Effects of herbivory and climate change factors on BVOC emissions from boreal conifers

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DISSERTATIONS | RAJENDRA PRASAD GHIMIRE | EFFECTS OF HERBIVORY AND CLIMATE CHANGE... | No 23

RAJENDRA PRASAD GHIMIRE

EFFECTS OF HERBIVORY AND CLIMATE CHANGE FACTORS PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND

Emission rates of biogenic volatile organic compounds (BVOCs) from boreal conifer forests are highly dependent on the severity

of biotic stress and abiotic climate change factors, which are of increasing importance in the context of climate warming in northern

Europe. This thesis assesses the individual and interactive effects of insect herbivory and climate relevant abiotic factors on BVOC

emissions from boreal conifers. The results contribute to current understanding of complex biosphere-atmosphere interactions

under climate change.

RAJENDRA PRASAD GHIMIRE

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AUTHOR: RAJENDRA PRASAD GHIMIRE

Effects of herbivory and climate change factors on BVOC emissions from boreal

conifers

Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences

No 236

Academic Dissertation

To be presented by permission of the Faculty of Science and Forestry for public examination in the Auditorium SN201 in Snellmania Building at the University of

Eastern Finland, Kuopio, on October, 21, 2016, at 12 o’clock noon.

Department of Environmental and Biological Sciences

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Grano Oy Jyväskylä, 2016

Editor: Research director Pertti Pasanen

Distribution:

University of Eastern Finland Library / Sales of publications P.O.Box 107, FI-80101 Joensuu, Finland

tel. +358-50-3058396 www.uef.fi/kirjasto

ISBN: 978-952-61-2251-9 ISBN: 978-952-61-2252-6 (PDF)

ISSNL: 1798-5668 ISSN: 1798-5668 ISSN: 1798-5676 (PDF)

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Author’s address: Rajendra Prasad Ghimire University of Eastern Finland

Department of Environmental and Biological Sciences P.O. Box 1627

70211 KUOPIO FINLAND

email:rajendra.ghimire@uef.fi

Supervisors: Professor Jarmo Holopainen, Ph.D.

University of Eastern Finland

email:jarmo.holopainen@uef.fi

Docent Minna Kivimäenpää, Ph.D.

email:minna.kivimaenpaa@uef.fi Professor Toini Holopainen, Ph.D.

email:toini.holopainen@uef.fi

Reviewers: Professor Christer Björkman, Ph.D.

Swedish University of Agricultural Sciences Department of Ecology

P.O. Box 7044 750 07 UPPSALA SWEDEN

email:Christer.Bjorkman@slu.se Professor Janne Rinne, Ph.D.

Lund University

Department of Physical Geography and Ecosystem Science Sölvegatan 12

S-223 62 LUND SWEDEN

email:janne.rinne@nateko.lu.se

Opponent: Professor Jaana Bäck, Ph.D.

University of Helsinki Department of Forest Sciences P.O. Box 27

00014 Helsinki FINLAND

email:jaana.back@helsinki.fi

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ABSTRACT

Boreal conifer forests emit substantial amounts of biogenic volatile organic compounds (BVOCs), particularly terpenoids, into the atmosphere. Conifers of the circumpolar zone are subjected to stress caused by insect outbreaks, and are greatly affected by warming, rising concentrations of tropospheric ozone and unbalanced nitrogen availability. Two dominant species of European boreal forests, Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies Karst.), have suffered increasingly from outbreaks of pine sawflies (Neodiprion sertifer Geoffroy, Diprion pini L., and Acantholyda posticalis Matsumura) and European spruce bark beetles (Ips typographus L.), respectively.

The aim of this thesis was to assess how insect herbivory and climate change factors affect BVOC emissions from boreal conifers.

More specifically, the first aim was to evaluate the quantity and composition of BVOCs from shoots and below-ground parts of young Scots pine subjected to needle damage by larvae of the diprionid sawflies N. sertifer and D. pini. The second aim was to determine the impact of bark beetle invasion on bark BVOC emissions of Norway spruce stands in natural forest sites. Multifactorial three-year-long open field experiments were conducted to assess the individual and interactive effects of herbivory and abiotic climate change factors (warming, ozone and soil nitrogen availability) on BVOC emissions of Scots pine. BVOC samples were collected using dynamic headspace sampling in the laboratory and field, with analysis by gas chromatography-mass spectrometry (GC-MS).

In the first experiment, feeding on Scots pine needles by N. sertifer larvae substantially increased the localized emission rates of total monoterpenes (MTs), sesquiterpenes (SQTs) and green leaf volatiles (GLVs) from damaged shoots. D. pini-feeding substantially increased the localized emission of total MTs from defoliated branches, but reduced the below-ground MT emission. Bark beetle attack in natural Norway spruce stands significantly increased the total emission of MTs, but decreased the emission of several SQTs. In the multifactorial open-field experiment, elevated temperature significantly increased the total emission of non-oxygenated monoterpenes (MT-nx), oxygenated monoterpenes (MT-ox) and SQTs. Elevated ozone in combination with higher nitrogen increased emissions of several MTs and total SQTs. Mild damage to Scots pine shoots by A. posticalis larvae significantly increased the localized emissions of total MT-ox and SQTs from the damaged shoots. In the last year of the open-field

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experiment, intensive damage to Scots pine shoots by A. posticalis notably increased the localized emission rates of total MT-nx, MT-ox, SQTs and GLVs from insect-damaged shoots and induced the systemic emission of total MT-nx, SQTs and GLVs in neighbouring branches during the feeding period. Effects of A. posticalis-feeding on localized VOC emissions of Scots pine shoots were significantly reduced by warming, but enhanced when both ozone and temperature were elevated simultaneously. Furthermore, the systemic emission rate of total MT-nx in response to A. posticalis-feeding was significantly increased when ozone was elevated during feeding and nitrogen was elevated around three months after the start of feeding.

Overall, results of this thesis suggest that herbivore damage to Scots pine shoots and Norway spruce tree trunks trigger BVOC emissions. Herbivory and climate change relevant abiotic factors, separately and in combination, alter the emission rates of BVOCs from Scots pine shoots. There is great potential for further increases in BVOC emissions of boreal conifer forests in northern Europe with predicted increases in insect outbreaks, temperature, ozone concentrations and nitrogen availability. The expected increases in biogenic VOC emissions may affect atmospheric chemistry and the global climate through the formation of secondary organic aerosols in the atmosphere.

Universal Decimal Classification: 504.7, 543.613.3, 543.635.7, 582.475.1, 582.475.3, 591.531.1

CAB Thesaurus: boreal forests; coniferous forests; volatile compounds;

terpenoids; monoterpenes; sesquiterpenes; Pinus sylvestris; Picea abies;

herbivores; herbivory; Neodiprion sertifer; Diprion pini; Ips typographus;

environmental factors; climate change; temperature; global warming; ozone;

nitrogen; field experimentation

Yleinen suomalainen asiasanasto: boreaalinen vyöhyke; havupuut; haihtuvat orgaaniset yhdisteet; terpeenit; mänty; kuusi; kasvinsyöjät; sahapistiäiset;

kaarnakuoriaiset; ympäristötekijät; ilmastonmuutokset; lämpötila;

lämpeneminen; otsoni; typpi; kenttätutkimus

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Acknowledgements

This study was conducted in the Department of Environmental and Biological Sciences at the Kuopio campus of the University of Eastern Finland. The field experiments were conducted at forest sites in the Iitti and Lahti Municipalities of southern Finland, and in the open-field site of the Research Garden of the Kuopio campus in central Finland. I wish to acknowledge financial support from the Academy of Finland (project no.

133322), University of Eastern Finland (spearhead project CABI, no. 931050), UEF Doctoral Programme in Biology of Environmental Change (project no. 4900207) and North Savo Regional Fund of the Finnish Cultural Foundation.

I express my sincere gratitude to my supervisors, Professor Jarmo Holopainen, Docent Minna Kivimäenpää and Professor Toini Holopainen for the continuous support all around the clock to complete my PhD study. As supervisors, their advice and inspiration concerning both my studies and research work have been priceless and beyond words. I wish to acknowledge them for their endless efforts, firm expertise and critical- thinking in all the stages of my PhD studies – from planning and designing of the experiments to the processes of writing scientific papers and the thesis summary.

I thank Jaana Rissanen for her help during laboratory work and field sampling. I am grateful to Timo Oksanen for his technical assistance in maintaining experimental equipment and setting- up of temperature and ozone exposure in the Ruohoniemi field site. Virpi Tiihonen, Aarne Lehikoinen, Hanna Valolahti, and other staff at the Department of Environmental and Biological Sciences and the Research Garden of Kuopio are appreciated for their help in field work and other practical matters.

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I sincerely thank the two reviewers of this thesis, Professor Christer Björkman and Professor Janne Rinne for their thorough and in-depth evaluation of the thesis and insightful comments to enhance its quality. I express my sincere thanks to Professor Jaana Bäck who accepted the invitation to act as my opponent for the public defence.

I am thankful to all the co-authors of the publications included in this thesis, Dr. Päivi Lyytikäinen-Saarenmaa, Minna Blomqvist, Dr. Anne Kasurinen, Dr. Elina Häikiö, Dr. Sirkka Sutinen and Juha-Matti Markkanen. I thank Dr. James Blande for editing the English language of this thesis.

I express my sincere gratitude to my parents. I am grateful to my wife and son for their patience, love and for the moral support they have given to me.

Kuopio, September 2016 Rajendra Prasad Ghimire

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LIST OF ABBREVIATIONS

BVOC Biogenic volatile organic compound

CH⁴ Methane

CCN cloud condensation nuclei CO² Carbon dioxide

DMAPP Dimethylallyl pyrophosphate DMNT 4,8-dimethylnona-1,3,7-triene DMS Dimethyl sulphide

ELVOC Extremely low volatility organic compound GC-MS Gas chromatography - mass spectrometry GHG Greenhouse gases

GLV Green leaf volatile GPP Geranyl diphosphate

IPCC Intergovernmental Panel on Climate Change IPP Isopentenyl pyrophosphate

IR-heater Infrared-heater LAI Leaf area index

LOX Lipoxygenase

MEGAN Model of emissions of gases and aerosols from nature

MeJA Methyl jasmonate

MEP 2-C-methyl-D-erythritol 4-phosphate MeSA Methyl salicylate

MT Monoterpene

MT-nx Non-oxygenated monoterpene MT-ox Oxygenated monoterpene MVA Mevalonic acid

NO² Nitrogen dioxide NO³ Nitrate radical N²O Nitrous oxide NO^x Nitrogen oxides

OVOC Other volatile organic compound

O³ Ozone

OH^- Hydroxyl radical

PET Polyethylene terephthalate ppb Parts per billion

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PTR-MS Proton transfer reaction mass-spectrometry SOA Secondary organic aerosols

SQT Sesquiterpene

TMTT 4,8,12-trimethyltrideca-1,3,7,11-tetraene UV-B Ultraviolet B radiation (280–320 nm)

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LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following original publications referred to in the text by chapter numbers 2–5.

Chapter 2 Ghimire RP, Markkanen JM, Kivimäenpää M, Lyytikäinen-Saarenmaa P and Holopainen JK (2013) Needle removal by pine sawfly larvae increases branch- level VOC emissions and reduces below-ground

emissions of Scots pine. Environmental Science and Technology 47: 4325–4332.

Chapter 3 Ghimire RP, Kivimäenpää M, Blomqvist M, Holopainen T, Lyytikäinen Saarenmaa P and Holopainen JK (2016) Effect of bark beetle (Ips typographus L.) attack on bark VOC emissions of Norway spruce (Picea abies) trees.

Atmospheric Environment 126: 145–152.

Chapter 4 Kivimäenpää M, Ghimire RP, Sutinen S, Häikiö E, Kasurinen A, Holopainen T and Holopainen JK (2016) Increases in volatile organic compound emissions of Scots pine in response to elevated ozone and warming are modified by herbivory and soil nitrogen availability.

European Journal of Forest Research 135: 343–360.

Chapter 5 Ghimire RP, Kivimäenpää M, Kasurinen A, Häikiö E, Holopainen T and Holopainen JK. Herbivore-induced BVOC emissions of Scots pine under warming, elevated ozone and increased nitrogen availability in an open- field exposure. Submitted manuscript.

The original articles have been reprinted with the permission of the copyright holders.

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AUTHOR’S CONTRIBUTION

In the first paper (chapter 2), Rajendra Prasad Ghimire (R. P. G.) planned the experiments in collaboration with his supervisors, conducted all the insect and VOC experiments with Minna Kivimäenpää and Juha-Matti Markkanen, and had primary responsibility for data analysis and writing the article. For the papers in the chapter 3 and 5, the author conducted the experiments in co-operation with his co-authors, and had the main responsibilities for the collections and analyses of samples, statistical analyses and writing the papers. In the article of chapter 4, the author had the main responsibility of insect experiments and VOC samplings and participated in writing the paper. In the papers of the chapter 4 and 5, the author was involved in the maintenance of the experimental set-up.

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1 Introduction

1.1 BOREAL FOREST REGION

The boreal forest or Taiga is the largest terrestrial biome on Earth, comprising about 30% of the total forest area (Taggart and Cross 2009) and 73% of the coniferous forest area worldwide (Machacova et al. 2016). It is primarily situated in the circumpolar zone between 50 and 70° N latitudes and covers parts of Russia, Alaska, Canada and Scandinavia (Soja et al.

2007). Characteristically, boreal forest consists of evergreen conifer species, including pine (Pinus), spruce (Picea), and fir (Abies) and the non-evergreen conifer larch (Larix), which are interspersed with trees of deciduous genera such as birch (Betula), aspen (Populus), willow (Salix), alder (Alnus) and rowan (Sorbus). In Scandinavia, the dominating evergreen conifers species are Scots pine (Pinus sylvestris) and Norway spruce (Picea abies). The boreal forest zone is the largest reservoir of soil carbon accounting for over 50% of the carbon stock (both biomass and soil carbon) of all vegetation (Kasischke 2000;

Taggart and Cross 2009), and regulates the global climate by affecting the carbon cycle and the radiation balance of the Earth.

1.2 CLIMATE CHANGE IN THE BOREAL REGION

The climate has widespread impacts on biological systems of the Earth, and increasing temperature, tropospheric ozone concentrations and nitrogen availability are important components of global climate change. For the period covering the past 800,000 years, total greenhouse gas (GHG) emissions were the highest (49 gigatonnes CO²-equivalents per year) between 2000 and 2010, and CO² emissions from fossil fuel combustion and industrial processes contributed about 78% to the total GHG emissions increase over the 2000–2010 period

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(IPCC 2014). With increasing concentrations of greenhouse gases, mainly CO², in the atmosphere, the average surface temperature of the Earth has increased by 0.85°C during the period 1880 to 2012 and temperatures are predicted to increase by 2°C by the end of the 21^st century and to continue rising in the 22^nd century (IPCC 2014). The period from 1983 to 2012 was the warmest of the last 1,400 years in the northern hemisphere and warming has been greatest in Scandinavia since 1980 (IPCC 2014). The ambient concentration of tropospheric ozone (O³) has increased from a level of 10 ppb prior to industrial times to the present level that is three-to four-fold higher in the northern hemisphere (Wittig et al. 2007; Hartmann et al. 2013). Annual anthropogenic nitrogen deposition has increased in the terrestrial ecosystems of the Earth since 1860, and more than one third of the total N-stock (27 Tg yr-¹) sequestered by terrestrial ecosystems in the period 2001–2010 was from human activities (Zaehle 2013). Current and predicted climate changes are not only for a change in mean climate parameters, but also for increases in the frequency and magnitude of extreme weather events (Kjellström 2004). For example, hot-temperature extremes (heat waves) and tropical nights (warmer nights above 20°C) have been increasingly frequent in northern Europe since 1950 (IPCC 2014).

Warming positively affects boreal forest ecosystems by increasing net N-mineralization and primary productivity and also by extending the growing season (Rustad et al. 2001;

Hyvönen et al. 2007; Allison and Treseder 2008). Ozone is a phytotoxic gas, the prevailing concentration of which reduces photosynthesis, growth and biomass production of boreal trees (Wittig et al. 2009; Ainsworth et al. 2012). Higher nitrogen availability promotes plant productivity and carbon uptake in the boreal forests (Ollinger et al. 2002; Högberg et al. 2003).

However, the functioning of boreal Scots pine forests is limited by low nitrogen availability due to both low N² fixation and low atmospheric N deposition (which is in the range of 1–3 kg N h^-1 y^-1) (Korhonen et al. 2013). Elevated CO²concentration increases net primary productivity of boreal trees by accelerating the rate

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of photosynthesis (Hyvönen et al. 2007). Elevated CO² promotes N-uptake by forest trees by increasing the below-ground allocation of organic carbon due to enhanced root growth and mycorrhizal abundance in the soil (Treseder 2004). Besides climatic impacts on vegetation, boreal forest itself may be a source of GHG emissions such as N²O and methane (CH⁴) from both the above-ground biomass (Machacova et al. 2013) and the forest floor (Pihlatie et al. 2007). Recently, Pinus sylvestris, a major boreal conifer species was found to emit two major greenhouse gases, N²O and CH⁴, from its shoots and stems (Machacova et al. 2016).

1.3 IMPORTANT INSECT HERBIVORES OF THE BOREAL FORESTS IN SCANDINAVIA

1.3.1 Biology of insect species

The European pine sawfly, Neodiprion sertifer Geoffroy (Hymenoptera: Diprionidae) and the Large pine sawfly, Diprion pini L. (Hymenoptera: Diprionidae) are severe pests of Scots pine (Pinus sylvestris L.) and outbreak in cycles. N. sertifer females usually lay 6–8 eggs in each of approximately 10–12 pine needles from late August to October and the eggs overwinter in needles. Larvae start to feed on mature previous- year needles of Scots pine during the first half of the summer season. Larvae pupate in soil in July and new adults emerge in August and September. D. pini has different timing to its life cycle. Adults overwinter as pupae in soil and emerge from soil from May to July. Adult females lay eggs on pine needles in June and July, and larvae hatch 3–4 weeks later. D. pini larvae consume all pine needle generations in the latter half of the growing season until they pupate from August to September.

Both species of diprionid sawflies produce only one generation per year and male larvae have five and female larvae six instars during their development (Lyytikäinen-Saarenmaa 1999;

Lyytikäinen-Saarenmaa and Tomppo 2002; Kurkela et al. 2005).

The great web-spinning pine sawfly, Acantholyda posticalis (Matsumura) (Hymenoptera: Pamphiliidae) is a harmful insect

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species of the pine forests of Asia, and central and eastern Europe (Vapaavuori et al. 2010; Voolma et al. 2009). A. posticalis females lay eggs on needles of young and mature Scots pine in June. Larvae cut the needles of all needle generations and feed on them inside a silken web that they make close to the branch bark. Larvae feed on needles for three to four weeks from June to July, and at the fourth larval instar stage they move to the soil where they stay for two to five years before a short pupation.

Larvae of this species pass through five or six instars (depending on the sexes). Adults emerge from the soil from May to early July, but due to the long developmental period in the soil they are of different ages (Voolma et al. 2009).

The European spruce bark beetle (Ips typographus L.

Coleoptera: Curculionidae) is the most severe pest of Norway spruce (Picea abies K.) in the whole of Europe and Eurasia (Chinellato et al. 2014; Öhrn et al. 2014). Adults of I. typographus attack the thick bark of the Norway spruce trunk in May and June, females lay eggs in feeding galleries under the bark surface and the larvae develop and feed on the phloem underneath it (Cognato 2015). Larvae pupate in their feeding galleries and emerging adults make exit holes in the bark in August and September and then fly away after emergence.

Adults dig holes in soil litter for over-wintering during colder seasons. I. typographus usually attack bark of weakened or dying spruce trees such as wind fallen trees. After population density increases, adult beetles are capable of attacking heathy trees in the surrounding area (Weed et al. 2015).

1.3.2 Insect outbreaks and climate change

There have been serious outbreaks of the two species of diprionid sawflies (Neodiprion sertifer and Diprion pini) in the pine forests of northern Europe, which have resulted in tree growth reduction, timber losses and substantial economic losses (Lyytikäinen-Saarenmaa 1999; Lyytikäinen-Saarenmaa and Tomppo 2002; Kurkela et al. 2005). A large-scale outbreak of D.

pini occurred in Scots pine forests of Finland during the period 1997–2001. The outbreak affected approximately 500,000 ha of

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the forest area in western, eastern and southern parts of the country (Lyytikäinen-Saarenmaa and Tomppo 2002). Heavy defoliation by N. sertifer and D. pini reduced tree growth in Scots pine stands by 38% and 94%, respectively, whereas the rates of tree mortality following outbreak by each species were respectively 4% and 30% (Lyytikäinen-Saarenmaa and Tomppo 2002). Outbreaks of D. pini are more severe than outbreaks of N.

sertifer due to its feeding on all-generation needles of pine and having the potential to fully defoliate the trees. Therefore, D.

pini outbreak has to be considered as a potential threat to both young and mature Scots pine forests of Scandinavia and other boreal regions. Sawfly performance may increase in northern Fennoscandia with increases in outbreaks on Scots pine projected to occur with climate warming (Niemelä et al. 2001).

Furthermore, primary damage caused to pine trees by sawfly outbreaks pose a risk to trees of secondary damage by other insects, e.g. bark beetles (Lyytikäinen-Saarenmaa and Tomppo 2002).

The web-spinning pine sawfly Acantholyda posticalis has recently undergone mass outbreak in Scot pine forests of Siberia, Estonia (Voolma et al. 2009) and Finland (Vapaavuori et al.

2010). The mass outbreaks of A. posticalis defoliated about 250 hectares of Scots pine stands growing on sandy soil on the Estonian island of Saaremaa in 2008 (Voolma et al. 2009). A.

posticalis outbreak occurred over an area of about 200 ha of Scots pine forest in western Finland during the dry summer of 2006. It caused serious damage to 30 ha of pine stands, and the seriously affected area was further increased in the summer of 2009, resulting in trees dying in up to 100 ha of the forest area (Vapaavuori et al. 2010). The northward spread of this sawfly species from, for example, Estonia (in the south), and covering the entire boreal forest zone is expected with current and projected global warming (Vapaavuori et al. 2010).

Outbreaks of the European spruce bark beetle (Ips typographus) have been found to increase at southern latitudes and with low elevation, and also with increasing summer temperature in Europe (Chinellato et al. 2014). I. typographus is a

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tree-killing bark beetle species which has the potential to undergo large-scale mass outbreaks. Less than 1% of over 600,000 bark beetle species (Curculionidae: Scolytinae) worldwide undergo broad-scale outbreaks (Raffa et al. 2008).

Global warming is the key driver of extreme weather events (droughts and storms) that result in an increased number of weaker, stressed and wind thrown trees in forest sites and largely increase the population of I. typographus by providing them necessary resources such as a food, shelter and suitable breeding materials. The total volume of Norway spruce trees killed by I. typographus outbreaks during the period of 1960–

2009 in Sweden was about 9 million m³, and the largest I.

typographus outbreak in Sweden during the period (1971–1981) killed about 4.5 million m³ of those spruce trees (Kärvemo and Schroeder 2010). The ‘Gudrun’ storm of southern Sweden in 2005 felled 50–75 million m³ of conifer trees (Bengtsson and Nilsson 2007); this was the largest single cause of timber damage in the country in the 20^th century and resulted in a massive outbreak of I. typographus in the Swedish forests. I.

typographus outbreaks are likely to increase in the boreal forest region with more severe forest damage predicted to occur with increases in global warming and the frequency and intensity of storms (IPCC 2014).

Temperature plays a key role in species composition and population dynamics of forest insects. Temperature influences insect populations by either directly affecting their phenology, behaviour, abundance and distribution or indirectly via other climate-induced changes mediated by other climate parameters (Bale et al. 2002). On the other hand, sensitivity of species to warming increases with trophic level, and higher level species benefit more from climate warming than insect herbivores, and this in the long-term may result in a balanced ecological response to climate warming (Berggren et al. 2009). Climate warming causes periodic increases in insect populations and enhances their outbreak potential (Dale et al. 2001). Populations of insect species adapted to the high latitude zone, mostly covered by boreal forests, have big advantages with climate

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warming due to increases in their fecundity rate, generations per year and survival, and by reduction in their mortality rate with warmer winters (Stange and Ayres 2010). Warming causes northward shift in the outbreak areas of forest defoliators due to a positive growth response of the boreal conifer treeline trees with warmth (Wilmking et al. 2004; Soja et al. 2007). Future outbreaks (for the period 2080–2100) of spruce budworms Choristoneura fumiferana Clem. (Lepidoptera: Tortricidae) are predicted to last for approximately 6 years longer and to cause 15% more defoliation than the current outbreaks (Gray 2008).

European spruce bark beetles (I. typographus) are expected to benefit from predicted climate warming with increases in their geographic range, number of generations per summer and number of intensive outbreaks (Jönsson et al. 2009). The attack rates of I. typographus have been found to increase with summer temperature in Europe (Chinellato et al. 2014). An increase in temperature of 5^oC increases the outbreak risk of N. sertifer in boreal pine forests due to a shortening of the larval development period by 37–41% (Kollberg et al. 2013). There is a likelihood of ‘damage chains’ in the ecosystem food-web, e.g.

the defoliation of pine trees by N. sertifer makes trees more susceptible to the attack by pine shoot beetles (Tomicus spp.) (Björkman et al. 2011).

1.4. BIOGENIC VOC EMISSIONS IN THE CONTEXT OF BOREAL CONIFER FORESTS

1.4.1 Characteristics of biogenic VOCs

Biogenic volatile organic compounds (BVOC) are non-methane hydrocarbons (Kesselmeier and Staudt 1999) and secondary metabolic compounds synthesized in plants, animals and microorganisms by different biochemical processes and are released into the Earth’s atmosphere. BVOCs comprise mainly isoprene, monoterpenes, other reactive VOCs and other VOCs according to their atmospheric lifetimes (Laothawornkitkul et al.

2009). They are also categorized into chemical groups such as terpenoids (a class of organic compounds composed of one or

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more isoprene units), phenylpropanoids/benzenoids, fatty acid derivatives and amino acid derivatives according to their biosynthetic origin (Dudareva et al. 2013). BVOCs, in terms of modelling their contribution to emissions of gases and aerosols in nature (MEGAN2.1), are divided into four main groups:

isoprene, terpenoids, stress VOCs and other VOCs (Guenther et al. 2012). Isoprene and monoterpenes are the most dominant species of BVOCs (Kesselmeier and Staudt 1999; Guenther et al.

2012). Of the 100,000 known plant-derived compounds, over 1,700 have been identified as BVOCs that are released from nearly 100 different plant families including both flowering plants and non-flowering gymnosperms (Knudsen et al. 2006;

Dicke and Loreto 2010). Although, terrestrial plants, in some cases, release up to 10% of the total carbon assimilated from atmospheric CO² during net primary production back into atmosphere as BVOCs (Peñuelas and Llusià 2003), a more typical figure is 1—4% (Kesselmeier et al. 2002; Pressley et al.

2005; Rinne et al. 2009).

Emissions of volatile compounds from plants can be constitutive (continuously synthesized compounds that are emitted from mesophyll cells and are dependent on temperature and light or compounds released via diffusion from specialized plant storage structures) (Grote et al. 2013; Niinemets et al. 2013) or induced (emissions of de novo synthesized volatiles activated in response to various stresses) (Paré and Tumlinson 1997;

Niinemets et al. 2013). Constitutively emitted BVOCs from plants have several ecological and metabolic functions. In conifers, volatile terpenes are constitutively released mainly from resin ducts terpene storage structures both in stressed and unstressed conditions but they can also be synthesized de novo in damaged or nearby tissues to fulfil a higher demand for defensive compounds to compensate for stress (Ghirardo et al.

2010). In addition to immediate stress-induced emissions, biotic stress also elicits emissions that trigger secondary induction responses in plants, which possibly affect systemic emission responses (Niinemets et al. 2013). Terpenoids are released both from above-ground and below-ground plant parts, such as

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stems (Martin et al. 2002), needles and foliage (Martin et al.

2002), and roots (Lin et al. 2007) of conifers.

1.4.2 Typical conifer VOCs

Conifers emit a diverse range of volatile terpene compounds from their above-ground parts. Monoterpenes dominate the volatile blend emitted by Scots pine, with SQTs constituting a minor component (Komenda and Koppmann 2002), while isoprene, monoterpenes and some SQTs are typical for Picea spp. (Hayward et al. 2004; Kivimäenpää et al. 2013;

Bourtsoukidis et al. 2014).

The most common monoterpenes emitted by Pinus sylvestris are -pinene, 3-carene, camphene, and -pinene (Komenda and Koppmann 2002), of which -pinene and 3-carene are the most abundant compounds in branch, canopy and above-canopy measurements (Räisänen et al. 2009; Bäck et al. 2012). Other dominant terpenes include the monoterpenes myrcene, limonene, -phellandrene and terpinolene, and the sesquiterpenes -muurolene, longifolene, -cadinene, - caryophyllene and -bourbonene, which were reported as shoot and bark emissions of young Scots pine seedlings (Heijari et al.

2011; Kovalchuk et al. 2015). The monoterpenes -pinene, 3- carene and -pinene and sesquiterpenes -bergamotene, - farnesene, -farnesene and -caryophyllene were emitted by branches of mature pine trees (Tarvainen et al. 2005; Helmig et al. 2007). The variation in monoterpene profiles emitted by Scots pine are known to be caused predominantly by genotypic variation between pine individuals (Komenda and Koppmann 2002) and pine provenances (Semiz et al. 2007). Furthermore, monoterpene diversity of Scots pine, particularly of the two dominant MTs, -pinene and 3-carene depends on the stand history and seed origin (Bäck et al. 2012).

Isoprene and the monoterpenes -pinene, camphene, limonene and -pinene were the most common VOCs in the shoot emissions of young Norway spruce seedlings (Blande et al. 2009; Kivimäenpää et al. 2013), while two other monoterpenes ( -phellandrene and 3-carene) and three

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sesquiterpenes (longifolene, longipinene and -bourbonene) were also reported in the study by Kivimäenpää et al. (2013). In addition to isoprene and monoterpenes, other compounds (including acetic acid, acetone, acetaldehyde, methanol) and OVOCs (hexanal and methyl salicylate) were detected in the foliar emissions of young Norway spruce (Filella et al. 2007). In addition to isoprene and the monoterpenes -pinene, 3-carene, camphene, limonene, -pinene and -phellandrene, total SQTs and other oxygenated BVOCs (including formaldehyde, ethanol, formic acid, methyl ethyl ketone, pinonaldehyde and caronaldehyde) were reported in the emissions of mature Norway spruce trees (Bourtsoukidis et al. 2014). Similarly, the monoterpenes tricyclene, -pinene, 3-carene, camphene and - pinene dominated the BVOC profiles of tree bark emissions of mature Lodgepole pine (Amin et al. 2012) and Engelmann spruce (Amin et al. 2013).

BVOCs produced by the belowground tissues of conifers are similar in diversity to those produced by aboveground organs.

Conifer roots were found to mainly emit volatile terpenoids which have important and diverse roles in the rhizosphere, and impact on soil ecology and atmospheric chemistry. The monoterpenes -pinene and -pinene were dominant VOCs in the root emissions of Pinus halepensis, whereas -pinene, camphene and limonene were major monoterpenes in the rhizosphere emissions of Scots pine (Lin et al. 2007). With the dominance of -pinene and 3-carene, other common monoterpenes detected in the rhizosphere emissions of Scots pine seedlings were camphene, sabinene, -pinene, myrcene, limonene, -phellandrene, -terpinene and terpinolene (Rasheed et al. manuscript). Mono-and sesquiterpenes and some non-isoprenoids were the common VOC species released from Pinus pinea roots with -pinene, -pinene and limonene the dominant MTs (Lin et al. 2007). Below-ground VOC emissions include not only the emissions from plant roots but also the emissions from dead organic matter and microorganisms. Methanol (a highly dominating compound) and some MTs were released from microbial decomposition of

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needle litter of Pinus contorta and Pinus ponderosa species (Gray et al. 2010). The monoterpenes -pinene, -pinene, camphene, 3- carene and limonene were detected in the needle litter emissions of Scots pine and Norway spruce during decomposition in a natural environment (Isidorov et al. 2010). Of the dominant MTs, -pinene was emitted from the roots and needle litters of Scots pine, whereas -pinene and myrcene were mainly found in the root and litter emissions of Norway spruce (Ludley et al.

2009).

1.4.3 Synthesis of biogenic VOCs

The two main precursors for the biosynthesis of volatile terpenoids are isopentenyl diphosphate (IPP) and its allylic isomer, dimethylallyl diphosphate (DMAPP) (McGarvey and Croteau 1995). Biosynthesis of BVOCs is driven by the energy provided by primary metabolism and depends on the availability of carbon and nutrients such as nitrogen and sulphur in plants. Primary metabolism in plants includes the production of fundamental compounds including fatty acids, amino acids, and sugars. Different BVOCs originate from different biosynthetic pathways. The lipoxygenase (LOX) pathway activates the biosynthesis of green leaf volatiles (GLVs) and methyl jasmonate (MeJA) in the cytosol (Matsui et al. 2012;

Dudareva et al. 2013). The methylerythritol phosphate (MEP) pathway is located in plastids of plant cells and is responsible for the synthesis of hemiterpenes (C⁵), monoterpenes (C¹⁰), diterpenes (C²⁰) and the homoterpene TMTT (4,8,12- trimethyltrideca-1,3,7,11-tetraene) (Pichersky et al. 2006). The mevalonic acid (MVA) pathway, which is mostly cytosolic, but also linked to endoplasmic reticulum and peroxisomes synthesizes volatile sesquiterpenes (C¹⁵), triterpenes (C³⁰) and the homoterpene DMNT (4,8-dimethylnona-1,3,7-triene) (Dudareva et al. 2013).

1.4.4 Role of BVOCs

Plants emit diverse and complex blends of VOCs, which have both metabolic and ecological functions. Interactions between

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organisms, such as plant-to-plant, plant-to animal or microbe and microbe-to-microbe interactions, are mediated by biogenic VOCs. Plants have developed physical and chemical defence systems to protect themselves against various biotic and abiotic stressors. BVOCs function in oxidative protection and thermal tolerance for plants (Peñuelas and Llusia 2003; Dudareva et al.

2006) and in signalling with other plants and organisms above- ground (Dicke and Baldwin 2010; Heil and Karban 2010;

Holopainen et al. 2013) and below-ground (Wenke et al. 2010).

BVOCs promote plant growth and reproduction (Knudsen et al.

2006; Dudareva et al. 2013), and defence against herbivores (Unsicker et al. 2009; Dicke and Baldwin 2010; Holopainen and Gershenzon 2010), pathogens (Huang et al. 2012) and abiotic stresses (Loreto and Schnitzler 2010; Possell and Loreto 2013).

Some of the key abiotic stress factors against which plants have to defend during their growth and developmental processes are high temperature (Possell and Loreto 2013), ozone (Pinto et al.

2010) and low soil nitrogen availability (Oliet et al. 2013).

1.4.5 BVOCs have impacts on Earth’s atmosphere and climate BVOCs, particularly mono-and sesquiterpenes and isoprene emitted by boreal ecosystems are highly reactive components of the lower atmosphere. BVOCs react with hydroxyl (OH) and nitrate (NO³) radicals and O³ (Atkinson and Arey 2003). In photochemical reactions with nitrogen oxides (NO^X), BVOCs yield ozone in the troposphere (Matyssek and Sandermann 2003; Pinto et al. 2010). Many BVOCs are highly reactive with ozone, which is decomposed in the lower atmosphere during O³ photolysis, and results in the formation of OH radicals that oxidise VOCs. Products of BVOC oxidation by ozone and OH radicals include extremely low volatility organic compounds (ELVOCs), which contribute to secondary organic aerosol (SOA) growth and the production of atmospheric nanoparticles and cloud condensation nuclei (CCN) (Hao et al. 2011; Ehn et al.

2014; Jokinen et al. 2015). The compounds -pinene, -pinene and limonene are the main BVOC species that result in the formation of highly oxygenated peroxy radicals (RO²) in

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oxidation reactions with O³and OH (Ehn et al. 2014; Rissanen et al. 2014; Jokinen et al. 2015). The formation of -pinene-derived ELVOC is much greater in ozonolysis reactions than in reactions with OH radicals, and the total yield of ELVOC from -pinene ozonolysis was higher than that from -pinene ozonolysis (Ehn et al. 2014).

The main natural aerosol precursor, dimethyl sulphide (DMS), is mainly obtained as oceanic emissions by marine algae (Watts 2000) and from BVOCs emitted from the terrestrial biosphere (Boucher et al. 2013). Globally, a major fraction of atmospheric SOA is expected to originate from biogenic VOCs, and the current atmospheric SOA budget is estimated to be 12–

1820 Tg y¹(Spracklen et al. 2011). BVOC emissions from boreal forests contribute almost 50% of cloud condensation nuclei at the regional scale (Spracklen et al. 2008). Modelling of monoterpene emission rates predicted that 10% defoliation in the boreal insect outbreak area would result in an increase of 480% in total particulate mass and 45% in cloud condensation nuclei at the global scale compared to the emissions of non- outbreak forest area (Joutsensaari et al. 2015). Excessive yield of organic aerosols in the atmosphere will affect temperature balance and global climate of the Earth by reflection and adsorption of solar and terrestrial radiation and through the alteration of cloud albedo over the boreal forest canopy (Spracklen et al. 2008).

1.5 EFFECTS OF CLIMATE CHANGE ON CONIFER VOC EMISSIONS

Environmental and climatic factors have direct effects on primary and secondary metabolic activities of plants and influence the volatile compounds released from both vegetative (Martin et al. 2002) and floral organs (Jakobsen and Olsen1994).

Plants are programmed to mitigate the negative effects of abiotic stress by the release of greater amounts of volatile compounds, mainly isoprene, monoterpenes and sesquiterpenes (Holopainen and Gershenzon 2010).

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1.5.1 Atmospheric CO²

Rising atmospheric CO² concentrations generally increase plants productivity and biomass and therefore enhance further production and emission of BVOCs. However, majority of the recent studies have shown that elevated CO²in a long-term decreased ecosystem level isoprene emission from deciduous plant species (Rosenstiel et al. 2003; Pegoraro et al. 2004). In response to elevated CO², monoterpene emissions from Quercus ilex were increased (Staudt et al. 2001), decreased (Loreto et al.

2001), but emissions from Pinus sylvestris (Räisänen et al. 2008), Ponderosa pine Pinus ponderosa and Douglas fir Pseudotsuga menziesii (Constable et al. 1999) were not significantly affected.

In general, elevated CO²as a single factor has not been found to increase BVOC emissions of conifers. However, combined long- term exposure to warming and elevated CO² is known to increase monoterpene emissions of Scots pine (Räisänen et al.

2008), which is more relevant to current climate change scenario.

1.5.2 Warming

In general, VOC emissions from plants increase with warming due to an increase in volatile vapor pressure and increased rates of their biosynthesis at elevated temperatures (Holopainen and Gershenzon 2010). Besides direct effects on biochemical reactions in BVOC-releasing metabolic pathways, warming also enhances VOC emissions from plants indirectly by lengthening growing seasons (Peñuelas and Staudt 2010). Climate warming (+ 4^oC) in combination with increased leaf area index (LAI) due to elevated atmospheric CO²(700 µmol mol^-1 CO²) is predicted to increase MT emissions from P. ponderosa trees by up to 80%

(Constable et al.1999). In field studies with semi-controlled environmental conditions, the emissions of mono-and sesquiterpenes from Scots pine branches clearly depended on seasonal cycle and increased with warming (Tarvainen et al.

2005), and the emissions of several SQTs from pine shoots exponentially increased with warming (Helmig et al. 2007). The emission rates of volatile compounds (monoterpenes, acetone,

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methanol, GLVs) from the foliage of young Norway spruce increased exponentially with elevated temperature in a laboratory study (Filella et al. 2007). The emission rates of isoprene and MTs from Sitka spruce (Picea sitchensis Bong.) branches increased with elevated temperatures, with isoprene emission reaching maximum at 35–38°C and MTs at 30°C in a laboratory study (Hayward et al. 2004). However, long-term warming by 2–6°C was reported to decrease BVOC emissions from 25-to 30 year-old Scots pine trees in a closed-top chamber experiment (Räisänen et al. 2008).

1.5.3 Ozone

Ozone sensitivity of conifer trees is lower than that of deciduous tree species, and Norway spruce is considered to be somewhat more tolerant to ozone than Scots pine (Prozherina et al. 2009).

Conifers respond to O³ stress by stomatal closure, which is an avoidance mechanism that limits O³uptake, and by biochemical detoxification, which is a chemical defence mechanism (Huttunen and Manninen 2013). Very few studies have shown clear effects of ozone on BVOC emissions of plants due to high reactivity of ozone with VOCs in plant leaves (Chameides 1989) and loss of ozone to sampling materials and enclosures by adsorption (Ortega and Helmig 2008) during exposure periods.

Plants are known to both increase and decrease BVOC emissions in response to ozone. In the case of conifers, ozone is commonly found to increase BVOC emissions of shoots. Monoterpene emissions of Scots pine were increased (1.4-fold) by ozone exposure at a level of 50 ppb for 8h/d (Heiden et al. 1999). Total terpene emissions of Norway spruce shoots were enhanced 2- fold in response to daytime exposure levels of 40 ppb (Kivimäenpää et al. 2013). Ozone-induced changes in conifer VOC emissions may be caused by its direct impact on plant biochemistry and physiology (Long and Naidu 2002) and by subsequent alteration to BVOC biosynthetic pathways. Ozone stress is known to activate chemical defences of plants including the release of BVOCs (Pinto et al. 2010).

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1.5.4 Soil nitrogen availability/deposition

Nitrogen fertilization promotes plant growth by enhancing primary productivity, but reduces MT-rich resin acid concentrations in the shoots of Scots pine saplings (Holopainen et al.1995). N-fertilization reduced terpene emissions from Pinus halepensis by 38% in a greenhouse experiment (Blanch et al.

2007). On the other hand, high N-availability enhanced both the foliar MT concentrations and MT emissions of Douglas-fir (P.

menziesii) throughout the year except the period of leaf expansion (Lerdau et al. 1995). Nitrogen uptake of conifers increases with elevated CO² due to increased mycorrhizal abundance (Treseder 2004). This will result in enhanced growth and net primary production of plants, leading to increased BVOC emissions in conifers. Furthermore, N-uptake in plants usually increases with temperature elevation due to enhanced rates of nitrification in temperature limited soil (Dalias et al.

2002). Therefore, the cold-acclimated boreal ecosystem is N- limited due to a slow rate of mineralization, nitrification and decomposition and the N-uptake rate in boreal conifers may increase in the presence of elevated CO²and warming.

1.6 EFFECTS OF HERBIVORE FEEDING ON CONIFER VOC EMISSIONS

Conifers have evolved several defence mechanisms to protect themselves from various biotic stresses in their natural environment. One such defence is mediated by the release of BVOCs, particularly mono-and sesquiterpenes and GLVs from above-ground and below-ground parts. Joutsensaari et al. (2015) reported increases in both the localized emissions of total BVOCs and the combined (localized and systemic) emissions of total MTs in N. sertifer-damaged Scots pine seedlings. Feeding- damage by Hylobius abietis on the bark of young Scots pine increased systemic emissions (emissions from intact parts of insect-damaged plants) of total MTs and SQTs from the whole shoot of pine seedlings (Heijari et al. 2011). Feeding damage by the same insect species enhanced the emission of MTs and SQTs

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from Pinus brutia seedlings (Semiz et al. 2016). Effective induction of phytogenic BVOCs during herbivore attack is one of the plants’ physio-chemical responses to reduce feeding stress and the associated negative consequences following damage.

After the onset of herbivore-feeding on conifers, a large release of MTs may be caused by rupturing of resin storage structures.

However, the emission rates of such induced BVOCs are variable within (among plant parts)-and between plants. The magnitude of herbivore-induced emissions in plants varies greatly with herbivore species, their feeding pattern and damage intensity (Williams et al. 1997; Dicke 2009; Niinemets 2010). This variation in emission responses, at least with the same species of plants and herbivores, may also depend on feeding damage rate, which may influence the rate of biosynthesis and emissions of different BVOCs.

In addition to the specialized storage tissues in needles, monoterpene synthesis also occurs in the resin canals in conifer bark (Martin et al. 2002). H. abietis damage on the bark of Scots pine seedlings enhanced localized bark emissions of total MTs and some SQTs (Heijari et al. 2011) and systemic shoot emissions of total BVOCs and total SQTs (Kovalchuk et al. 2015).

Mature Lodgepole pine (Pinus contorta var. latifolia Engelm.) and Engelmann spruce (Picea engelmannii Parry ex Engelm.) trees attacked by the mountain pine beetle (Dendroctonus ponderosae) had increased bark emissions of total BVOCs (Amin et al. 2012, 2013). The constitutive defence mechanisms of conifer bark usually include the thickening of bark, resin synthesis and formation of lignin around phloem. However, the inducible defence of conifers in response to herbivore attack is characterized by the excessive formation of resins and secondary metabolites with the dominance of terpenoids on the bark surface.

Monoterpenes have been found to dominate the root and rhizosphere emissions of pines (Smolander et al. 2006; Lin et al.

2007), although the impacts of herbivore feeding on below- ground emissions of conifer BVOCs have not been studied earlier. Resin (a mixture of volatile monoterpenes and

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sesquiterpenes and non-volatile diterpenes) is produced in the secretory epithelial cells of conifer resin ducts and the genus Pinus has the most advanced resin canal system in conifers covering needles, wood and bark (Trapp and Croteau 2001).

Pine roots also have resin ducts for resin storage (Rubiales et al.

2008), which might be the primary source of rhizosphere BVOC emissions. Sticky resin of conifers has been known to mediate defence in stressed conditions (Trapp and Croteau 2001).

Increased flow of resin to the herbivore-attacked sites of stem bark was found to drain resin storage for several meters through the closed resin duct system of conifers (Trapp and Croteau 2001). Increased flow of resin to damage sites might be the primary defence response in Pinus as terpene synthesis genes in stem bark were not even activated at herbivore damage sites (Kovalchuk et al. 2015). However, it is not known if resin is channelled from pine stem to the roots in the event of root damage.

1.7 OBJECTIVES AND HYPOTHESES

The aims of this thesis were:

1. To elucidate the effect of needle-feeding by sawfly larvae on the emission of BVOCs by Scots pine.

2. To understand the impact of bark beetle invasions on the emission of BVOCs from bark of Norway spruce.

3. To determine the effects of herbivory and climate change relevant abiotic factors, both individually and in combination, on the BVOC emissions of Scots pine.

The specific aims of the studies included in this thesis were:

1. To evaluate the quantity and composition of BVOCs induced from shoot and below-ground parts of young Scots pine in response to needle damage by larvae of the diprionid sawflies Neodiprion sertifer and Diprion pini under laboratory and field conditions (chapter 2).

2. To determine if feeding-damage by the European spruce bark beetle Ips typographus in Norway spruce stands

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increases the emission rates of bark VOCs from the attack sites, and if the emission rates are correlated with mean beetle attack density (chapter 3).

3. By conducting a multifactorial three-year-long (2011–2013) open field experiment, the aim was to investigate how;

a. warming of 1^oC above the ambient level, elevated ozone of 1.5 times the ambient concentration, increased soil nitrogen availability of 120 kg N ha^-1y^-1 over background levels and mild herbivory by the web-spinning pine sawfly Acantholyda posticalis, separately and in combination, affect localized BVOC emission rates of Scots pine seedlings in 2012 and late spring 2013 (chapter 4), and

b. intensive feeding by the same herbivore species alone, and in interaction with the same climate change factors at the same exposure levels (as in 3a), affect localized and systemic BVOC emissions of Scots pine seedlings in 2013 (chapter 5).

The following hypotheses were tested in the studies contained in this thesis:

1. Feeding damage by N. sertifer and D. pini induces localized shoot BVOC emissions and systemic shoot-and below- ground BVOC emissions in young Scots pine seedlings (chapter 2).

2. I. typographus attack in Norway spruce stands enhances BVOC emissions from the tree bark surface, and the emission rates increase with mean beetle attack density (chapter 3).

3. Localized BVOC emissions from the shoots of young Scots pine seedlings increase separately with warming, elevated ozone, increased soil nitrogen and mild feeding by A.

posticalis larvae and also in combinations of two or more of these factors (chapter 4).

4. Intensive biotic stress due to A. posticalis feeding enhances localized and systemic BVOC emissions from the shoots of Scots pine seedlings and the emissions further increase in

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the interactions with abiotic climate change factors (chapter 5).

1.8 SUMMARY OF THE EXPERIMENTS

The first study (chapter 2) was conducted in environment controlled plant growth chambers at the Kuopio campus of the University of Eastern Finland (UEF) where Scots pine seedlings were infested with Neodiprion sertifer larvae, and in the open- field site of the Research Garden of Kuopio campus (62^o 53´ N, 27^o 37´ E, and 80 m asl) with seedlings infested with Diprion pini larvae. Three sites were chosen for the second study (chapter 3);

(site 1) a Norway spruce stand attacked by Ips typographus located at Haapa-Kimola of Iitti municipality in southern Finland (60ô53´ N, 26ô 20´ E, 69 m asl), (site 2) an I. typographus- free control site at Ruohoniemi, Kuopio, central Finland, and (site 3) eight urban forest stands infested with bark beetles in the city of Lahti (60ô 59´ N, 25ô 39´ E, 105 m asl) in southern Finland.

The third study (chapter 4) and the fourth study (chapter 5) were performed in an open-field exposure site incorporating warming, elevated ozone exposure and higher nitrogen availability in the Ruohoniemi field site of the Kuopio campus, where pine seedlings were infested with Acantholyda posticalis larvae.

BVOC samples were collected by pulling headspace air through steel tubes filled with Tenax TA and Carbopack B adsorbents using the dynamic headspace sampling technique in the laboratory and field sites. The BVOC samples were analyzed by gas chromatography-mass spectrometry (GC-MS) with samples desorbed by thermal desorption. The summaries of all the studies and the experiments therein are shown in Table 1.

The map in Figure 1A shows the study locations and the insect outbreak areas. The temperature and ozone exposure set-up is shown in Figure 1B. BVOC samplings from shoot-and tree bark of plants are shown in Figures 1C and 1D, respectively.

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ble 1. Summary of the experiments related to each topic/study. Chapter number refers to original scientific publications which contain detailed riptions of the related experimental work. earch topics/studiesExperiment typesExperimental sitesTreatmentsTimeChapters cts of sawfly (N. sertifer andD. ) feeding on shoot-and below- ound BVOC emissions of Scots pine dlings Growth chamberKuopio campus, UEF, Finland (62o 53´ N, 27o 37´ E)

N. sertifer-damageJune 20102 Open-fieldResearch garden of Kuopio campus, UEF, Finland (62o 53´ N, 27o 37´ E)

D. pini-damageAugust 2010 Effects of bark beetle (I. graphus) invasion on bark VOC s of Norway spruce stands

Field (forest site)Haapa-Kimola of Iitti Municipality, southern Finland (60o53´ N, 26o 20´ E)

I. typographus-damageJune and August 20113 Field (urban forest sites)Ruohoniemi, Kuopio, central Finland (62o 53´ N, 27o 37´ E)

Control siteAugust 2011 Field (urban forest sites)Lahti Municipality, southern Finland (60o 59´ N, 25o 39´ E) I. typographus-damageJune and August 2012

Effects of herbivory and climate change factors on BVOC emissions from boreal conifers

uef.fi

Dissertations in Forestry and Natural Sciences

RAJENDRA PRASAD GHIMIRE

RAJENDRA PRASAD GHIMIRE

Effects of herbivory and climate change factors on BVOC emissions from boreal

conifers

Acknowledgements

Contents

1 Introduction