absolute terpene contents of conifers are influenced by soil parameters, and their relative proportions are affected by genetic variables influenced by environmental factors (Hiltunen et al., 1975a; Yazdani et al., 1985; Semiz et al., 2007).

3.1. Chemotypes of spruces and pines 3.1.1. Spruces (Papers II and III)

Chemical analyses of spruces showed that emissions from unwounded seedlings and pieces of phloem obtained from them differed considerably (Figure 2 in II). Therefore, the following discussion about spruce chemotypes will be based solely on data obtained from analyses of whole plant emissions from unwounded spruce seedlings.

There were substantial variations in the profiles of volatiles released by the unwounded Norway spruce seedlings. However, of 80 examined most belonged to either the “limonene” or the “bornyl acetate” chemotype (46% and 15% of the seedlings, in which the proportions of limonene and bornyl acetate in their volatiles amounted to 31-63% and 35-54%, respectively; Figure 2). The monoterpene hydrocarbon camphene has been found to dominate solvent extracts of spruce needles by several authors (Borg-Karlson et al., 1993; Persson et al., 1993; Persson et al., 1996), while camphene together with bornyl acetate are the main monoterpenes in ageing spruce needles according to Merk et al. (1988). Thus, as mentioned above, apparent discrepancies between data presented in previously published studies and Paper II could be due to the use of seedlings from different localities or populations, or to age-effects on needle terpenes (Merk et al., 1988; Persson et al., 1993). In addition, different sampling (e.g. solvent extraction or headspace analyses) and analytical methods have been used to characterize various plant chemotypes, further complicating attempts to compare data obtained by different authors (II; Persson et al., 1996; Orav et al., 1996).

The occurrence and inheritance of “3-carene” and “α- + β-pinene spruces” (II) has been discussed (Esteban et al., 1976), but due to the lack of information about the inheritance of

12

limonene, bornyl acetate and estragole it is difficult to tell whether the examined seedlings are representative of the respective chemotypes (II). Figure 2. PCA score plots characterizing different types of Norway spruce seedlings, based on relative amounts of compounds in volatiles obtained from unwounded seedlings (peak areas from GC-MS analyses, normalized to 100%). The sizes of circles are related to the relative proportions of bornyl acetate, limonene and estragole in plots A, B and C, respectively.

Spruce seedlings of limonene and bornyl acetate types released different relative proportions of volatiles (see Appendix 2A). The limonene and bornyl acetate released by unwounded “limonene spruces” amounted to 48% and 13% of their measured volatiles, while the corresponding proportions for “bornyl acetate seedlings” of similar age were 25% and 38%, respectively. Due to the differences in the relative proportions of limonene and bornyl acetate, there were substantial differences in the emission profiles of the unwounded seedlings (T-test, P < 0.01 for both limonene and bornyl acetate).

Some unwounded spruces also released more sesquiterpenes than the others (Figure 3). For example, a high proportion of longifolene was present in the emissions of one “3-carene spruce”, out of two representatives (see Appendix 2A). Therefore, a more detailed evaluation was carried out to assess whether the release of sesquiterpenes is chemotype-specific. For this purpose a PCA plot was prepared based on the relative amounts of all identified volatiles (Table 2 in II), except sesquiterpenes, amounts of which were summed, and plotted as a group. The positions of chemically different seedlings were defined by dummy variables (0, 1).

The PCA analysis showed high proportions of sesquiterpenes (16 – 31%) to be present in the emissions of some unwounded individuals of both bornyl acetate and 3-carene types (Figure 3). Hence as a conclusion, during the active growth of the first year Norway spruce seedlings, changes in terpene biosynthesis lead to high proportions of sesquiterpenes being released more often by some individuals of “bornyl acetate spruces” and by rare “3-carene-” and “α- + β-pinene spruces”.

1.5 -1.0 PC 1, 34%

-1.0

PC

2, 2

8%

1.5 A

1.5-1.0 PC 1, 34%

B

1.5-1.0 PC 1, 34%

C

13

Figure 3. PCA score plot based on the volatiles (see Table 2 in II) of 22 – 27 weeks old unwounded spruce seedlings, in which proportions of sesquiterpenes are summed, the size of the circles is related to the relative proportion of grouped sesquiterpenes, and the arrows show directions associated with different types of seedlings. Black circles, 22-week-old seedlings; grey circles, 27-week-old seedlings.

3.1.2. Pines (Paper III)

Differences in the terpene contents of mature specimens of spruces and pines have been reported by several authors (Borg-Karlson et al., 1993; Persson et al., 1993; Persson et al., 1996; Sjödin et al., 1996). The studies reported in Papers II and III extended these findings by showing that considerable differences are present even in early stages of seedling development (II, III). Indeed, different chemotypes of pines could be distinguished by analyzing the composition of terpenes released by unwounded seedlings and pieces of phloem from them when they were still less than six weeks old (III).

Various analytical methods have been applied to characterize the chemotypes of pines (Hiltunen et al., 1975a; Hiltunen et al., 1975b; Sjödin et al., 1996; Sjödin et al., 2000; Yassaa and Williams, 2007), but the chemotypes distinguished using the SPME-method were consistent with the published data.

Among 99 unwounded pine seedlings, 83% belonged to 3-carene and 16% to α-pinene chemotypes (Figure 4A, 4B) (Hiltunen et al., 1975a, Hiltunen et al., 1975b; Yazdani et al., 1985; Manninen et al., 2002; Thoss et al., 2007). In the emissions of one unwounded seedling unusual proportions of limonene and α-pinene (almost equal) were detected (Figure 4C), while phloem obtained from it released mostly α-pinene (74% of total volatiles; Figure 2 in III). Intriguingly, this is consistent with the finding by Yazdani et al. (1985) of pines that have relatively high

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PC 1, 41%

PC2,

30%

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-1.0 PC 1, 41% 1.5 -1.0 PC 1, 41% 1.5

proportions of limonene in their emissions in native populations located close to latitude 68ºN in Sweden.

Figure 4. PCA score plots characterizing different types of Scots pine seedlings based on relative amounts of compounds in volatiles from unwounded seedlings (peak areas from GC-MS analyses normalized to 100%). The sizes of circles are related to the relative proportions of 3-carene, α-pinene and limonene in plots A, B and C, respectively. White circles, 3.5 to 11.5 week-old plants; black circles, 35 to 43 week-old seedlings. Arrows indicate directions of positions associated with 3.5 and 43 weeks old pines.

Like spruce seedlings, pine seedlings differ in the monoterpene hydrocarbon profiles (Sjödin et al., in 2000) and, in accordance with expectations, the analyses presented in Paper III of unwounded seedlings and pieces of phloem from them showed that 3-carene and α-pinene were the main terpenes in pines of 3-carene and α-pinene chemotypes, respectively (Figure 2 in III). When bouquets of seedlings of similar age were compared, the emissions of “3-carene seedlings” had higher proportions of sabinene, p-cymene, γ-terpinene, terpinolene, 2-methyl-6-methylene-1,7-octadien-3-one, geranyl acetone, α-longipinene, α-copaene and sativene than “α-pinene pines”, and lower proportions of limonene, β-phellandrene, (E)-β-ocimene and (E)-β-caryophyllene (see appendix 2B). Hence, differences in the oxygenated monoterpene and sesquiterpene contents can also be found in the volatiles emitted from pines of different types.

3.1.2. Resistance of different chemotypes of spruce and pine seedlings to Hylobius abietis (Papers II and III)

Conifer chemotypes are of significant potential practical interest because of the differences in their resistance to herbivores (Wheeler and Ordung, 2005; Thoss and Byers, 2006). For example, pines that produce high amounts of (-)-α-pinene have been found to be attacked more often by I. typographus beetles than others (Almquist et al., 2006), and correlations have been found between levels of (S)-cis-verbenol, an aggregation pheromone, extracted from the hindgut of male I. typographys beetles and the proportions of (-)-α-pinene in phloem tissues of attacked spruces (Lindström et al., 1989).

The main volatile in most of the spruce seedlings we examined was (-)-limonene, a known repellent of pine weevils (Nordlander, 1990; Nordlander, 1991; Wibe et al., 1998). In addition,

A

1.5 -1.0 PC 1, 41%-1.0

P

C 2

, 25%

1.

5

A B C

15

bornyl acetate and estragole activate both olfactory and gustatory receptors of H. abietis (Wibe et al., 1997; Klepzic and Schlyter, 1999). Thus, the most resistant of the highly diverse spruces to this serious pest could be the rare estragole type, or seedlings that release high amounts of limonene or bornyl acetate. However, no data on the behavior of H. abietis have been acquired as yet to test this hypothesis.

It has been reported (Leather et al., 1994) that pine weevils prefer Scots pines to Norway spruces. The reason for this could be related to the higher proportion of (+)-α-pinene, an attractant of pine weevils, than (-)-limonene in the emissions of undamaged pines of 3-carene and α-pinene chemotypes (Figure 2 in III) (Nordlander, 1990; Nordlander, 1991; Wibe et al., 1998). The responses of herbivores to plants are related to the composition of the plants’ bouquets of volatiles (see chapter 1), however 3-carene also elicits a strong antennal response in H. abietis, albeit not as strongly as α-pinene and limonene (Wibe et al., 1997). Investigations into the resistance of pines of different chemotypes to pine weevils could provide indications of the suitability of 3-carene as a pest resistance marker of pines.

3.2. Relative proportions of conifer volatiles 3.2.1. Spruce seedlings (Papers I and II)

Data about the spruce seedlings discussed in chapter 3.1.1 show the high diversity of spruce chemotypes in orchards of Saleby and Österfärnebo (II). Despite the high standard deviations of the relative proportions of both individual and grouped volatiles, evaluation of the data obtained from all of the spruce types showed there were considerable differences between the emissions of seedlings of different ages (II).

Grouping of the seedlings according to their age demonstrated that limonene and bornyl acetate dominated the emissions from seedlings of the respective types as they aged, throughout the monitoring period, but the changes in the relative proportions of some terpenes varied amongst different types of spruces (Figure 5). For example, as the seedlings aged the proportion of limonene in the volatiles of “limonene spruces” seedlings increased, while the proportion of bornyl acetate in the volatiles of “bornyl acetate spruces” declined. In addition, there were differences between these chemotypes in the changes in proportions of volatiles classified as “other MT” (Table 2 in II; Figure 5). There was also an apparently limonene chemotype-specific increase in the proportion of estragole by the end of the seedlings’ active growth-phase (27 weeks). However, due to scarcity of these seedlings their volatile contents did not differ statistically significantly from those released by “bornyl acetate spruces”. In contrast to the other volatiles, changes in the sesquiterpenes released were similar in both “limonene” and “bornyl acetate seedlings” (Figure 5).

16

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n=18 n=7 n=12 n=5 n=4 n=3

7 - 10 weeks 22 - 27 weeks 35 - 43 weeks 7 - 10 weeks 22 - 27 weeks 35 - 43 weeks

"limonene spruces" "bornyl acetate spruces"

Rel

ativ

e pr

opor

tions

, %

α-pinene limonene 3-carene other MTborneol bornyl acetate geranyl acetone other MT-OSqT estragole methylthymylether

Figure 5. Relative proportions of volatiles (means ± SD) released by unwounded spruce seedlings (volatiles in each age group normalized to 100%). 7-10 weeks and 22-27 weeks old spruce seedlings analyzed during the active growth-phase; 35 and 43 weeks old seedlings analyzed during the winter/early spring.

The composition of spruce volatiles was also influenced by the time of year that the

seedlings were sown (III). For instance, sesquiterpenes were the main compounds in the whole plant bouquets of unwounded 1-year-old conventional spruces (sown in March, analyzed in the following spring; I), while limonene dominated the emissions of 43-week-old seedlings (also called 1-year-old seedlings, but sown in July and analyzed in the following spring; II). In section 3.1.1 a high proportion of sesquiterpenes was shown to be characteristic of rare α- + β-pinene- and 3-carene chemotypes. However, too few replicates were analyzed to draw definitive conclusions about whether or not the sesquiterpene contents of older seedlings may be chemotype specific.

The composition of volatiles stored in phloem tissues differed between seedlings of differing ages (Figure 6). The main volatiles detected in the phloem emissions from 7-10 weeks old seedlings were (3Z)-hexenal and borneol (Figures 6). Detectable emission of α-pinene and limonene started after the 20th week of growth, and by the end of the growth-period (the 27th week), levels of GLVs were sub-detectable in the phloem tissues (Figure 6). Phloem emissions of wintering seedlings contained relatively high proportions of sesquiterpenes. However, by the 43rd week the phloem pieces again released GLVs. In addition, phloem emissions of seedlings kept in an outdoor area (OA) analyzed in May had higher proportions of GLVs than those obtained from seedlings kept in a freezer, showing that their phloem tissues were in a “spring phase”.

Comparison of the phloem volatiles of conventional seedlings (I) and 43-week-old OA spruce seedlings (II), showed that GLVs were only abundant in the latter (II), presumably because of the difference in the time they were sown (see above, in this section). Before in-wintering, seedlings sown in March (I) have more time to develop than those sown in July (II).

17

α-Pinene, β-pinene, α-longipinene and longifolene were found to be the main phloem volatiles. In addition, camphene was present, together with α-pinene, in phloem tissues of some spruces. Although no attention was focused on the enantiomeric composition of camphene and β-pinene during the analyses, the correlations found between the (+)-enantiomers and (-)-enantiomers of α- and β-pinene, camphene and α-pinene, in spruce tissues support their occurrence (Sjödin et al., 2000). (-)-α-Pinene and (-)-β-pinene are also reportedly co-products of MeJA-activated pinene synthase (Martin et al., 2004). Finally myrcene, 3-carene, β-phellandrene, limonene and terpinolene were detected as minor constituents of phloem emissions of some spruce individuals.

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(3Z)-hexenal (E2)-hexenal (3Z)-hexen-1-ol α-pinenelimonene other MT borneol bornyl acetateother MT-O SqT methythymylether

Figure 6. Relative proportions of phloem volatiles (means ± SD). Peak areas of compounds obtained in GC-MS analyses of samples representing each age are normalized to 100%. M and J, seedlings sown in March and July respectively; OA, seedlings derived from the outdoor area; F, seedlings from the freezer.

Phloem samples from some spruces released solely longifolene (enantiomers not separated),

while others also released α-longipinene and small proportions of longicyclene or cyclosativene. Thus, at least two enzymes appear to be involved in the production of longifolene. Among minor sesquiterpenes small amounts of (E)-β-caryophyllene co-existed with α-humulene (see also section 3.4.2), and germacrene D was detected in samples in which β-bourbonene and δ-cadiene were present.

Growth phase

Winter / early spring phase

18

3.2.2. Pine seedlings (Paper III)

During ageing the composition of volatiles emitted from unwounded pines changed more than those of the main types of spruces (Figure 7). Geranyl acetone, which is known to be produced from nerolidol (Boland et al., 1998), was a dominant compound in the volatiles of 3.5-week-old seedlings, but present only in minor amounts in the emissions of older seedlings of various chemotypes. The relative amounts of α-pinene and 3-carene increased in pines of the 3-carene chemotype, but increases in both α-pinene and limonene were detected in representatives of the α-pinene chemotype (Figure 7).

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α-pinene 3-carene limonene other MT geranyl acetoneother MT-O longifolene other SqT others

Figure 7. Relative proportions of volatiles (means ± SD) released by unwounded pines of 3-carene and α-pinene chemotypes (peak areas of compounds obtained in GC-MS analyses for each seedling age/age range normalized to 100%).

Since no information is available on the effects of geranyl acetone on the behavior of forest pests, it is impossible to predict whether the youngest “3-carene pines” would be more resistant than the older ones. However, the occurrence and dose of 3-carene reportedly affect survival rates of conifers (Rocchini et al., 2000; Pasquier-Barre et al., 2001). Behavioral or field experiments are warranted to find out whether the survival rates of “3-carene” pine seedlings could be related to the amounts of 3-carene they release (see section 3.1.2).

High amounts of GLVs were only found in the wounded phloem samples of 3.5-week-old pines (Table 3 in III; Figure 4 in III). During ageing of “3-carene pines”, the relative proportion of the main terpene increased and the phloem tissues of wintering pines released relatively high proportions of SqT (Figure 4 in III).

19

3.3. Enantiomeric composition of α-pinene and limonene 3.3.1. Spruce seedlings (Papers I and II)

Despite the existence of different chemotypes, the relative proportions of α-pinene and limonene enantiomers released by unwounded spruce seedlings of different ages and developmental stages were similar (Table 4 in II). In the age range of 22-43 weeks, the proportion of (-)-α-pinene declined from 81% to 74%, while that of (-)-limonene varied only between 73% and 79% (amount of (-)-enantiomers divided by the sum of (-)- and (+)-enantiomers, in percent) (Table 4 in II). The data on the enantiomeric composition of α-pinene of unwounded spruces presented in Paper I and II show that there were no significant changes in the enantiomeric composition of α-pinene released during the first year, but there was a marked decline in the proportion of the (-)-enantiomer released by conventional seedlings (to 64%; T-test, P < 0.04). The proportion of (-)-limonene in the bouquets of unwounded conventional seedlings (I) was 68%, which was not significantly different from the proportions detected in the emissions of young spruce seedlings (II).

In addition to the major changes observed among the phloem volatiles of spruces (Paper II), ageing of seedlings also affected the proportions of α-pinene enantiomers (Figure 8).

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Figure 8. Relative proportions of (-)-α-pinene and (-)-limonene enantiomers – calculated by dividing 2D-GC peak areas of the (-)-enantiomer by the summed peak areas of (-)- and (+)-enantiomers, normalized to 100% – released by pieces of phloem from spruce seedlings (means ± SD). F, winter-phase seedlings from the freezer.

Dividing the results obtained from 22 and 27 weeks old seedlings (n=14) into groups

showed that the phloem tissues initially released less (-)-α-pinene than (+)-pinene, but during the following four weeks the relative importance of the enantiomers reversed and the proportion of (-)-α-pinene increased from 40% to 80%. The analyses of 28-week-old seedlings kept in the freezer (II) and conventional seedlings (I) detected no significant changes in the enantiomeric composition of α-pinene (Figure 8).

Ageing of seedlings also influenced the proportions of limonene enantiomers (Figure 8). Initially, 36% of limonene emitted from wounded spruce phloem seedlings consisted of (-)-

20

limonene enantiomer, but during the following four weeks the proportions of the two enantiomers became almost equal, that of the (-)-limonene increasing to 55%. Furthermore, data obtained from the conventional seedlings indicate that the ratio of limonene enantiomers continued to change at least until the beginning of their second year of growth, when the proportion of the (-)-limonene was 71% (Figure 8). The proportions of (-)-α-pinene and (-)-limonene in needles of mature Norway spruces reportedly decrease during ageing (Persson et al., 1993). Thus, the relative rates of biosynthesis of different monoterpenes clearly change with the age of the tissues.

Both the unwounded and wounded conventional seedlings (I) released similar enantiomeric proportions of (-)-α-pinene and (-)-limonene. However, in absolute terms, injured seedlings emitted much more combined α-pinenes than limonenes (Figure 9). Therefore, one of the reasons why conventional spruces are attacked more by weevils could be related to the attractive α-pinenes masking the smell of the repellent (-)-limonene from healthy spruces (Nordlander, 1990; Nordlander, 1991).

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Figure 9. Relative proportions of α-pinene and limonene enantiomers (means ± SE) released by unwounded and wounded conventional seedlings. The amounts of (-)- and (+)-enantiomers of α-pinene and limonene are normalized to 100% for each plant type.

3.3.2. Pine seedlings (Paper III)

In the analysis of chiral monoterpenes in pine seedlings the enantiomeric composition of the various chemotypes was initially compared. The only significant observed effects of ageing of pine seedlings on the enantiomeric proportions of the volatiles were in the proportions of α-pinenes released by unwounded “α-pinene pines” (Figure 6A in III). For example, 32% of the α-pinene emitted by unwounded 3-carene chemotype pines consisted of (+)-α-pinene, on average, throughout the whole year of monitored growth, while the proportions released by “α-pinene pines”, increased considerably, from 30% (active growth-phase) to 48% (winter/early spring-phase) (P = 0.02). In contrast, 58% of the limonene released by unwounded seedlings of both types consisted, on average, of (-)-limonene throughout the year (Figure 6B in III).

On average, 20% and 52% of the α-pinene and limonene released from the phloem samples from “3-carene pines” consisted of (-)-α-pinene and (-)-limonene, respectively (Figure 6A and 6B

21

in III). Phloems of seedlings of the α-pinene chemotype in the active growth phase emitted considerably larger proportions of (-)-α–pinene (53%) than their wintered and in-wintered counterparts (24%) (P < 0.01) (Figure 6A in III). Due to the large degree of scatter in the data, no statistically significant difference was found between the composition of limonene released by the phloem tissues of “α–pinene seedlings” (64% of (-)-limonene) (Figure 6B in III).

Finally, data obtained from mini-pines (III) were compared to data obtained from 2- and 3-year-old seedlings (Figure 10). The proportions of α-pinene enantiomers emitted by “3-carene pines” of the three different ages were similar; the (-)-enantiomer consistently accounting for ca. 23% and 12% of the α-pinenes emitted by unwounded seedlings and pieces of their phloem, respectively. Statistically significant differences were found for the composition of limonene.

Figure 10. Relative proportions of (-)-α-pinene and (-)-limonene – calculated by dividing the peak areas of (-)-enantiomers obtained in 2D-GC analyses by the sum of (+)- and (-) enantiomer peak areas, normalized to 100% (means ± SE) – released by pine seedlings of 3-carene chemotype. The enantiomeric composition of the limonene released by unwounded 2 and 3 years old pines was similar, but that of 1 and 2 years old pines was significantly different (T-test, P = 0.03).

According to results obtained from the phloem samples of the pine seedlings, the relative proportions of the limonene enantiomers changed with age, as found for spruce seedlings (Figures 8). T-tests proved that the proportions of (-)-limonene released by phloem samples from 1-year-old-pines were significantly lower than those in the phloem emissions of 3-year-old pines.

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Emissions of unwounded seedlings

Emissions of phloem pieces

22

3.4. Biosynthesis of terpenes of young seedlings 3.4.1. Monoterpenes (Paper IV)

Several authors have shown emissions of compounds to be related to the activities of corresponding enzymes (Dudareva et al., 2000a; Martin et al., 2003), to substrate concentrations (Bartram et al., 2006) and/or to synthase concentrations (Dudareva et al., 2000a). Therefore, peak areas and relative amounts of compounds were also used in correlation analysis as an alternative means to explore the biosynthetic links among the terpenes as primary or induced constituents of resin (Sjödin et al., 2000; Fäldt et al., 2001).

The results, discussed below, are in accordance with the age-related changes observed in the chemical composition of the seedlings (Figures 5-10). Despite of different chemotypes among mini-seedlings of Norway spruce and Scots pine mini-seedlings (II, III), the enantiomers of α-pinene and limonene correlated (Figure 1, 2 in IV). The highest correlation was found between limonene enantiomers released by unwounded spruce and pine seedlings (Figure 1, 2 in IV). In earlier investigations a correlation between the enantiomers of α-pinene is shown for Cuban pine (Pinus cubensis, Fäldt et al., 2001). For loblolly pine (Pinus taeda) α-pinenes are published as co-products of (-)-β-pinene synthase (Phillips et al. 2003). Due to a few number of spruces replicates per chemotype, it was difficult to evaluate, whether the emission of monoterpenes is chemotype specific. However plotting of α-pinene enantiomers; and of (+)-α-pinene and (+)-limonene of unwounded spruces of 22 and 27 weeks showed the highest value of (+)-α-pinene to represent the spruce of α-pinene/β-pinene chemotype, while the next highest value belonged to a seedling releasing relatively 12% of estragole (Figure 1 in IV). In spite of existence of a few pines of α-pinene chemotype among seedlings of 9.5 and 11.5 weeks, the correlation between (+)-α-pinene and (+)-limonene released by unwounded pines and their phloem pieces seemed to be depend on the pine chemotype, id est. on the genetics of pines (Figure 2 in IV).

Sjödin et al. (2000) have shown the co-emissions of selected monoterpenes to be tissues-specific. In our studies, the unwounded pine seedlings of α-pinene chemotype released similar amounts of α-pinene and limonene as the phloem tissues of respective pines. Thus the co-emission of (+)-α-pinene and (+)-limonene was found to be tissue specific (Figure 2 in IV). High distribution of the aforementioned volatiles in the phloem emissions of “α-pinene pines” suggests the involvement of additional enzymes producing (+)-α-pinene and (+)-limonene as by-products of some other monoterpenes (Martin et al., 2004). In case of 3-carene pines the emissions of α-pinene and limonene from unwounded seedlings and their phloems were too different. Therefore it was difficult to make any conclusions, whether the co-existence of α-pinene and limonene enantiomers is tissue-specific also for “3-carene pines”.

3.4.2. Sesquiterpenes (Paper IV)

The main findings of the sesquiterpene analyses were as follows. Longifolene and (E)-β-caryophyllene were the main sesquiterpenes of unwounded 22 and 27 week-old spruces of both

23

limonene and bornyl acetate types (see Appendix 2A). There was also evidence of a co-occurrence of α-longipinene and longifolene (Figure 3 in IV), in accordance with findings that a gene is induced by MeJA treatment in Norway spruce which encodes an enzyme that catalyses the biosynthesis of both longifolene and α-longipinene (Martin et al., 2004). Additionally in 1998 Steele et al. proposed these two sesquiterpenes to be derived from a common intermediate. Distribution of data of 7 – 10 weeks old spruces in Figure 3 (IV) suggested the possible existence of two different enzymes, which might release α-longipinene and longifolene in different ratios. Surprisingly during ageing of seedlings the release of those compounds changed (Figure 3 in IV). During the studies the enantiomers of sesquiterpenes were not analyzed (König et al., 1999), thus no conclusion can be drawn about whether or not any co-occurrence was enantiomer-specific.

In pines a strong correlation between (E)-β-caryophyllene and α-humulene has been detected (Semiz et al., 2007), and a similar correlation was found in the needle emissions of young unwounded spruce seedlings (Figure 11). We also found a correlation of (E)-β-caryophyllene and α-humulene while evaluating the data of unwounded pines and of phloem tissues of spruces. Caryophyllenes are discussed to be synthesized via a common intermediate (Steele et al., 1998; Picaud et al., 2006). However, their high release from Norway spruce and Scots pine mini-seedlings might be caused by (a) caryophyllene enzyme(s) present in the needle and phloem tissues of seedlings. Figure 11. Correlation plots of sesquiterpenes (GC-MS peak areas) of spruce seedlings.

0

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4. Volatile profiles of infested spruces 4.1. Volatiles released by spruces attacked by mites (Papers V and VI)

Stress reactions triggered by mites have been investigated in angiosperms (Dicke et al., 1990; Takabayashi et al., 1994, Arimura et al., 2000a, Arimura et al., 2000b; Takabayashi et al., 2000; Kessler and Baldwin, 2001; Arimura et al., 2004; Van Den Boom et al., 2004; Arimura et al., 2005; Himanen et al., 2005), and conifer defense responses to mites have been found to be dependent on the density of the mites in the spruce dwarf white spruce Picea glauca (Kielkiewicz et al., 2005; Puchalska, 2006). Therefore, in the studies reported in Papers V and VI interest was focused on the bouquets of Norway spruces that were being severely attacked by mites.

Mites feeding on the foliage of spruces induced the emission of several terpenes that were not detected in whole plant emissions of healthy clones 1091, 1090, 72 and 1321 (Figure 12; Table 1 in both V and VI). The releases of de novo compounds are probably related to the increased activity of corresponding synthases (Martin et al., 2003; Miller et al., 2005).

In addition to the main stress-induced volatiles – linalool, (E,E)-α- and (E)-β-farnesene – and unidentified compounds, the attacked spruces released (Z)-β-ocimene, 2,6-dimethyl-2,6-octadiene, cis- and trans-linalooloxides, citronellal, geraniol, geranial, trans-α- and trans-β-bergamotene, and both (Z)-β- and (Z,E)-α-farnesene (Table 1 in both V and VI). (E)-α-Bisabolene was released by the healthy clones 1091, 1090 and 72, but infestation of mites increased its emission (Table 1 in V). Farnesenes together with linalool and (E)-α-bisabolene were also the defence-related compounds produced by MeJA-treated and weevil-attacked Sitka spruces examined by Martin et al. (2003) and Miller et al. (2005). In contrast, abiotically stressed pines (kept under continuous light-regime of summer) released MeSA, (E)-β- and (E,E)-α-farnesene, but no linalool was detected (Kännaste, unpublished data).

The bouquet of compounds synthesized de novo also included GLVs and aromatic volatiles (Table 1 in both V and VI). Among them (3Z)-hexen-1-ol, (3Z)-hexenyl acetate and MeSA are common plant volatiles that are often released following herbivore attack or air pollution damage (Heiden et al., 2003; Van Den Boom et al., 2004; Himanen et al., 2005; Vuorinen et al., 2005). They have also been found to induce indirect defense reactions in neighboring, intact plants (Shulaev et al., 1997; Arimura et al., 2001).

Stress reactions of plants induced by specific compounds present in the oral secretions of herbivores (Arimura et al., 2005) are known to be herbivore- and plant clone-specific (Takabayashi et al., 1991; Takabayashi et al., 1994; Himanen et al., 2005; Vuorinen et al., 2005; Delphia et al., 2007). Due to difficulties in controlling the population of mites (V and VI), large variations were found in the abundance of many compounds synthesized de novo. Nevertheless, statistical analysis demonstrated that there were clone-specific patterns in the relative amounts and peak areas of linalool, MeSA, trans-α-bergamotene, (E)-β-farnesene, (E,E)-α-farnesene and β-sesquiphellandrene (Table 2 in V). In addition, in spruces of clone 1321 releases of linalool and (E)-β-farnesene were Nalepella sp.-specific (Figure 12; Table 2 in VI).

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Figure 12. Representative chromatograms of five years old spruces of clone 1321. Identified compounds – 1:,6-methyl-5-heptene-2-one; 2, 3-carene; 3, limonene; 4, 2,6-dimethyl-2,6-octadiene; 5, (-)-linalool; 6, methyl salicylate (MeSA); 7, butyl carbitol acetate (background compound); 8, vanillin; 9, unidentified compound 119 (100), 93 (90), 41 (59), 80 (52), 79 (50); 10, (E)-β-farnesene; 11, (E)-α-bergamotene; 12, (E,E)-α-farnesene; 13, (E)-α-bisabolene; 14, unknown SqT (MW 218).

4.1.1. Enantiomeric composition of chiral monoterpenes in the whole plant emissions (Papers V and VI)

The enantiomeric composition of certain terpenes did not seem to change during mite infestation. For instance, ca. 80% of the limonene released by both healthy and mite-infested spruces of clone 1321 consisted of the (-)-enantiomer (VI). Furthermore, a similar proportion (88%) of (-)-limonene was released by MeJA-activated (-)-limonene synthase originating from Norway spruce in a study presented by Martin et al. (2004).

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The proportions of the linalool enantiomers produced and released in responses to the two species of mites were similar – ca. 95% of the (-)-enantiomer in both cases, in the experiments in which induced spruce defenses were examined, despite the use of different Norway spruce genotypes (V, VI), Synthases producing similar proportions of the linalool enantiomers have also been identified in both Norway and Sitka spruces stressed by MeJA and/or weevils (Martin et al., 2004; Miller et al., 2005).

4.1.2. Enantiomeric composition of chiral monoterpenes stored in the phloem tissues of spruces attacked by mites (Paper V)

Feeding by pine weevils on the stem of Sitka spruces (P. sitchensis) is known to trigger defense reactions in the needles of the plants (Miller et al., 2005). The emissions of defense compounds from non-attacked parts of a tree might be induced either by an airborne signal from the wounded site or by certain rapidly migrating messenger compounds, e.g. a protein, ACC, ethylene or MeSA (Hudgins and Franceschi, 2004; Krekling et al., 2004, Park et al., 2007), that (inter alia) increases in the activity of monoterpene synthases e.g. (+)-3-carene, (-)-limonene and (-)-α/β-pinene synthases (Fäldt et al., 2003; Martin et al., 2004).

The composition of monoterpenes stored in the phloem tissues of spruces was analyzed by piercing the phloem of spruces then collecting emitted volatiles. PCA score plot of the 2D-GC data showed that although the emissions of the three clones were significantly different (Figure 4 in V), the relative composition of the selected terpenes released by phloem samples of mite-attacked and healthy clones were similar (Figure 13).

Figure 13. PCA score plot based on the relative amounts of selected monoterpenes released upon piercing the stem base of spruces. Circles, clone 1090 (the most resistant clone); x-marks, clone 72; triangles, clone 1091 (the most susceptible clone). Arrows indicate the distribution between selected monoterpenes and spruce grafts.

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The susceptible clone 1091 had the highest proportion of 3-carene and the most resistant clone released the highest proportion of (-)-limonene (Figure 4 in V). Thus, in contrast to fungal attack (Tao, personal communication), feeding by mites on the spruces does not seem to affect the relative composition of terpenes stored in the stem phloem.

4.2. Correlations amongst induced terpenes (Paper VI)

Terpenes are biosynthesized by several terpene synthases, but initially all monoterpenes are derived from GPP and sesquiterpenes from FPP (see section 1.1.1).

Subjecting the data obtained from mite-infested spruces (clone 1321) to correlation analysis allowed us to suggest some possible biosynthetic links for terpenes (Scheme 3; Figure 2 in VI). For instance, although there is no information about the biosynthetic routes of geraniol, its aldehyde, 2,6-dimethyl-2,6-octadine and (Z)-β-ocimene in spruces, the correlation plots indicated their release to be related to (-)-limonene (Scheme 3). Among the volatiles considered here, myrcene is the only one that has been described as a by-product of limonene syntheses (Martin et al., 2004).

(-)-Limonene and myrcene seem to be involved in the biosynthesis of geranial and its corresponding alcohol (Scheme 3; Figure 2 in VI). An oxidation or reduction could explain the higher correlation found between (-)-limonene and geraniol (R2=0.88) than between (-)-limonene and geranial (R2=0.53).

The oxidation or reduction step is consistent with the high correlation coefficient between the plotted peak areas of geraniol and geranial (R2=0.93) detected in the emissions of Nalepella sp.-infested spruces (V). A similar biosynthetic route reportedly occurs in basil, in which nerol is putatively derived via the oxidation of geraniol to the corresponding aldehyde (Iijima et al., 2006).

In addition, (Z)-β-ocimene and 6-methyl-5-hepten-2-one may be involved in the production of geranial (Figure 2 in VI). In partial support of this hypothesis the biotransformation of monoterpenes by fungi or fungal preparations has been intensively investigated, due to the increasing demands for natural aroma compounds (Devi and Bhattacharyya, 1977; Busmann and Berger, 1994; Demyttenaere and Pooter, 1996; Demyttenaere and Kimpe, 2001). High yields of 6-methyl-5-hepten-2-one can be produced from citral (E-isomer known as geranial; Z-isomer as neral) by Penicillium digitatum, supporting the high correlation we found between geranial and 6-methyl-5-hepten-2-one (R2=0.99).

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(-)-limonene myrcene

2,6-dimethyl-2,6-octadiene

(Z)-β-ocimene

O

6-methyl-2-hepten-2-one

OOH

(Z,E)-α-farnesene

geranialgeraniol

OPP

GPP

OPPFPP

2,6-dimethyl-2,6-octadiene

PLASTIDS CYTOSOL

Scheme 3. Proposed biosynthetic relations of stress induced terpenes.

Plotting the terpenes on the correlation plots against each other demonstrated that the release of the acyclic monoterpene 2,6-dimethyl-2,6-octadiene, which is present in grapes (Rocha et al., 2007), correlated to the one of (Z,E)-α- and (E,E)-α-farnesene. This was surprising, and difficult to explain, since the biosynthesis of mono- and sesquiterpenes are generally believed to occur via different pathways (Dudareva et al., 2006) (Scheme 1). On the other hand, there are several known examples of larger molecules, like farnesenes, being degraded into smaller compounds (Kaiser, 1993; Boland et al., 1998; Dudareva et al., 2006; Krings et al., 2006). Thus, the 2,6-dimethyl-2,6-octadiene detected in the headspace of stressed spruces may have originated from the MEP-pathway and the degradation of (Z,E)-α- and (E,E)-α-farnesene.

4.3. Biosynthetic relations of induced aromatic compounds (Papers IV and V)

In addition to MeSA the bouquets of spruces infested by Nalepella sp. and Oligonychus ununguis contained minor amounts of several aromatic compounds such as benzyl alcohol, benzoic acid, methyl benzoate, benzaldehyde, 1,2- and 1,3-dimethoxybenzene, benzyl acetate,

29

vanillin, estragole, (E)-anethole and eugenol. All of these plant compounds are derived from phenylalanine (Dudareva et al., 2006). Benzyl alcohol and its corresponding acid have been previously identified in extracts of conifer needles (Isidorov et al., 2006), and methyl benzoate has been detected as an odor constituent of female Norway spruce flowers (Borg-Karlson et al., 1985). Furthermore, the occurrence of vanillin is probably related to increased production of lignin (Jourdes et al., 2007). Therefore, we have several reasons to suggest that feeding of mites increases the activity of PAL, which is involved in lignification, as pathogens and fungi have also been shown to do (Campbell and Ellis, 1992; Lange et al., 1994; Cvikrová et al., 2006).

High correlations between MeSA and methyl benzoate, benzyl alcohol and benzoic acid were found both between and within mite-infested Norway spruces clones 1090, 1091 and 72 (Figure 14), indicating that they are synthesized by the same pathways, probably involving homologous enzymes to those found in angiosperms (Dudareva et al., 1998; Dudareva et al., 2004). Figure 14. Correlations between (peak areas from MS-chromatograms) of aromatic compounds detected among the bouquets of mite-infested Norway spruces.

4.4. Impact of induced spruce volatiles on pine weevils, Hylobius abietis (Papers V and VI)

The impacts of selected spruce stress volatiles on pine weevils were evaluated by two types of tests. In feeding bioassays conducted as described by Bratt et al. (2001) and Borg-Karlson et al. (2006), the feeding deterrent effects of GLVs were observed (Figure 15A). While in the behavior tests the orientation towards spruce stress volatiles was studied (Figures 15B and C).

In the feeding test (Figure 15A) a cut piece of pine was used. The opened areas, where weevils were allowed to feed, were loaded with a solution of a test compound (5 mM) or with a solvent (methanol) as a control. The behavior of the weevils was examined in multi-choice arenas

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(24 traps) using dispensers loaded with MeSA, (E)-β-farnesene or (-)-linalool as bait; or the combined odor of each compound and a cut spruce twig. In the first part of an experiment the effect of stress compounds was examined (six traps per dispenser of a compound and six traps without any odor). In the second part a freshly cut Norway spruce twig was suspended in each of the traps. Hence the trap of a spruce twig without a dispenser was used as a control.

Figure 15. Bioassays with the pine weevils (Hylobius abietis). A, feeding experiment; B, a trap with a dispenser in a multi-choice arena; C, multi-choice arena with traps.

In the feeding experiment (Figure 15A) the effects of (3Z)-hexenol or (3Z)-hexenyl acetate were investigated, and both compounds were found to be repellent even after 6 hours from the start of an experiment. (3Z)-hexenol was identified as one of the GLVs released by phloem tissues from young conifer seedlings (II; III), hence the volatile may serve as a marker for resistance to weevils.

In the behavior studies (Figure 15B and C) MeSA alone did not affect the weevils; whereas its combination with conifer twig odor (monoterpenes) was repellent (Figure 4 in VI). Thus, we could speculate that infested spruce grafts with high amounts of MeSA may be less attractive to the weevils than healthy ones. On the other hand, MeSA released by a stressed spruce may serve as a messenger for parasitoids and carnivorous mites (see section 1.2) or as an egg-laying deterrent (De Boer and Dicke, 2004; Ulland et al., 2008).

(E)-β-farnesene when tested alone was attractive to both female and male weevils (Figure 3 in VI). However, when the compound was offered together with spruce odor, no attraction of weevils was observed. This might have been due to the high concentration of highly attractive monoterpenes released from the cut twigs, which may have masked the effect of farnesene. The

B

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31

attractiveness of (E)-β-farnesene without background odor to weevils shows that the seedlings might be threatened by weevils if the release of monoterpenes including (-)-limonene is decreased. Like MeSA, (E)-β-farnesene may also be involved in indirect plant defenses (Mumm and Hilker, 2005).

The catches of traps with (-)-linalool did not differ considerably from the catches of controls. However, the dispensers released about 10 times more of this compound than the stressed spruces (VI), hence the behavior of weevils could be related to its dose. (-)-Linalool in combination with the spruce odor was repellent for both sexes of weevils (Figure 4 in VI). Thus, depending of the composition of a spruce’s odor, linalool and MeSA may prevent the trees from being attacked by pine weevils.

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33

5. Conclusions The main goals of the studies underlying this thesis were to investigate pest resistance markers of spruce and pine mini-seedlings and to assess why mini-seedlings are attacked by pine weevils less severely than conventional seedlings. Resistance markers might be induced in mite-infested conifers, thus stress reactions of spruces attacked by mites and the effects of stress volatiles of pine weevils were studied.

Based on the volatiles, several chemotypes were found among the spruce and pine mini-seedlings. Resistance to weevils may be related, at least partly, to the high chemical diversity among unwounded spruce mini-plants. The known repellent (-)-limonene and an antifeedant, bornyl acetate, were major compounds in the emissions of most of the spruce seedlings.

Considerable differences were found between the phloem emissions of spruces and pines. Comparison of conifer seedlings at various times during their active growth-phase showed GLV contents to be high only in the phloem of the youngest pines. Moreover, due to rapid changes in the biosynthesis of terpenes, the chemotypes of pines could be distinguished from the 5th week of their development. In spruces, α-pinene and limonene were first detected in the phloem emissions of 22-week-old seedlings.

Severe problems in plant nurseries can be caused by mites, which also trigger changes in the composition of spruce volatiles when they feed on seedlings. The release of de novo compounds is known to be related to several factors, including light, age of the needles and the plant species. Thus, given the high chemodiversity of Norway spruces, in order to obtain a clear overview of the spruce defenses induced by mites, selected spruce clones were studied instead of seedlings, and the results showed that the bouquets of “new” volatiles were spruce genotype- and mite species-specific!

(-)-Linalool, α- and β-farnesenes and MeSA were detected as the main stress volatiles. In addition, several aromatic compounds, some of which are known to be antifeedants of pine weevils, were present in minor amounts.

Bioassays were performed to study the effects of spruce stress volatiles. In feeding tests GLVs were found to be antifeedants, which may affect the feeding behavior of H. abietis towards spruce seedlings less than 20 weeks old. In behavior tests (E)-β-farnesene was found to be attractive to weevils when offered alone, while the combination of MeSA or (-)-linalool with a wounded spruce odor was repellent. Hence, in clearcut areas the survival of stressed spruce seedlings, the bouquets of which are dominated by (E)-β-farnesene, could be more endangered by weevils than healthy ones, while MeSA and (-)-linalool might increase the resistance of seedlings.

In future, more detailed behavior experiments of weevils with a mixture of all three selected stress compounds and/or with stressed spruces could be of interest to see inter alia whether the stress volatiles of different doses mask each other, and to identify the phase of stress development at which spruces become attractive to weevils. In addition, studies of stressed pines could be of interest, to see whether the production of stress compounds is conifer species-specific.

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Acknowledgements First of all I would like to express my gratitude to my supervisor, Prof. Anna-Karin Borg-

Karlson, for accepting me as a PhD student and for involving me in interesting projects, as well for her support and inspiring enthusiasm throughout the time I’ve spent at KTH. I also thank the late Dr. Enno Mõttus of the Estonian University of Life Sciences, who provided me with knowledge about semiochemicals and encouraged me by securing the PhD position abroad for me.

I would like to acknowledge FORMAS, the Carl Trygger Foundation, the Leader+ initiative (financed by EU structural funds) and finally the Estonian Archimedes Foundation for financing my studies.

I would also like to thank Lars Lundqvist, Dr. Katinka Pålsson and all the co-authors of the papers this thesis is based upon for collaboration and valuable comments regarding presentation of the data.

Every person in the Ecological Chemistry group at KTH is investigating different subjects. Thus, it has been interesting and inspiring to attend the presentations where various problems and results related to chemical ecology have been discussed. I thank all the present and former members of our research group.

I would also like to express my gratitude to people in the administration, and all the other students and researches in the Organic Chemistry section for the help they provided in various ways.

Most of all I would like to thank all my family, especially my mother, Tõnu, Mari and Maivo for endless care. Without your support it would have been difficult to reach so far. Thank you for being next to me!

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49

Appendixes

Appendix 1 The author’s contribution to the papers I performed the 2D-GC analyses and wrote a minor part of the manuscript II performed most of the experiments and wrote the manuscript III performed most of the experiments and wrote the manuscript IV planned the work, performed most of the experiments and wrote the manuscript V performed the 2D-GC analyses and wrote most of the manuscript VI performed the GC-MS and 2DGC analyses, prepared and tested the dispensers and wrote

most of the manuscript

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Appendix 2A Relative amounts (means ± SD) of compounds released by unwounded spruce seedlings

* - camphor co-eluted with an unknown oxygenated monoterpene

Limonene type Bornyl acetate type 3-carene type Compunds 22 – 27 weeks

n=7 22 – 27 weeks

n=4 22 weeks

n=1 27 weeks

n=1

α-Pinene 2.67 ± 2.09 3.26 ± 1.34 2.52 4.34 Camphene 2.92 ± 0.86 3.45 ± 1.06 1.56 1.50 β-Pinene 0.31 ± 0.48 0.20 ± 0.40 0.91 10.49 Myrcene 3.59 ± 2.50 2.93 ± 1.49 1.42 0.76 α -Phellandrene 0.14 ± 0.30 0.33 ± 0.65 0.33 0.24 3-Carene 0.26 ± 0.29 0.48 ± 0.35 15.55 16.23 p-Cymene 3.59 ± 3.19 3.00 ± 2.88 1.58 1.18 β-Phellandrene 2.24 ± 1.31 1.25 ± 0.61 5.40 10.77 Limonene 48.37 ± 8.49 25.06 ± 5.14 10.80 3.20 γ-Terpinene 1.64 ± 0.57 0.91 ± 0.21 1.00 0.95 o-Cymenene 2.46 ± 1.79 0.85 ± 0.72 1.40 0.68 Terpinolene 0.81 ± 0.58 0.60 ± 0.66 3.81 2.43 cis-Rose oxide 0.17 ± 0.45 - 0.04 - Unknown MT-O, B 94,79; M 152

0.07 ± 0.12 - 0.05 -

Camphor* 0.77 ± 0.83 1.09 ± 1.35 0.10 0.87 Camphene hydrate 0.05 ± 0.13 0.23 ± 0.29 0.36 - Borneol 1.83 ± 1.21 1.77 ± 0.53 0.93 0.54 Unknown MT-O, M 69, 84; M 152

0.24 ± 0.32 0.10 ± 0.13 1.24 0.60

Terpinen-4-ol 0.27 ± 0.34 0.21 ± 0.25 0.35 0.84 Cymen-8-ol 0.38 ± 0.66 0.16 ± 0.32 1.76 - α-Terpineol 0.22 ± 0.48 0.50 ± 0.49 - - Estragole 4.00 ± 3.78 1.12 ± 1.42 13.91 2.15 Fenchyl acetate 0.02 ± 0.05 0.15 ± 0.18 0.15 - Methylthymylether 1.12 ± 1.46 0.92 ± 1.01 1.07 7.35 Carvone 0.42 ± 0.52 0.11 ± 0.16 0.25 - Piperitone 2.18 ± 4.55 0.18 ± 0.36 0.10 - Bornyl acetate 12.93 ± 7.78 38.13 ± 3.53 20.43 3.28 Unknown MT-O B 93, 136, 121 0.03 ± 0.09 0.17 ± 0.34 - - Unknown MT-O B 121, 136, 93 2.49 ± 2.16 3.65 ± 2.87 2.63 0.46 Citronellyl acetate 0.34 ± 0.66 1.00 ± 0.98 1.13 - α-Longipinene 0.42 ± 0.49 0.48 ± 0.40 1.14 10.69 β-Bourbonene 0.25 ± 0.38 0.85 ± 0.65 0.36 0.87 Longifolene 1.36 ± 0.90 2.45 ± 2.14 0.85 18.32 (E)-β-Caryophyllene 1.28 ± 1.21 1.24 ± 0.80 2.90 1.24 trans-α-Bergamotene 0.21 ± 0.41 0.60 ± 1.09 - - Geranyl acetone 0.50 ± 0.97 0.12 ± 0.25 0.18 - α-Humulene 0.23 ± 0.40 0.79 ± 0.60 1.79 - (E)-β-Farnesene 0.34 ± 0.52 0.28 ± 0.55 0.30 - α-Muurolene 0.16 ± 0.22 0.38 ± 0.49 0.36 - δ-Cadinene 0.71 ± 1.28 0.97 ± 0.97 1.31 -

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Appendix 2B Relative amounts (means ± SD) of compounds released by unwounded pine seedlings

9.5 weeks old pine seedlings

3-carene chemotype n=21

α-pinene chemotype n=4 Compunds

emission of unwounded seedlings

emission of phloem pieces

emission of unwounded seedlings

emission of phloem pieces

(3Z)-Hexenal - 0.56 ± 0.94 - 0.18 ± 0.18 (3Z)-Hexenol - 0.26 ± 0.82 - 0.02 ± 0.04 α-Thujene 0.20 ± 0.21 0.20 ± 0.16 - 0.03 ± 0.05 α-Pinene 6.96 ± 5.66 28.79 ± 13.02 57.04 ± 15.62 79.79 ± 5.88 Camphene 0.23 ± 0.28 0.33 ± 0.22 1.18 ± 0.94 0.88 ± 0.11 Sabinene 1.56 ± 0.62 3.66 ± 3.41 0.07 ± 0.08 0.09 ± 0.07 β-Pinene 0.59 ± 0.44 2.27 ± 2.08 4.85 ± 0.84 9.16 ± 4.86 Myrcene 6.39 ± 2.21 2.92 ± 0.80 6.86 ± 3.83 1.38 ± 0.33 3-Carene 38.30 ± 11.76 50.30 ± 11.96 2.41 ± 1.36 3.69 ± 3.23 p-Cymene 2.07 ± 1.14 0.13 ± 0.14 1.42 ± 1.50 0.01 ± 0.02 β-Phellandrene 1.45 ± 0.52 1.15 ± 0.54 4.24 ± 2.16 0.80 ± 0.34 Limonene 1.03 ± 0.56 1.02 ± 1.51 4.16 ± 1.31 2.16 ± 1.20 (E)-β-Ocimene 1.44 ± 1.70 - 3.35 ± 2.76 - γ-Terpinene 2.54 ± 1.40 0.61 ± 0.21 1.28 ± 1.27 0.03 ± 0.04 p-Cymenene 0.64 ± 0.44 - - - o-Cymenene 2.63 ± 0.92 - 0.67 ± 1.05 - Terpinolene 6.44 ± 3.62 6.87 ± 1.86 0.10 ± 0.20 0.63 ± 0.42 Unknown 43(100), 109(64), 41(56), 95(52), 64(47), 91(42), 137(35)

0.95 ± 0.62 - - -

p-Mentha-1,3,8-triene 0.44 ± 0.37 - - - 2-Methyl-6-methylene-1,7-octadien-3-one

0.79 ± 0.54 - 0.16 ± 0.18 -

p-Mentha-1,5-diene-8-ol 0.31 ± 0.32 - - - α-Terpineol 1.54 ± 1.20 - 1.69 ± 2.37 - Verbenone 0.12 ± 0.21 - 0.27 ± 0.33 - trans-Chrysanthenyl acetate 1.12 ± 1.01 - - - Methythymylether 0.89 ± 0.67 0.10 ± 0.20 2.20 ± 2.44 0.02 ± 0.05 MT-O 93(100), 136(51), 121(48), 43(28)

2.44 ± 1.88 0.06 ± 0.09 - -

MT-O 121(100), 136(76), 93(75), 43(33)

7.62 ± 5.73 0.23 ± 0.24 2.46 ± 0.63 -

α-Longipinene 3.67 ± 6.97 0.22 ± 0.27 0.85 ± 0.79 0.24 ± 0.15 α -Copaene 0.19 ± 0.44 - 0.08 ± 0.16 0.01 ± 0.02 Sativene 0.16 ± 0.37 - 0.05 ± 0.11 0.02 ± 0.04 Longifolene 1.11 ± 1.21 0.20 ± 0.26 1.07 ± 1.12 0.81 ± 1.12 (E)-β-Caryophyllene 2.55 ± 1.70 0.11 ± 0.19 2.96 ± 1.56 0.05 ± 0.05 Geranyl acetone 2.58 ± 6.36 - 0.19 ± 0.22 - α -Humulene 0.16 ± 0.29 - 0.14 ± 0.28 - Germacrene D 0.01 ± 0.07 - - - α–Muurolene 0.34 ± 0.48 - 0.26 ± 0.38 - δ-Cadinene 0.52 ± 0.56 - - -