Rhizosphere processes of scots pine seedlings under growth conditions of future climate and shoot herbivory

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Dissertations in Forestry and Natural Sciences

MUHAMMAD USMAN RASHEED

Rhizosphere Processes of Scots Pine Seedlings Under Growth Conditions of Future

PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND

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RHIZOSPHERE PROCESSES OF SCOTS PINE SEEDLINGS UNDER GROWTH CONDITIONS OF

FUTURE CLIMATE AND SHOOT HERBIVORY

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Muhammad Usman Rasheed

RHIZOSPHERE PROCESSES OF SCOTS PINE SEEDLINGS UNDER GROWTH CONDITIONS OF

FUTURE CLIMATE AND SHOOT HERBIVORY

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

No 403

University of Eastern Finland Kuopio

2020

Academic dissertation

To be presented by permission of the faculty of Science and Forestry for public examination in room SN 200, in Snellmania building at the University of Eastern Finland, Kuopio, on 1^st of December

2020, at 12 00.

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Grano Oy Jyväskylä, 2020

Editor: Research Director Pertti Pasanen Distributor: University of Eastern Finland Library

www.uef.fi/kirjasto

ISBN: 978-952-61-3650-9 (print/nid.) ISBN: 978-952-61-3651-6 (PDF)

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

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Author’s address: Muhammad Usman Rasheed, M.Sc.

University of Eastern Finland

Department of Environmental and Biological Sciences P.O. Box 1627, FI-70210, KUOPIO, FINLAND email: usman.rasheed@uef.fi

Supervisors: Senior Researcher Anne Kasurinen, Ph.D.

Department of Environmental and Biological Sciences P.O. Box 1627, FI-70210, KUOPIO, FINLAND email: anne.kasurinen@uef.fi

Docent Minna Kivimäenpää, Ph.D.

Department of Environmental and Biological Sciences P.O. Box 1627, FI-70210, KUOPIO, FINLAND email: minna.kivimaenpaa@uef.fi

Emeritus Professor Toini Holopainen, Ph.D.

Department of Environmental and Biological Sciences P.O. Box 1627, FI-70210, KUOPIO, FINLAND email: toini.holopainen@uef.fi

Reviewers: Assistant Professor Amy Trowbridge, PhD.

University of Wisconsin-Madison Department of Entomology

646 Russell Laboratories, 1630 Linder Drive, MADISON, WI, USA

email: amtrowbridge@wisc.edu

Senior Research Fellow Ivika Ostonen, PhD.

University of Tartu

Institute of Ecology and Earth sciences Ülikooli 18, 50090 Tartu, ESTONIA email: ivika.ostonen@ut.ee

Opponent: Professor Håkan Wallander, PhD.

Lund University Department of Biology

P.O. Box 117, 221 00 LUND, SWEDEN email: hakan.wallander@biol.lu.se

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ABSTRACT

Boreal forests are the largest terrestrial biome. Scots pine (Pinus sylvestris L.) is the most abundant tree species in European boreal forests. Anthropogenic activities have led to a simultaneous increase in global mean surface temperature, tropospheric ozone (O3) concentration and atmospheric nitrogen (N) deposition to soil. Warming is expected to increase herbivore outbreaks and enable the herbivore spread across the northern boreal forests. Thus, increased warming, tropospheric O3 concentration, N availability in soil, and herbivory damage are all important factors that will affect the boreal forests in the future.

Warming and N deposition may increase the carbon (C) sink capacity of the Scots pine forests, but this increasing effect of warming and N deposition may be transient, especially in the southern limits of boreal forests. Scots pine roots and their symbiotic mycorrhizal fungi respond to N deposition in a dose-dependent manner. Generally, low rates of N deposition may increase C allocation to roots and mycorrhizal fungi, while high rates of N deposition may decrease it. Scots pine trees are relatively more susceptible to O3 exposure compared to other boreal coniferous species, e.g. Norway spruce. Phytotoxic effects of O3 on Scots pine include decreased photosynthesis, early needle senescence, and altered C allocation pattern between shoots and roots. Ozone does not penetrate deep in the soil, but it is expected to have indirect effects via shoots on rhizosphere processes. Shoot herbivory may decrease seedling growth and may also have a decreasing effect on mycorrhizal colonization in Scots pine trees.

This thesis studied the effects of warming, N addition to soil, O3 and shoot herbivory on Scots pine seedlings based on two field experiments (exp. 1 and exp. 2). In exp. 1, potted Scots pine seedlings, growing in mixture of sand and peat, were exposed to warming (air c.

1.0C, soil c. 0.7C above ambient), high (120 kg N ha^-1 yr^-1) rates of N addition to soil, increased O3 concentration (×1.5 ambient level of ~40 ppb) and herbivory by needle feeding larvae of the web-spinning pine sawfly (Acantholyda posticalis Matsumura). In exp. 2, Scots pine seedlings, growing in forest soil were planted in field plots, and exposed to air warming (air c. 0.5C, soil c. 4.0C above ambient), moderate (30 kg N ha^-1 yr^-1) rates of N addition to soil, herbivory by the bark feeding large pine weevil (Hylobius abietis L.). The seedlings were exposed to these abiotic and biotic factors in single as well as combined exposure for three growing seasons. The effects of altered growth conditions were reported from shoot and root dry weight (DW), root mass fraction (RMF), root morphology, concentration of phenolic compounds in roots, mycorrhizal colonization, mycorrhizal morphotypes, rhizosphere soil and root fungal biomass, bacterial biomass in rhizosphere and rhizosphere BVOC emissions.

Seedling growth stimulation was observed due to warming and N addition in both experiments. Growth stimulation in combined exposure to warming and moderate N addition was lower than growth stimulation in single exposure regimes in exp. 2. High N addition in exp.

1 caused a preference of shoot growth over root growth; and had decreasing effects on length proportions of fine roots and mycorrhizal fungi which were not observed with moderate N addition. Increases in root ramification index (RRI, root tips m^-1) and mycorrhizal colonization were observed due to O3 treatment. Mycorrhizal colonization decreased due to both

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needle (exp. 1) and bark herbivory (exp. 2). Needle feeding increased length proportions of some fine root fractions but bark herbivory generally decreased length proportions of fine roots. O3 exposure at ambient N levels in soil decreased Fungi-to-bacteria ratio (F:B-ratio) in rhizosphere. Rhizosphere BVOC emissions generally decreased due to all the experimental treatments in both experiments. All four treatments showed interactive effects whereby the effect of one treatment was modified in combined treatment (e.g. the decreasing effect of moderate N addition on mycorrhizal colonization was neutralized by warming treatment). In general, both field experiments showed that the interactive effects became more common with the duration of exposure.

These findings suggest that the future projections might give an overestimate of C sequestration in Scots pine trees, if based on single factor manipulation like warming or N addition experiments. The general decrease in rhizosphere level BVOC emissions due to altered growing conditions in the future will have implications on root growth, mycorrhizal colonization and trophic level interactions e.g. might alter the susceptibility of the roots to herbivory. The results also suggest that warming, N addition, O3 and herbivory may alter the root morphology and decrease mycorrhizal colonization in roots, potentially leading to a less efficient root system which may be detrimental to young Scots pine stands in the future.

Keywords: Acantholyda posticalis Matsumura, Hylobius abietis L., mycorrhizal fungi, nitrogen addition, Pinus sylvestris L., rhizosphere BVOCs, root morphology, root phenolics, soil PLFAs, tropospheric ozone, warming.

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List of abbreviations

BVOCs Biogenic volatile organic compounds

C Carbon

CFC Chlorofluorocarbon

CH4 Methane

C:N-ratio Carbon-to-nitrogen ratio

CO2 Carbon dioxide

DW Dry weight

EMF Ectomycorrhizal fungi EMM Extramatrical mycelia F:B-ratio Fungi-to-bacteria ratio

GHG Greenhouse gas

GLVs Green leaf volatiles HNO3 Nitric acid

H2SO4 Sulphuric acid

N Nitrogen

NHx NH3 + NH4+

NH3 Ammonia gas

NH4+ Ammonium ion

n-MT Non-oxygenated monoterpene

NO Nitric oxide

NO2 Nitrogen dioxide

NOx Oxides of nitrogen

O3 Ozone

o-MT Oxygenated monoterpene PLFA Phospholipid fatty acids ppb Parts per billion

PSC Plant secondary compound RMF Root mass fraction ROS Reactive oxygen species RRI Root ramification index

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SOM Soil organic matter

SQT Sesquiterpene

SRA Specific root surface area SRL Specific root length VWC Volumetric water content

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List of original publications

This thesis includes the following original research articles referred to in the text as chapters 2-4.

Chapter 2 Muhammad Usman Rasheed, Anne Kasurinen, Minna Kivimäenpää, Ra- jendra Ghimire, Elina Häikiö, Promise Mpamah, Jarmo K. Holopainen &

Toini Holopainen (2017) The responses of shoot-root-rhizosphere continuum to simultaneous fertilizer addition, warming, ozone and herbivory in young Scots pine seedlings in a high latitude field experiment. Soil Biol- ogy and Biochemistry 114: 279-294

Chapter 3 Muhammad Usman Rasheed, Riitta Julkunen-Tiitto, Minna Kivimäenpää, Johanna Riikonen & Anne Kasurinen (2020) Responses of soil-grown Scots pine seedlings to experimental warming, moderate nitrogen addition and bark herbivory in a three-year field experiment. Science of the Total Environment 733: 139110

Chapter 4 Muhammad Usman Rasheed, Minna Kivimäenpää & Anne Kasurinen. Bi- ogenic volatile organic compounds emissions from rhizosphere of Scots pine (Pinus sylvestris) seedlings exposed to warming, moderate N addition and bark herbivory by large pine weevils (Hylobius abietis). Submitted manuscript.

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Author’s contribution

Muhammad Usman Rasheed (MUR), measured the shoot and root DW, extracted PLFAs from rhizosphere soil, calculated the RMF, SRL, SRA, RRI, mycorrhizal colonization and length proportions of fine roots in exp. 1. MUR also analyzed exposure data (i.e. O3 concentration, air and soil temperature, soil moisture, and amount of rain) from the field in exp. 1. In exp. 2, MUR participated in general maintenance the field experiment, collection of exposure data, herbivore collection and herbivory exposure in the field. MUR also participated in harvesting for samples at the end of exposure season, rhizosphere BVOC sampling, measured shoot and root DW, analyzed exposure data from the field, extracted ergosterol from roots, extracted PLFAs from soil, performed root microscopy for mycorrhizal morphotypes, calculated RMF, SRL, SRA, RRI, mycorrhizal colonization, length proportions of fine and coarse roots, and calculated the emission rates of BVOC in exp. 2. MUR performed statistical analyses on the data presented in the research articles; is the first author in the original research papers (chapters 2 & 3) and the manuscript (chapter 4) included in this thesis.

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Acknowledgements

This work was conducted at the Department of Environmental and Biological Sciences at the University of Eastern Finland, Kuopio campus. One of the field experiments (exp. 1) was conducted at the open-air exposure field of University of Eastern Finland in Ruohoniemi. The second experiment (exp. 2) was conducted at the open-air exposure field at Natural Resources institute (Luke), in Suonenjoki. I wish to acknowledge the Unversity of Eastern Finland for providing the facilities and the funding (Spearhead project CABI) for conducting this research. I would also like to acknowledge the Academy of Finland (project numbers 133322, 266542, 272939 and 303785), Kuopion Luonon Ystävän Yhdistys (KLYY), Olvi Foundation, Finnish Cultural Foundation and Faculty of Science and Forestry, University of Eastern Finland for their generous financial support that enabled this research.

I would like to express my gratitude towards my supervisors, Senior Researcher Dr. Anne Kasurinen, Docent Dr. Minna Kivimäenpää and Emerita Professor Dr. Toini Holopainen for the opportunity to pursue my PhD, constructive comments on the manuscripts, their guidance through the research work, and encouragement throughout the process of PhD work. I would like to thank Dr. Rajendra P. Ghimire, Dr. Elina Häikiö, Dr. Promise Mpamah, Emeritus Professor Dr. Jarmo K. Holopainen, Emeita Professor Dr. Riitta Julkunen-Tiitto and Senior Researcher Dr. Johanna Riikonen for their contribution to the manuscripts as co-authors. I would like to thank technicians Mr. Timo Oksanen, Mr. Jukka Laitinen and Mr. Pasi-Yli Pirillä for their technical advice and help in maintaining the field experiments. I also thank Dr. Päivi Tiiva as well as laboratory technicians Ms. Jaana Rissanen and Ms. Virpi Tiihonen for helping in harvest and sample collection at the end of each experiment season. I extend a special thanks to Dr. Patrick G. Tisza for checking grammar of this thesis.

I would like to extend my gratitude to Assistant Professor Dr. Amy Trowbridge and Senior Research Fellow Dr. Ivika Ostonen for agreeing to evaluate this thesis. I also thank Professor Dr.

Håkan Wallander for agreeing to be the opponent for the public defense of this thesis.

I am grateful to my family and friends, in Pakistan and in Finland, who created a support system for me outside the workplace wihout which the journey of my doctoral studies would have been impossible.

Muhammad Usman Rasheed Kuopio November 2020

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1.7.3 Research Questions ... 47 2 The Responses of Shoot-root-rhizosphere continuum to simultaneous fertilizer addition, warming, ozone and herbivory in young scots pine seedlings in a high latitude field experiment ... 51 3 Responses of soil-grown Scots pine seedlings to experimental warming, moderate nitrogen addition and bark herbivory in a three-year field Experiment ... 53 4 Emissions of biogenic volatile organic compounds (BVOCs) from rhizosphere of Scots pine (Pinus sylvestris) seedlings exposed to warming, moderate N addition and bark herbivory by large pine weevil (Hylobius abietis) ... 55 5 General Discussion and Conclusions... 57 5.1 SUMMARY OF MAIN FINDINGS ... 57 5.2 CARBON ASSIMILATION AND RMF RESPONES TO CLIMATE CHANGE AND HERBIVORY ... 60 5.2.1 Interactive Effects of Warming and N Addition on Growth Parameters 60 5.2.2 Moderate and High N Addition Effects on C Allocation to Root Systems

... 61 5.2.3 Ozone and Herbivory Effects on Shoot and Root Variables ... 62 5.3 ROOT MORPHOLOGY VARIABLES, MYCORRHIZAL FUNGI AND SOIL MICROFLORA ... 63 5.3.1 Effects of Moderate and High N Addition on Root Morphology ... 63 5.3.2 Warming Effects on Root Morphology and Mycorrhizal Fungi ... 64 5.3.3 Ozone Effects on Root Morphology, Mycorrhizal Fungi and Soil Bacteria

... 66 5.3.4 Root Morphology Variables and Mycorrhizal Fungi Respond Differently to Bark and Needle Herbivory ... 67 5.3.5 Interactive Effects of O3 Exposure and Needle Herbivory on RRI ... 68 5.3.6 Treatment Effects on Soil Fungi and Rhizobacteria ... 68 5.4 RESPONSES OF SECONDARY COMPOUNDS IN RHIZOSPHERE ... 69 5.4.1 Herbivory and Warming Effects on Root Phenolics ... 69 5.4.2 Warming, N Addition, Ozone and Herbivory Effects on Rhizosphere BVOCs ... 70 5.5 CONCLUSIONS AND FUTURE DIRECTIONS ... 72 6 References ... 74

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1 Introduction

1.1 BOREAL FORESTS

Boreal forests are characterized by cold continental and subarctic climate, and are the largest terrestrial biome comprising around 30% of the world’s forested area with circumpolar distribution, primarily between latitudes 50 to 60N (Taggart and Cross 2009, Thiffault 2019).

The climate of boreal forests is characterized by long winters and short summers, typically lasting three month or less (Taggart and Cross 2009, Thiffault 2019).

Boreal forests represent a significant carbon (C) sink, assimilating atmospheric carbon dioxide (CO2), thereby reducing radiative forcing and mitigating climate change (Lemprière et al. 2013, Holmberg et al. 2019). In Finland, boreal forests are the largest sink of greenhouse gases (GHGs), absorbing over 2.5 Tg of CO2 equivalent yr^-1 of GHGs (Official Statistics of Finland 2019). The vegetation in boreal forests is a significant C pool, stem wood being the largest aboveground pool with a high C sink capacity (Schimel et al. 2001, Liski et al. 2003, Gough et al. 2008). The slowly decomposing plant litter to the soil surface also represents a significant C pool, e.g. old boreal forests of Central Siberia are estimated to have equal amounts of C stored in vegetation and in plant litter on soil surface (Verdova et al. 2018). The soil in boreal forests is generally a mix of sandy, acidic mineral soil and peat soil, and is estimated to sequester more C as soil organic matter (SOM) than temperate and tropical forests together (Gough et al. 2008, Taggart and Cross 2009, DeLuca and Boisvenue 2012). The mean C content of boreal soils is estimated to be 343 t C ha^-1(including 14 t C ha^-1 of root biomass), compared to C content of 96 t C ha^-1 and 123 t C ha^-1 from temperate and tropical soils respectively (Brunner and Godbold 2007). Due to low temperatures and pH, boreal soils have low nitrogen (N) availability, compared to e.g. temperate soils (DeLuca and Boisvenue 2012, Thiffault).

Pine (Pinus) and spruce (Picea) are the most abundant coniferous trees in boreal forests (Taggart and Cross 2009). Scots pine (Pinus sylvestris L.) is the commonest and an econom- ically valuable tree species of the European boreal forests (DeLuca and Boisvenue 2012, Thiffault 2019). Scots pine is also widespread, being native to UK, Norway, Sweden, Finland, and Russia (Howard 1944). Scots pine has an apical meristematic shoot growth pattern, grows

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at a slow to medium growth rate of about 30 to 60 cm per year, and mature trees reach an average height of 35 m (Marinich and Powell 2017). In mature Scots pine trees, the aboveground part may account for about 85% of the whole tree biomass (Helmisaari et al. 2002).

1.1.1 Root System and Morphology in Boreal Trees

Roots anchor trees in soil, are the primary organs of water and nutrient absorption, and are also a C storage organ (Kalliokoski 2011). Root structures vary among tree species (Cannon 1949). Generally, a primary root forms branches that spread horizontally and vertically and define the spatial extent of the roots and rhizosphere (the region of soil that is influenced by roots; Hartmann et al. 2008, Kalliokoski 2011). Root branches are sometimes described relative to each other based on order number. Either the primary root originating from the seed or the root tips (that harbor root hair or mycorrhizal associations) are assigned as the first order root, and the rest of the roots are given a higher order relative to the chosen first order root (Cannon 1949, Pregitzer et al. 2002, Dajon and Reubens 2008). The choice of first order root is generally based on the types or part of the root system in question (Dajon and Reubens 2008).

Root branches can also be classified by their diameter, typically the roots with diameter (Ø)  2 mm are considered fine roots while Ø > 2mm roots are considered coarse roots (Dajon and Reubens 2008). The diameter-based classification is arbitrary, as root-diameter and -function relationships vary among tree species, and even within the fine roots of Ø  2 mm, roots of different diameters may have different functions (Pregitzer et al. 2002, Guo et al. 2008).

Coarse roots form the skeleton of the root system, while the fine roots are the main organs for water and nutrient absorption (Kalliokoski et al. 2008, Zadworny et al. 2016) as well as form symbiotic associations with mycorrhizal fungi (Smith and Read 2008). Fine root biomass and root mass fraction (RMF), i.e. ratio of total root dry mass to total seedling dry mass (g g^-1), are used to assess the C distribution across trees and are used as indicators of root efficiency (Cudlin et al. 2007, Qi et al. 2019). Mean RMF from the boreal evergreen gymnosperm trees has been reported as 0.115, which is relatively low compared to that of temperate evergreen gymnosperms, which is 0.287 (Qi et al. 2019). In young Scots pine, however, the RMF may be as high as 0.25; and seedlings may allocate over 40 % of photosynthates for root production (Helmisaari et al. 2002). Root morphological parameters, e.g. root ramification index (RRI,

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root tips m^-1), specific root length (SRL, length of fine roots per unit of root DW, m g^-1) and specific root surface area (SRA, fine root surface area per unit of root DW, m² g^-1) are also considered indicators of root health and soil exploration efficiency (Ostonen et al 2006, Ostonen et al. 2007a, Ostonen et al. 2007b, Dajon and Reubens 2008, Kramer-Walter et al.

2016). Although the nutrient and water uptake efficiency have not been directly studied, con- sidering that fine roots are the primary organs of water and nutrient uptake and that the absorption is proportional to contact area between roots and soil, an increase in fine root biomass, RMF, RRI, SRA and SRL may indicate an increased soil exploring efficiency. All the root parameters stated above are subject to environmental influences, representing adaptive strategies by trees to increase C sequestration capacity under changing environmental conditions (Kramer-Wallertz et al. 2016). Scots pine has taproot system that reach depths of 1.5- 3.0 m, and its lateral roots tend to develop symmetrically around the main root (Laitakari 1929, Marinich and Powell 2017). For studies where the focus is fine root morphology (including this thesis), root tips are usually chosen as the first order roots and the rest of the roots system is allocated a higher order number with respect to root tips (Dajon and Reubens 2008).

1.1.2 Mycorrhizal Fungi and Rhizosphere Microflora in Boreal Forests

Most of the roots of coniferous trees within the boreal region, including Scots pine, form symbiotic associations with ectomycorrhizal fungi (EMF; Smith and Read 2008). EMF are structurally diverse. The anatomy of EMF symbiotic interactions has been studied for less than 10% of ectomycorrhizal species (Agerer 2006). Generally, the EMF symbiosis is distin- guished from other putatively symbiotic plant-fungi interactions based on characteristic morphological features i.e., mantle grown around short roots (root tips forming mycorrhizal symbiosis) and Hartig net (hyphae) (Brundrett 2004, Smith and Read 2008, Tedersoo et al. 2010).

Hartig net is a complex network of fungal hyphae that ramify extensively and grow between (but not inside) the root epidermal and cortical cells (Tedersoo et al. 2010). The Hartig net provides a large surface area and acts as the nutrient and C exchange interface between EMF and the host, whereby the host trees exchange photosynthetic C products for soil resources from EMF (Tedersoo et al. 2010). It is estimated in lab-based experiments that trees infected with EMF may allocate up to 1/3 more C below-ground when compared to non-EMF infected trees (Cairney 2012). The mantle around short roots provides physical protection to the short

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roots and can be a site for C storage (Peterson et al. 2004, Cairney 2012). Most EMF infect a broad range of host species, but some species are specific to host trees (Mitchell 1993). For example, Suillus-type EMF are considered to be Pinaceae (pine family) specific root associ- ates (Peterson et al. 2004, Tedersoo et al. 2010).

Most EMF form extramatrical mycelia (EMM) (e.g. Ascomycetes EMF is an exception) growing out from the EMF mantle into the soil (Agerer 2006, Cairney 2012). In some EMF the hyphae aggregate to form hydrophobic rhizomorphs behind the growing end, while the hydrophilic hyphae explore the soils to absorb nutrients, extending the reach of roots to farther soil resources (Peterson et al. 2004, Smith and Read 2008, Cairney 2012, Ekbald et al. 2013, Zhang et al. 2019). Given that mycorrhizal symbiosis increases nutrient availability to roots, mycorrhizal colonization and mycorrhizal diversity should also be considered indicators of root efficiency (Agerer 2006). In EMF, EMM are also sites of oxidative and hydrolytic enzyme activity associated with SOM degradation, indicating that not only EMM absorb available nutrients in soil but also digest labile or recalcitrant C in SOM (Phillips et al. 2014).

EMM also explore the soils for new root tips to colonize and possibly connect plant roots, facilitating inter-plant C and nutrient movement (Cairney 2012). C storage capacity of EMF is reported to be sensitive to changing climatic factors e.g. surface level CO2 concentration and N concentration in soil, but the C sink capacity of EMF is less understood, especially in the changing growth conditions of boreal forests (Treseder and Allen 2000). For the studies included in this thesis, root ergosterol concentration, mycorrhizal colonization of root tips (by counting mycorrhizal tips under microscope) and mycorrhizal morphotyping (studying the morphotype of mycorrhizal roots under microscope) were used as measures of EMF symbiosis in the roots of young Scots pine. Ergosterol is a membrane lipid almost exclusively found in fungi (Mille-Lindbolm et al. 2004). Ergosterol from the roots was extracted using the method introduced by Nylund and Wallander (1992) with minor modifications described in Kasurinen et al (2001).

In addition to the symbiotic mycorrhizal fungi, rhizosphere of trees in boreal forest soils are host to soil bacteria and fungi (e.g. saprophytic fungi), whose primary function is to recy- cle litter entering the soil from above- and belowground (Bardgett 2005). It is estimated that soil bacteria and saprophytic fungi carry perform approximately 95% of the decomposition of organic matter in Scots pine forests (Persson et al. 1980). In addition to decomposition of SOM, bacteria in rhizosphere may also assist mycorrhizal fungi by promoting the formation of mycorrhizal symbiosis or maintaining the established symbiosis (Lladó et al. 2017). It is

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estimated that bacteria are the most abundant microflora in soil (Lladó et al. 2017), with nearly 4.4 thousand taxa per kilogram of soil (Curtis et al. 2002). Despite the vast diversity of soil bacteria, fungi dominate the forest soils in terms of biomass (Bardgett 2005, Högberg et al 2007a, Joergensen and Wichem 2008). Persson et al. (1980) estimated bacterial biomass of 39 g DW m^-2 against the fungal biomass of 120 g DW m^-2 in a 120-year-old Scots pine forest.

Fungi-to-bacteria ratio (F:B-ratio) is an indicator of soil C sink capacity; a higher F:B- ratio indicates a higher C storage capacity (Deng et al. 2016, Malik et al. 2016). Root exudates, litter return to the soil, physiochemical characteristics of soil, and climate change influence the composition and abundance of soil fungi, soil bacteria and consequently, the F:B-ratio (Lladó et al. 2017, Fernández-Alonso et al. 2018). The future climate scenario is also expected to alter the abundance, diversity, and spatial distribution of soil fungi, including EMF with profound consequences on SOM (Mitchell 1993, Smith and Read 2008, Ekbald et al. 2013, Santalahti et al. 2016). For the studies included in this thesis, the relative proportions of phospholipid fatty acid (PLFA) biomarkers was used as a measure of fungal and bacterial abundance in rhizosphere soil. PLFA analysis is a sensitive and fast technique for assessing the living microbial community in soil (Kim et al. 2016). PLFAs were extracted from the rhizosphere soil using the method introduced by Bligh and Dyer (1959).

1.1.3 Plant Secondary Compounds in Boreal Forests

Plants constitutively produce an array (estimated to be around 200,000) of chemically and structurally diverse plant secondary compounds (PSCs) (Holopainen et al. 2018). The PSCs may constitute up to one-third of the dry mass in woody trees and the distribution of these compounds may vary in different parts of the tree, e.g. concentration of catechins is lower in the roots than in stems of juvenile seedlings of boreal woody trees (Obst 1998, Keski-Saar and Julkunen-Tiitto 2003). The PSCs are classified into several groups such as phenolics (approximately 8,000 known compounds) and terpenoids (approximately 30,000 known compounds) based on their chemical structure (Holopainen et al. 2018).

Phenolic compounds are a group of non-volatile PSCs important in chemical defense within plant tissue against biotic and abiotic factors (Holopainen et al. 2018, Metsämuuronen and Siren 2019). Phenolic compounds have been extracted from leaves/needles, stem and roots of juvenile seedlings, and mature trees from boreal forests (Keski-Saari and Julkunen-

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Tiitto 2003, Ghimire et al. 2018, Metsämuuronen and Siren 2019). Despite the composition of phenolic extracts from leaf/needle, stem and roots varying within the tree, the compounds generally have antibacterial, antioxidative, insecticidal or fungicidal properties (Metsämuuro- nen and Siren 2019), e.g. flavonoids (sub-category of phenolic compounds) in needles of Scots pine are suggested to have insecticidal effects on sawfly larvae (Larsson et al. 1992, Vihakas 2014, Vihakas et al. 2015). Phenolic compounds pinosylvin, pinocembrin and pinosylvin-monomethylether extracted from Scots pine phloem have been shown to have fungicidal properties (Bois et al. 1999). Catechin, another phenolic compound with possible an- timicrobial effects, has been isolated from needle, bark, and roots of Scots pine (Metsämuuro- nen and Siren 2019). Another phenolic compound, protocatechuic acid, which has antibacterial activity, has been isolated from roots of Norway spruce (Picea abies L.) and Scots pine (Chao and Yin 2009, Tiiva et al. 2019, Metsämuuronen and Siren 2019). Tannins (sub-category of phenolics) and flavonoids have also been shown to inhibit enzyme activity in soil, preventing C and N mineralization and inducing toxicity against soil microbes in boreal forests including Scots pine forests (Rauha et al. 2000, Kanerva et al. 2008). The concentration of phenolic compounds is sensitive to changing climate, e.g. warming will decrease the concentration of phenolic compounds in stems of boreal trees (Holopainen et al. 2018).

The PSCs with high vapor pressure at room temperature (low boiling point) are commonly referred to as biogenic volatile organic compounds (BVOCs) (Dudareva et al. 2013). BVOCs comprise isoprene, monoterpenes (MTs), sesquiterpenes (SQTs, sometimes considered semi- volatiles) and other non-methane volatile hydrocarbons (Dudareva et al. 2006, Dudareva et al. 2013). BVOCs are released into the environment from leaves/needles, cones, stem, and roots of the plants (Rasmann and Agrawal 2008, Bäck et al. 2012, Delroy et al. 2016, Holo- painen et al. 2018, Šimpraga et al. 2019). Soil and root microflora (including EMF) and plant litter also emit BVOCs (Isidorov and Jdanova 2002, Leff and Fierer 2008, Bäck et al. 2010, Penuelas et al. 2014, Kivimäenpää et al. 2018).

BVOCs serve several functions in forests, often as a blend e.g., they act as chemical sig- nals for plant-plant (e.g. inter- and intraspecific interactions), plant-animal/insect (e.g. herbivores, pollinators or parasitoids), plant-microbe (e.g. microflora of soil) and microbe-microbe (e.g. soil bacteria and fungi) communication (Dudareva et al. 2013, Šimpraga et al. 2019).

BVOCs promote growth in plants and increase their thermal tolerance (Penuelas and Llusia 2003, Šimpraga et al. 2019). BVOCs also play an important role in defense against insect herbivores (Holopainen 2004, Rasmann and Agrawal 2008, Holopainen and Gershenzon

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2010, Delroy et al. 2016). It is suggested that root BVOCs may be involved in inter and intraspecific root recognition below-ground and may have allelopathic effects on nearby roots of plants of other species (Delroy et al. 2016). BVOCs emitted by soil fungi in rhizosphere help prevent tree roots from infection by pathogenic fungi e.g. isolates of saprobic fungi Trametes versicolor suppress the growth of pathogenic fungi Armillaria spp. (Šimpraga et al. 2019).

Trees infected by pathogenic fungi may alter root BVOCs to recruit antifungal bacteria to the rhizosphere (Šimpraga et al. 2019). BVOCs from EMF (e.g. auxin, ethylene) can induce lateral root formation and branching of the lateral roots (Ditengou et al. 2015). SQTs from EMF are also involved in ramification of fine roots and formation of mycorrhizal symbiosis (Di- tengou et al. 2015). The profile of the plant litter BVOCs generally resembles that of the living plants but also consists of compounds produced by microbes decomposing the litter, however, there can be variations in composition of litter BVOCs depending on the diversity of decom- posers degrading it and the extent of degradation (Tang et al. 2019). Plant litter has been suggested to be the second largest source of BVOCs in the forests after vegetation (Mäki et al. 2019, Tang et al. 2019). The functions of litter BVOCs are less understood compared to those of foliar BVOCs. It is suggested that litter BVOCs may have allelopathic effects, i.e.

inhibiting germination or growth of competing understory vegetation (Šimpraga et al. 2019).

BVOCs emissions of Scots pine are usually dominated by MTs with α-pinene, δ-3-carene, - terpene, tricyclene, α-thujene, -phellandrene and -pinene being some of the most commonly found compounds from the shoot and rhizosphere (Lin et al. 2007, Bäck et al. 2012, Ghimire et al. 2013).

An estimated 1000 Tg C yr^-1 is released into the atmosphere globally in the form of BVOCs (Guenther et al. 2012). BVOCs can get oxidized in the atmosphere which condense on and contribute to the growth of small particles in air, forming secondary aerosols (Penuelas and Llusia 2003, Spracklen et al. 2008). Aerosols in the atmosphere generally have a cooling effect as they disperse solar radiation on their own, or by serving as nuclei for cloud formation (Penuelas and Llusia 2003, Spracklen et al. 2008). It is estimated that the cooling effect of secondary aerosols from boreal BVOCs emissions gives a significant negative feedback to climate warming (Spracklen et al. 2008). Another sink of atmospheric BVOCs is forest soil itself (Tang et al. 2019, Rinnan and Albers 2020). Trowbridge et al. (2020) have shown that soils, especially soils dominated by mycorrhizal fungi (whereby 85% of basal area was com- prised of trees associated with mycorrhizal fungi), can be a sink for atmospheric BVOCs e.g.

methanol, acetone, and isoprene, during growing season and fall. At present, there is no direct

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evidence that mycorrhizal fungi absorb BVOCs, but soil microflora appears to have the capacity to mineralize the BVOCs from aboveground, which is likely an important way the ecosystem can balance the amount of compounds with low reactivity in the atmosphere e.g.

methanol (Tang et al. 2019, Rinnan and Albers 2020). In a changing climate, BVOCs emissions from forests are expected to change, e.g., warming or herbivory may increase the terpene emissions from boreal trees, but there is knowledge gap as to the significance of soils as sinks (or sources) of BVOCs, and how will their sink capacity change with changing climate (Hol- opainen et al. 2018, Rinnan and Albers 2020).

1.2 NEEDLE AND BARK HERBIVORY, AND ITS EFFECTS ON BOREAL FORESTS

Trees in boreal forests are exposed to numerous herbivorous insects in their lifetime e.g. needle feeding and/or bark feeding insects (Lindelöw and Björkman 2001). Climate warming is generally expected to increase the insect damage by increasing the frequency of outbreaks, the feeding period, early emergence, the number of life cycles per summer and delayed over- wintering (Logan et al. 2003, Schwartzberg et al. 2014, Wainhouse et al. 2014). Thus, damage caused by needle and bark feeding herbivores is expected to increase in the northern boreal forests in the future, as climate of the northern latitudes becomes warmer (Logan et al. 2003, Schwartzberg et al. 2014). Feeding of foliage decreases foliar biomass (Myers and Sarfraz 2017). Herbivory creates a competition for the allocation of C resources of the tree between the host tree and the feeding herbivore (Haukioja and Honkanen 1997). In addition, needle feeding also deprives the tree of photosynthetic machinery (Haukioja and Honkanen 1997).

Some important needle feeding herbivores of boreal coniferous trees are larvae of the great web-spinning pine sawfly (Acantholyda posticalis Matsumura), the European sawfly (Neodi- prion sertifer Geoffroy) and the large pine sawfly (Diprion pini L.; Larsson and Tenow 1984, Day and Leather 1997, Voolma et al. 2009); and some common bark feeding herbivores of boreal coniferous trees are large pine weevil (Hylobius abietis L.), mountain pine beetle (Den- droctonus ponderosae Hopkins) and European spruce bark beetle (Ips typographus L.) (Lång- strom and Day 2004, Toivanen et al. 2009, Rosenberger et al. 2019). In exp. 1, larvae of the

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great web-spinning sawfly and in exp. 2, large pine weevils were used to study the effects of herbivory on young Scots pine seedlings.

The great web-spinning pine sawfly is an important pest of the pine forests of Europe (Voolma et al. 2009, 2016). The first to third instar larvae of the great web-spinning pine sawfly feed on pine needles of all generations (Ghimire 2015). The sensilla ultrastructure of great web-spinning pine sawfly reveal structures indicating both, chemo- and mechanical re- ceptors for identifying the host plant and potential mates (Yuan et al. 2013). Outbreaks of great web-spinning pine sawfly have been reported from Central and Eastern Europe, Central Asia and Siberia, including mass outbreaks, spanning over 200 hectares of Scots pine forests reported in Western Estonia and Western Finland (Voolma et al. 2009, 2016 Vapaavuori et al. 2010).

Large pine weevil is one of the most important pests of pine seedlings in the boreal region (Långström and Day 2004, Fedderwitz et al. 2018). Large pine weevils locate their hosts using constitutively emitted host BVOCs (Nordlander et al. 1986, Šimpraga et al. 2019). They feed on the bark of seedlings and crowns of mature trees including Scots pine (Örlander et al. 2000, Långström and Day 2004, Wallertz et al. 2014). Intense feeding of conifers by the large pine weevil leads to seedling mortality (Örlander and Nilsson 1999, Wallertz et al. 2014) and the roots of freshly killed or dying trees are used as sites for oviposition (Norlander 1990).

1.2.1 Effects of Herbivory on Tree Roots

It has been shown that simulated herbivory by clipping of needles in Scots pine as well as feeding by polyphagous herbivore the European tarnished plant bug (Lygys rugulipennis Pop- pius) leads to a decrease in total root mass and an increase in RMF (Manninen et al. 1998b, Lyytikäinen-Saarenmaa and Tomppo 2002, Kuikka et al. 2003, Hodar et al. 2008). Needle clipping translates into decreased C sequestration, and in turn less C is available for growth investment in root, thus decreasing the root growth (Lyytikäinen-Saarenmaa 1999, Hodar et al. 2008). The increase in RMF, however, indicates a preference of root growth over that of shoot growth (Hodar et al. 2008). Recently, an increase in proportion of fine roots (Ø 1.5-2.0 mm) was reported after short-term (1-day) feeding of large pine weevils on Scots pine seedlings (Tiiva et al. 2019). Root morphology responses to shoot herbivory are less studied than root biomass accumulation and allocation patterns in plants in response to herbivory.

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Increased RMF and length proportion of fine roots under herbivory pressure suggest that trees focus on growth and maintenance of the absorptive structures since an efficient roots system would enable better nutrient accumulation to compensate for herbivore damage.

1.2.2 Effects of Herbivory on Mycorrhizal Fungi and Rhizosphere Microflora

Shoot herbivory effects mycorrhizal fungi; an interaction that has been studied to a limited extent (Gange 2007). Shoot herbivory has been shown to decrease mycorrhizal colonization (including EMF), abundance, and diversity (Gehring and Whitham 1991, 2002, Del Veccio et al. 1993, Gehring et al. 1997, Kuikka et al. 2003). However, the opposite has also been reported; a few studies showed that herbivory may also lead to an increase in mycorrhizal colonization (Wamberg et al. 2003, Gange 2007). In agreement, a recent study found an increase in mycorrhizal colonization of roots after 1-day of bark feeding by the large pine weevils in Scots pine seedlings (Tiiva et al. 2019).

Responses of mycorrhizal abundance and diversity are dependent on the susceptibility of host species to herbivory, mycorrhizal species, and the level of herbivory (Gange et al. 2007, Sthultz et al. 2009, Gehring et al. 2014). At low levels of herbivory, plants can increase their rate of photosynthesis and compensate (or even overcompensate) for foliar loss due to herbivory (Gange et al. 2007). The increased C assimilation due to increased photosynthesis may result in increased C allocation to roots. Shoot herbivory has been shown to increase root exudation (Holland et al. 1996). Mycorrhizal symbionts may exploit the increased availability of C resources in the root exudates resulting in increased mycorrhizal colonization (Holland et al. 1996). At high levels of herbivore damage, increasing the rate of photosynthesis cannot compensate for biomass losses, consequently, C becomes a limited resource, leading to decreased C supply to mycorrhizal fungi, thus, decreasing mycorrhizal colonization (Gange et al. 2007). Any characterization of the intensity of herbivory has to be specific to the herbivore- plant-mycorrhizal interaction (Gange et al. 2007). Lyytikäinen-Saarenmaa et al. (1999) have defined artificial defoliation of < 25% of needle removal in Scots pine as ‘low’ damage.

The effects of shoot herbivory on soil saprophytic fungi and bacteria may be mediated via plant litter as plant litter is a C source for saprophytic fungi and bacteria (Lindo et al. 2013, Bonanomi et al. 2017, Fanin et al. 2019). Nutrient (N and labile C) quantity of plant litter has been shown to increase due to foliage feeding by the weevil Deporaus betulae L. from

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northern boreal forests, populated with downy birch (Betula pubescens Ehrh.), Scots pine and Norway spruce (Metcalfe et al. 2016). If herbivory alters the plant litter quality (either increasing or decreasing the nutrient content), then herbivory may lead to changes in soil microflora composition, e.g. increased labile C content in plant litter due to herbivory may increase the saprophytic fungi that decompose labile C.

1.2.3 Effects of Herbivory on Plant Secondary Compounds

One of the most widely studied role of plant PSCs is that of defence against herbivore attack.

It is proposed that the production of PSCs evolved in plants as a defence mechanism against herbivory and pathogens (Wink 2003, Metsämuuronen and Siren 2019). Herbivore feeding or even oviposition by insects may induce changes in gene transcription leading to altered (usually increased) biosynthesis of plant PSCs (Dicke 2009, Metsämuuronen and Siren 2019).

Regulation of PSC synthesis is dependent on the herbivore species involved and the duration of exposure (Dicke 2009, Niinemets 2010). The signal inducing stimulus for PSC biosynthesis upregulation is often transmitted from the feeding parts of the herbivore (Dicke et al. 2009, Dicke and Baldwin 2010, Šimpraga et al. 2019).

Foliar feeding by chewing insects may lead to an immediate release of green leaf volatiles (GLVs), while an increase in MTs and SQTs emission usually follows with a short delay and lasts for up to several hours (Šimpraga et al. 2019). Moderate feeding by larvae of the pine sawfly on Scots pine seedlings has been reported to increase foliar emissions of non-oxygenated monoterpenes (n-MTs), oxygenated monoterpenes (o-MTs), SQTs and GLVs (Ghimire et al. 2013, Kivimäenpää et al. 2016, 2017). Bark feeding by the large pine weevils has also been shown to increase MT and SQT emissions from shoots of pine trees (Heijari et al. 2011, Kovalchuk et al. 2015, Kari et al. 2019, Semiz et al. 2016). Tiiva et al. (2018) also observed an increase in MT emission rates form shoots in response to the large pine weevils feeding on Scots pine, but only when weevil-damaged seedlings were simultaneously exposed to warming and moderate (30 kg N ha^-1 yr^-1) N addition to soil.

Foliar damage due to herbivory can induce changes in belowground BVOC emissions (Trowbridge and Stoy 2013). Changes in root BVOC emissions may also alter the PSC composition of neighbouring plants, priming them against herbivore attack (Huang et al. 2019).

Leaf feeding by the geometrid moth species (Erannis defoliaria Clerck and Agriopis

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aurantiaria Hübner), and bark feeding by the large pine weevils did not affect root BVOC emissions from silver birch (Betula pendula Roth) (Maja et al. 2014). Tiiva et al. (2019) did not report any significant changes in rhizosphere BVOCs after bark feeding by the large pine weevils. Contrastingly, Ghimire et al (2013) reported a decrease in rhizospheric MT emissions after needle feeding by larvae of the pine sawflies on Scots pine while shoot level BVOC emissions increased.

A direct defence strategy against herbivore feeding is to increase the amount of toxic non- volatile PSCs in the tissue (Dicke et al. 2009). Larvae of gypsy moths feeding on leaves of black poplar (Populus nigra L.) have been shown to increase synthesis of phenolic compounds in the damaged leaves; it is also proposed that the induced defence may extend to undamaged leaves, stem and roots (Babst et al. 2009, Boeckler et al. 2013). Concentration of catechins, a phenolic compound, has been reported to increase in inner bark of Norway spruce following bark beetle attack (Metsämuuronen and Siren 2019). Bark herbivory has also been shown to increase concentration of phenolic compounds in the roots of Scots pine seedlings (Morreira et al. 2012, Tiiva et al. 2019). Although the defensive role of non-volatile PSCs is well established (Metsämuuronen and Siren 2019), changes in tissue-level concentration of these compounds, especially in the roots in responses to shoot herbivory, is still sparse.

1.3 CLIMATE WARMING AND ITS EFFECTS ON BOREAL FOR- ESTS

Global warming is the most prominent feature of climate change. Anthropogenic activities such as industrial processes, agriculture, power generation and wastewater, produce GHGs like CO2, nitrogenous oxides (NOx), methane (CH4), and chlorofluorocarbons (CFCs) (Allen et al. 2018). GHGs absorb and retain infrared radiation from the solar energy leading to warming of the atmosphere (Allen et al. 2018). Global mean surface temperatures have increased by over 1C compared to the mean temperatures prevailing during pre-industrial times (1850- 1900) (Allen et al. 2018). The trend of global warming is continuing despite mitigation efforts on a global scale. Climate models predict mean surface temperatures will increase by at least 0.5C relative to the currently prevailing surface temperatures by 2030 (Allen et al. 2018).

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Global warming is already pronounced in the northern boreal regions (north of 60N; Al- len et al. 2018). Boreal forests have responded to warming by accelerated growth, increasing the C sink capacity of the forests (Liski et al. 2003, Kauppi et al. 2014, Ruiz-Pérez and Vico 2020). However, despite a general trend of accelerated growth, trees in the boreal region, including Scots pine, have been deemed susceptible to excess warming (3-4C above prevailing average temperature), partly due to the dryness associated with increasing temperatures (Kellomäki 1995, Reich and Oleksyn 2008, Romero-Olivares et al. 2017, D'Orangeville et al.

2018, Ruiz-Pérez and Vico 2020). Low temperature is a defining characteristic of the climate in the boreal region, but now warming is moving the subarctic climate northwards, and with it, the boreal forests are projected to migrate northwards, or even face extirpation (Kellomäki et al. 2008, Tang and Beckage 2010, Fisichelli et al. 2014, Ruiz-Pérez and Vico 2020).

1.3.1 Effects of Warming on Tree Roots

Root responses towards increasing temperature among boreal coniferous species have been ranging from accelerated root growth (Kasurinen et al. 2012, Leppälammi-Kujansuu et al.

2014), to decreased root growth (Bergner et al. 2004, Bronson et al. 2008). The apparent decrease in root growth in response to warming of up to 5 C, reported by Bronson et al. (2008), was due to a decrease in fine root biomass, and it was suspected that this decrease in biomass was an indirect effect of warming, mediated through increased N availability in warmed soil.

More recently, Lehtonen et al. (2016) reported a negative correlation between temperature and fine root biomass in Finnish boreal forests; they also suggested that trees may develop more fine roots in the colder Northern Finland compared to warmer Southern Finland due to low soil N concentration in colder regions. It is likely that the decrease in root growth reported by Bergner et al. (2004) due to warming of 1 C was also mediated through increased N availability in soil in their warming experiment.

According to a global synthesis of root-shoot biomass allocation patterns in different bi- omes, RMF in the boreal trees has an inverse linear relationship with annual mean temperature, i.e. as the mean annual temperature increases, the RMF in the boreal trees decreases (Qi et al. 2019). The decrease in RMF due to warming implies that warming may stimulate shoot growth more than root growth in boreal trees. Warming has also been reported to induce production of thicker roots indicated as a decrease in fine root production (Zadworny et al. 2016),

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and a decrease in SRL in Scots pine trees (Ostonen et al. 2007a, 2007b, Zadworny et al. 2017).

Decrease in SRL due to warming has also been reported from seedlings of boreal deciduous species silver birch (Kasurinen et al. 2016).

1.3.2 Effects of Warming on Mycorrhizal Fungi and Rhizosphere Microflora

A decrease in EMF hyphal count in soil in response to warming has been reported, however it is speculated that the use of epifluorescence microscopy with fluorescein isothiocyanate as stain for fungal hyphae gives an underestimate of the hyphal count (Berg et al. 1998). In addition, several studies from boreal soils have reported an increase in fungal biomass and fungal diversity, including that of mycorrhizal fungi in response to warming (Clemmensen et al.

2006, Allison and Treseder 2008, Leppälammi-Kujansuu et al. 2014, Vega-Frutis et al. 2014, Treseder et al. 2016). The F:B-ratio has also been reported to increase in response to warming (Rinnan and Bååth 2009). The long-term changes in soil C reserves are closely dependent on how the soil microflora will respond to changing climate.

Warming, especially in combination with drying, has the potential to decrease abundance as well as activity of microflora in boreal soil (Allison and Treseder 2008). In boreal forests, bacterial abundance and biomass have not been reported to change due to warming, but the bacterial diversity has been reported to change in response to warming (Berg et al. 1998, Lladó et al. 2017). A warming experiment lasting 10-years revealed a shift towards N-mineralizing bacterial species; the shift in bacterial diversity being driven by N-rich root exudates (Zhang et al. 2016). Soil fungal species acclimatize faster than soil bacteria to increasing temperatures (Pietikäinen et al. 2005, Coucheney et al. 2013). Long-term warming (≥ 20 years) can cause a shift in bacterial diversity towards species capable of degrading complex C resources in soil (Lladó et al. 2017). It is likely that in a warming climate, the overall bacterial biomass in soil will not change significantly; but the diversity of soil bacteria will change such that the species that are less tolerant of increasing temperatures will be replaced by species more adapted to higher temperatures (Lladó et al. 2017).

1.3.3 Effects of Warming on Plant Secondary Compounds

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Warming usually increase BVOCs emissions from foliage (Penuelas and Staudt 2010, Holo- painen et al. 2018). Increase in BVOCs emissions is partly due to increased biosynthesis of BVOCs (Kesselmier and Staudt 1999, Loreto and Schnitzler 2010, Holopainen et al. 2018), and partly due to increased vapor pressure as a result of warming and also due to the increased temperature-dependent release of BVOCs from storage structures, e.g. in pine trees (Grote and Niinemets 2008, Ghirardo et al. 2010, Penuelas and Staudt 2010, Holopainen et al. 2010, Holopainen et al. 2018). BVOCs emissions from roots and rhizosphere are less widely studied compared to foliar BVOCs emissions (Holopainen et al. 2018). A decrease in MTs and non- isoprene based BVOCs emissions rates from rhizosphere of Scots pine in response to warming has been reported recently (Tiiva et al. 2019). Earlier in this thesis, it was discussed that soils may act as sinks for BVOCs like methanol, acetone, and isoprene; but warming has been also shown to increase soil BVOC influx, especially in the soils dominated by ECM (Trowbridge et al. 2020). Given that soils dominated with ECM are a sink for BVOCs, especially under warming treatment (Trowbridge et al. 2020), the decreased rhizospheric BVOC emissions reported by Tiiva et al. (2019), can be attributed to increased respiration rates of BVOC mineralizing soil microbes in rhizosphere under warming (Rinnan and Albers 2020).

Previously, non-volatile PSCs, including phenolic compounds were assumed to follow the carbon-nutrient balance, i.e. increased photosynthates will be utilized in synthesis of C-rich PSCs if nutrient availability limits C investment in growth (Bryant et al. 1983, Holopainen et al. 2018). However, many phenolic compounds, including flavonoids and tannins, have been reported to respond in an opposite fashion (Lindroth 2012). Warming has been reported to decrease the concentration of phenolic compounds in leaves of hybrid aspen (Kosonen et al.

2012), as well as in needles and stem of Norway spruce (Zhang et al. 2018). In Scots pine seedlings, total concentration of phenolic compounds in the stem of the newest growth tended to decrease due to warming, however, the concentration of total phenolic compounds from older stem tended to increase in response to warming (Ghimire et al. 2019). Phenolic compounds from root extracts and their role in root protection are well reported, e.g. from Scots pine and Norway spruce (Metsämuuronen and Siren 2019). Concentration of several phenolic acids in root exudates, with possible inhibitory effects on microbial activity in rhizosphere, have been reported to increase in response to warming in subalpine coniferous forests (Qiao et al. 2014). The effects of climate change on concentration of root phenolic compounds is still relatively under-reported.

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1.4 TROPOSPHERIC OZONE AND ITS EFFECTS ON BOREAL FORESTS

Ozone is considered a secondary pollutant as it is not directly released into the environment by anthropogenic activities. Nitrogenous oxides (NOx), CH4, and unburnt hydrocarbons in the presence of UV-radiation lead to in situ synthesis of O3 in the troposphere (Monks et al. 2015).

An increase in O3 precursor molecules in the troposphere has led to an increase in O3 concentration in the troposphere (Jenkin and Clemitshaw 2000, Wittig et al. 2007, Cooper et al. 2014, Monks et al. 2015). Background concentrations of O3 in the troposphere have increased from around 20-30 parts per billion (ppb) to over 50 ppb in the populated regions of North America, Europe, Middle East, South Asia and East Asia (Cionni et al. 2011, Cooper et al. 2014, EMEP 2016). The rate of increase of tropospheric O3 concentration in the northern hemisphere has decreased since 1990s (Vingarzan 2004), but the expected O3 concentrations downwind of the highly industrialized and polluted regions for the end of 21^st century can be over 50 ppb depending on emission scenarios (Wittig et al. 2009, Cionni et al. 2011). O3 in the troposphere adds to surface warming as it is also a GHG (Wittig et al. 2009).

O3 has been termed as one of the most important phytotoxic air pollutants (Wittig et al.

2007, Agathokleous et al. 2020). O3 is a reactive gas; it can oxidize molecules on the surfaces of e.g. needles (Monks et al. 2015). O3 enters the leaves and needles through stomata, where it forms reactive oxygen species (ROS) that are toxic to plants (Wittig et al. 2007, Monks et al. 2015). Susceptibility and responses towards O3 exposure vary between tree species (Runeckles and Krupa 1994, Sandermann 1996, Wittig et al. 2007, Valkama et al. 2007, Wit- tig et al. 2009). Boreal coniferous species are considered to be less susceptible to O3 damage than deciduous species, however, among the coniferous species, Scots pine is more susceptible to O3 when compared to Norway spruce (Prozherina et al. 2009, Huttunen and Manninen 2013). O3 exposure can reduce stomatal conductance leading to reduced photosynthesis, reduced growth, and early needle senescence (Paoletti and Grulke 2005, Augustaitis et al. 2007, Huttunen and Manninen 2013, Büker et al. 2015, Li et al. 2017). In the mesophyll tissue, O3

forms ROS which may cause oxidative stress, alters gene expression, protein synthesis and signalling pathways, leading to tissue damage; and it may trigger defence mechanisms similar to herbivory stress (Vahala et al. 2003, Baier et al. 2005, Jaspers and Kangasjärvi 2010, Büker et al. 2015, Vainonen and Kangasjärvi 2015).

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1.4.1 Effects of Ozone on Tree Roots

Acute O3 exposure may decrease photosynthesis and cause localized cell death; and if the O3

exposure continues, the effects of O3 can accumulate and may alter root and shoot growth as well as C allocation patterns between root and shoot (Manning 1995, Andersen 2003, Wang et al. 2016). Phytotoxic effects of O3 are dependent on O3 concentration (Wittig et al. 2009).

Total shoot and root biomass accumulation has been reported to decrease in response to O3

exposure of approximately 40 ppb, however, the harmful effects of O3 were more pronounced in shoot leading to an average increase in RMF (Wittig et al. 2009). A higher concentration of approximately 100 ppb of O3 became more harmful for roots compared to shoots resulting in an average decrease of RMF (Wittig et al. 2009). Ozone exposure of about ×1.5 to ×2 ambient levels (elevated level: 40 to 60 ppb of O3) has been reported to decrease fine root biomass and short roots in boreal trees (Kasurinen et al. 1999, 2005, Wang et al. 2016). The decreasing effect of O3 on root biomass and RMF are attributed to decreased C assimilation due to decreased photosynthesis, increased metabolic costs for damage repair in shoot and decreased phloem loading (Cudlin et al. 2007). Based on meta-analysis including studies from lab and field experiments by Cudlin et al. (2007), root parameters (fine root length, SRL or mycorrhizal colonization) is not necessarily an effective indicator of harmful effects of O3

exposure, as the overall effects of O3 exposure on the root parameters in the meta-analysis was zero, i.e. no overall effect.

1.4.2 Effects of Ozone on Mycorrhizal Fungi and Rhizosphere Microflora

Ozone can alter the microbial community composition in the soil (Agathokleous et al. 2020).

O3 effects on roots and microflora are considered to be indirect (mediated through shoot and leaf litter returned to soil) as O3 does not penetrate deep in soil (Diaz et al. 1996, Kasurinen et al. 1999, Kainulainen et al. 2000, Aneja et al. 2007, Agathokleous et al. 2020). Thus, while O3 is harmful to bacteria (Lehtola et al. 2001), there may not be any direct effects of tropospheric O3 on soil bacteria (Phillips et al. 2002). Increased concentration of O3 (×2 ambient, ranging between 20-60 ppb) resulted in higher presence of gram-positive bacteria in boreal peat soil, compared to peat at ambient O3 dominated by gram-negative bacteria (Mörsky et al.

2008). Mörsky et al. (2008) did not report a decline in total bacteria biomarkers, which

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indicates no decrease in total bacterial abundance. Gram-negative bacteria dominate the upper layers of soil and are closely associated with roots, possibly relying on labile C from root exudates (Högberg et al. 2007, Kramer and Gleixner 2008). O3 disrupts the C assimilation and results in decreased C allocation to roots (Wittig et al. 2009), which may result in decreased C availability for rhizobacteria, decreasing their abundance or driving the change in diversity. Decreasing effects of O3 exposure have also been reported on bacterial signature PLFA analysis from rhizosphere soil of European beech (Fagus sylvatica L.), silver birch, Norway spruce and Scots pine (Kanerva et al. 2008, Pritsch et al. 2009).

Mycorrhizal fungi are more susceptible to O3 exposure compared to e.g. saprophytic fungi, as they are directly dependent on C supply from roots (Wang et al. 2016). There are several studies from boreal species, including Scots pine, indicating a decrease in mycorrhizal colonization, abundance, diversity, or altered diversity of mycorrhizal fungi after O3 exposure (Perez-Soba et al. 1995, Manninen et al. 1998a, Edwards and Zak 2011, Wang et al. 2015, 2016). Some studies, however did not observe any significant effect of O3 exposure on mycorrhizal fungi associated with boreal trees; and even reported a transient increase in mycorrhizal abundance after short-term O3 exposure (Rantanen et al. 1994, Manninen et al. 1998a, Kasurinen et al. 1999, Häikiö et al. 2009). Despite the susceptibility of mycorrhizal fungi towards O3, Cudlin et al. (2007) have concluded that EMF in boreal and temperate regions may not be very sensitive to realistic O3 exposure (×2 ambient concentration). Soil fungal- and bacterial community responses towards O3 exposure may also be dependent on soil properties (Chen et al. 2019).

Total fungal biomass in boreal soil, based on PLFA biomarkers, has been reported to increase (Mörsky et al. 2008) or to not change (Pritsch et al. 2009) in response to O3 exposure.

Pritsch et al. (2008) reported an increase in total microbial biomass in the rhizosphere of O3- exposed beech seedlings over one vegetation period; they also reported no change in dissolved organic C supply to the soil over the one growing season. It is likely that the increase in microbial biomass reported by Pritsch et al. (2008) was due to increased amount of plant litter returned to the soil which was indicated by the increased cellobiohydrolase and glucosidase (complex C digesting enzymes) activity in the soil. PLFA analysis cannot differentiate between EMF and other e.g. saprophytic soil fungi (Zelles 1999), thus the increase in fungal biomass based on PLFA biomarkers, reported by Mörsky et al. (2008) and Pritsch et al.

(2009), cannot be associated with possible changes in EMF abundance.

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1.4.3 Effects of Ozone on Plant Secondary Compounds

Ozone induces defence responses in plants (Vainonen and Kangasjärvi 2015). One of the defence responses in boreal trees is increased biosynthesis of PSCs (Holopainen et al. 2018).

This leads to altered composition of constitutively synthesized secondary compounds as well as synthesis of stress induced secondary compounds (Holopainen et al. 2018). Concentration of non-volatile PSCs, like phenolics (e.g. flavonoids and tannins) have been reported to increase in the foliage of deciduous boreal trees when exposed to O3 (Holopainen et al. 2018).

An increase in secondary compounds in response to O3 exposure has not been observed in conifers including Scots pine (Kainulainen et al. 1998, Manninen et al. 2000, Riikonen et al.

2009, Riikonen et al. 2012), except for stilbenes that accumulate in Scots pine sapwood, phloem and needles (Metsämuuronen and Siren 2019). Increased O3 (×1.5 ambient level of

~40 ppb) has been reported to decrease concentration of phenolic compounds in Scots pine stem (Ghimire et al. 2019). There is lack of research on the effects if O3 on root level BVOCs emissions or non-volatile PSCs in boreal coniferous trees. Recently, an increase in the concentration of total phenolic compounds in roots of crop plants, wheat (Triticum aestivum L.) and wild spinach (Chenopodium album L.), was reported (Gosh et al. 2020).

Induced changes in BVOCs emissions in response to O3 exposure are considered to be concentration-dependent and also dependent on duration of exposure (Valkama et al. 2007, Penuelas, Josep and Staudt 2010, Blande et al. 2014). O3 exposure may induce BVOCs similar to the emissions induced by herbivore damage (Blande et al. 2014). However, it is unlikely that O3 will have any direct effects on rhizosphere BVOCs emissions, as O3 does not penetrate deep into the soil (Pinto et al. 2010). O3 may also react with BVOCs in the atmosphere, cuticle, and air space between mesophyll tissue (Jud et al. 2016). No changes in terpene concentrations in Scots pine and hybrid larch needles under O3 exposure have been observed (Manninen et al. 2000, Mochizuki et al. 2017). However, emissions of MTs, e.g. 3-carene, sabinene, cam- phene and terpinolene, o-MTs, GLVs as well as the emission of non-isoprene based BVOCs, have been reported to increase from shoots of Scots pine and Norway spruce in response to O3 exposure (×1.4 to ×1.5 ambient level of in the range 32-40 ppb) (Kivimäenpää et al. 2013, 2016, Ghimire et al. 2017). No changes in rhizosphere level BVOC emissions were observed in rapeseed (Brassica napus L.) in response to chronic and over 100 ppb O3 exposure (Acton et al. 2018).

Rhizosphere processes of scots pine seedlings under growth conditions of future climate and shoot herbivory

Dissertations in Forestry and Natural Sciences

MUHAMMAD USMAN RASHEED

Rhizosphere Processes of Scots Pine Seedlings Under Growth Conditions of Future

RHIZOSPHERE PROCESSES OF SCOTS PINE SEEDLINGS UNDER GROWTH CONDITIONS OF

FUTURE CLIMATE AND SHOOT HERBIVORY

Muhammad Usman Rasheed

RHIZOSPHERE PROCESSES OF SCOTS PINE SEEDLINGS UNDER GROWTH CONDITIONS OF

FUTURE CLIMATE AND SHOOT HERBIVORY

ABSTRACT

List of abbreviations

List of original publications

Author’s contribution

Acknowledgements

CONTENTS

1 Introduction

1.1 BOREAL FORESTS

1.2 NEEDLE AND BARK HERBIVORY, AND ITS EFFECTS ON BOREAL FORESTS

1.3 CLIMATE WARMING AND ITS EFFECTS ON BOREAL FOR- ESTS

1.4 TROPOSPHERIC OZONE AND ITS EFFECTS ON BOREAL FORESTS