The Biocontrol Agent Phlebiopsis Gigantea: Efficacy and Impacts on the Stump Bacterial Biota and Conifer Tree Defences

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The Biocontrol Agent Phlebiopsis gigantea: Efficacy and Impacts on the Stump Bacterial Biota and Conifer tree Defences

Hui Sun

Department of Forest Sciences Faculty of Agriculture and Forestry University of Helsinki and Biological Interactions Graduate School

ACADEMIC DISSERTATION

To be presented, with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public criticism in lecture room B6, B-building (Laotkartanonkaari 7), on November 18th 2011, at 12 o’clock noon.

Supervisors: Professor Fred O. Asiegbu Department of Forest Sciences University of Helsinki, Finland

Dr. Risto Kasanen Department of Forest Sciences University of Helsinki, Finland Finnish Forest Research Institute (METLA)

Pre-examiners: Professor Richard Hamelin Faculty of Forestry, Forest Sciences Centre 3032-2424 Main Mall UBC Vancouver, BC Canada V6T 1Z4

Dr. Ari Hietala The Norwegian Forest and Landscape Institute, N-1431, Ås, Norway

Opponent: Professor Pierluigi Bonello Department of Plant Pathology The Ohio State University, USA

Custos: Professor Fred O. Asiegbu Department of Forest Sciences University of Helsinki, Finland

Cover: Necrosis, cell death assay and lignified cell analysis in the necrotic tissues in Norway spruce inoculated with H. parviporum or P. gigantea as well as interaction between the two fungi.

ISSN 1795-7079 ISBN 978-952-10-7260-4 (back) ISBN 978-952-10-7261-1 (PDF) http://ethesis.helsinki.fi

Helsinki Universtiy Printing house Helsinki 2011 2

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CONTENTS

ABBREVIATIONS LIST OF ORIGINAL ARTICLES ABSTRACT

1. INTRODUCTION ...... 9

1.1 Heterobasidion root rot disease ...... 9

1.2 Disease management ...... 11 1.2.1 Control of Heterobasidion infections ...... 11 1.2.2 Silvicultural methods ...... 11 1.2.3 Stump removal ...... 11 1.2.4 Stump treatments with chemicals ...... 12 1.2.5 Biological control ...... 12

1.3 Phlebiopsis gigantea as a biological control agent ...... 13 1.3.1 Biology of P. gigantea...... 13 1.3.2 History of P. gigantea as a biological control agent ...... 13 1.3.3 The mechanism of P. gigantea as a biocontrol agent against H. annosum root rot ...... 13 1.3.4 Induced resistance as a mechanism for the biological control ...... 14 1.3.5 Properties of P. gigantea related to biocontrol efficacy ...... 14 1.3.6 Mating system of P. gigantea and potential for use of breeding approach ...... 15 1.3.7 Risk assessment on the potential for P. gigantea to acquire necrotrophic habit ...... 16

1.4 Impact of P. gigantea treatment on microbial diversity in the stump ...... 16

1.5 Next generation sequence (454 pyrosequencing) ...... 18

2. AIMS OF THE STUDY ...... 19

3. MATERIALS AND METHODS...... 20

4. RESULTS AND DISCUSSION ...... 21

4.1 Variation in the biocontrol properties of P. gigantea against Heterobasidion infection (I) ...... 21

4.2 Use of a breeding approach to improve the biocontrol properties of P. gigantea (II) ...... 22 4.2.1 Fruiting of progeny isolates ...... 22 4.2.2 Properties of the progeny and parental isolates of P. gigantea ...... 22

4.2.3 Biocontrol efficacy of the progeny and parental isolates of P. gigantea ...... 23

4.3 The potential of P. gigantea to acquire necrotrophic capability (III) ...... 24

4.4 Induced resistance as a mechanism of P. gigantea control against Heterobasidion root rot (III)...... 25

4.5 The impacts of treatment with biocontrol fungus (Phlebiopsis gigantea) on bacterial biota of Norway spruce stumps (IV) ...... 27 4.5.1 Bacterial community richness and diversity in stumps of P. abies ...... 27 4.5.2 P. gigantea treatment and bacterial diversity at different stages of stump decay ...... 27 4.5.3 Interaction between site, age of stump and P. gigantea treatment on the bacterial biota ...... 28

5. CONCLUSIONS AND PERSPECTIVES ...... 32

ACKNOWLEDGEMENTS ...... 34

REFERENCES ...... 36

ABBREVIATIONS

AMOVA Analysis of molecular variance AMP Antimicrobial peptide cDNA Complementary deoxyribonucleic acid DAHPS Deoxy-d-arabino-heptulosonate phosphate synthase ESTs Expressed sequence tags GO Gene ontology MEA Malt extract agar OTUs Operational taxonomic units PCR Polymerase chain reaction PR-10 Pathogenesis- related-10 rDNA Ribosomal deoxyribonucleic acid rRNA Ribosomal ribonucleic acid RT-PCR Reverse transcriptase polymerase chain reaction SAMS S-adenosyl-L- methionine synthetase

LIST OF ORIGINAL PUBLICATIONS

The present thesis is based on the following original articles, which will be referred to by the Roman numerals in the text (I-IV):

I. Sun H, Korhonen K, Hantula J, Kasanen R. (2009). Variation in properties of Phlebiopsis gigantea related to biocontrol against infection by Heterobasidion spp. in Norway spruce stumps. Forest Pathology 39 (2): 133-144.

II. Sun H, Korhonen K, Hantula J, Asiegbu FO, Kasanen R. (2009). Use of a breeding approach for improving biocontrol efficacy of Phlebiopsis gigantea strains against Heterobasidion infection of Norway spruce stumps. FEMS Microbiol Ecology 69 (2): 266-73.

III. Sun H, Paulin L, Alatalo E, Asiegbu FO. (2011). Response of living tissues of Pinus sylvestris to the saprotrophic biocontrol fungus Phlebiopsis gigantea. Tree Physiology 31: 438–451.

IV. Sun H, Terhonen E, Koskinen K, Paulin L, Kasanen R, Asiegbu FO. (2011). The impacts of treatment with biocontrol fungus (Phlebiopsis gigantea) on bacterial biota of Norway spruce stumps (submitted).

OTHER ARTICLES NOT INCLUDED IN THIS THESIS

I. Anthony C. Mgbeahuruike, Hui Sun, Petra Fransson, Risto Kasanen, Geoffrey Daniel, Magnus Karlsson, Frederick O Asiegbu. (2011). Screening of Phlebiopsis gigantea isolates for traits associated with biocontrol of the conifer pathogen Heterobasidion annosum. Biological Control 57: 118–129.

II. Risto Kasanen, Eeva Terhonen, Saija Huuskonen, Hui Sun, Antti Uotila. (2011). Infection rate of conifer stumps by Heterobasidion species in Central Finland. Scandinavian Journal of Forest Research 26 (5): 404–412.

III. Eeva Terhonen, Teresa Marco, Hui Sun, Risto Jalkanen, Risto Kasanen, Martti Vuorinen and Fred Asiegbu. (2011). The effect of latitude, season and needle-age on the mycota of Scots pine (Pinus sylvestris) needles in Finland. Silva Fennica (In press).

ABSTRACT

Phlebiopsis gigantea has been for a long time known as a strong competitor against Heterobasidion annosum and intensively applied as a biological control agent on stump surfaces of Picea abies in Fennoscandia. However, the mechanism underlying its antagonistic activity is still unknown. A primary concern is the possible impact of P. gigantea treatment on resident non-target microbial biota of conifer stumps. Additional risk factor is the potential of P. gigantea to acquire a necrotrophic habit through adaptation to living wood tissues.

This study focused on the differential screening of several P. gigantea isolates from diverse geographical sources as well as the use of breeding approach to enhance the biocontrol efficacy against H. annosum infection. The results showed a significant positive correlation between growth rate in wood and high biocontrol efficacy. Furthermore, with aid of breeding approach, several progeny strains were obtained that had better growth rate and control efficacy than parental isolates.

To address the issue of the potential of P. gigantea to acquire necrotrophic capability, a combination of histochemical, molecular and transcript profiling (454 sequencing) were used to investigate the interactions between these two fungi and ten year old P. sylvestris seedlings. The results revealed that both P. gigantea and H. annosum provoked strong necrotic lesions, but after prolonged incubation, P. gigantea lesions shrank and ceased to expand further. Tree seedlings pre-treated with P. gigantea further restricted H. annosum-induced necrosis and had elevated transcript levels of genes important for lignification, cell death regulation and jasmonic acid signalling. These suggest that induced localized resistance is a contributory factor for the biocontrol efficacy of P.gigantea, and it has a comparatively limited necrotrophic capability than H. annosum.

Finally, to investigate the potential impact of P. gigantea on the stump bacterial biota, 16S rDNA isolated from tissue samples from stumps of P. abies after 1-, 6- and 13-year post treatment was sequenced using bar-coded 454 Titanium pyrosequencing. Proteobacteria were found to be the most abundant at the initial stages of stump decay but were selectively replaced by Acidobacteria at advanced stages of the decay. Moreover, P. gigantea treatment significantly decreased the bacterial richness at initial decay stage in the stumps. Over time, the bacterial community in the stumps gradually recovered and the negative effects of P. gigantea was attenuated. Overall, the results were considered relevant and provide additional insight on the risk assessment as well as environmental impact on the long-term use of P. gigantea in the control of Heterobasidion root rot in conifer forests.

1. INTRODUCTION

1.1 Heterobasidion root rot disease

Heterobasidion root rot, caused by the closely related fungal species belonging to the Heterobasidion annosum complex, is a common and economically the most important disease of conifer trees in the boreal and temperate forests of the Northern Hemisphere (Woodward et al., 1998; Asiegbu et al., 2005). The yearly economic losses from devaluation of timber and growth reduction are estimated to at least 790 million euros in Europe (Woodward et al., 1998; Asiegbu et al., 2005). The economic loss due to the decay in Finland alone is estimated at 50 million euros annually (Finland´s National Forest Programme 2015, 2008). In addition, the costs of the preventive chemical or biological control of the disease during cuttings are estimated at five million euros per year (Finnish Statistical Yearbook of Forestry 2009).

H. annosum complex has a wide geographical distribution particularly in many parts of Europe, North America, China and Japan (Dai and Korhonen, 1999; Dai et al., 2003; Ota et al., 2006). It was long considered to be a single biological species with a wide host range. However, mating experiments determined that many different intersterile groups (ISGs) were present within the species complex and these ISGs showed very high host specificity in terms of specialized host preferences (Capretti et al., 1990; Chase and Ullrich, 1988; Korhonen et al., 1992; Niemelä and Korhonen, 1998). Therefore, H. annosum complex was initially classified as P- (with host preference for Scots pine), S- (Norway spruce), and F-type (silver fir) based on their host specificity. New species names of H. annosum sensu stricto (s.s.), H. parviporum and H. abietinum have been given for the European P, S and F ISGs, respectively (Korhonen, 1978; Capretti et al., 1990). Similarly, the North American S and P groups have also been given new names, H. occidentale and H. irregulare, respectively (Otrosina and Garbelotto, 2010). The European and North American H. annosum populations form different clades as detected by the population genetic analyses (Fabritius and Karjalainen, 1993; Kasuga et al., 1993; Stenlid et al., 1994; La Porta et al., 1997; Garbelotto et al., 1998; Vainio and Hantula., 1999; Kasuga and Mitchelson, 2000; Johannesson and Stenlid, 2003; Maijala et al., 2003). In addition, all the three ISGs occur in Europe and two (P and S) in North America. Different populations exist within intersterile groups and some intersterile groups have multiple group types. However, the ISGs still show differences in distribution and host preferences despite the wide host ranges.

In Europe, H. annosum s.s. mainly attacks Scots pine (Pinus sylvestris), but also infects many other conifers (Picea abies and Juniperus communis) and some deciduous trees, such as birch (Betula) (Korhonen, 1978; Korhonen and Piri, 1994). The distribution area of the P group covers almost all of the Europe except the driest areas in the south and the coldest pine forests in the north (Korhonen, 1978; Capretti et al., 1990; Korhonen and Stenlid, 1998; Laporta et al., 1997; Stenlid, 1987).

H. parviporum occurs mostly on Norway spruce (P. abies) in many parts of Europe, but also attacks Abies sibirica in north-eastern Europe (Korhonen, 1978; Korhonen et al., 1997; Korhonen et al., 2005; Dai et al., 2006). The S group appears to occur following the natural range of P. abies.

H. abietium has been mainly found in fir forest (Abies alba). However, it can also colonize other coniferous and deciduous trees, including P. abies. However, its distribution in Europe is more restricted than that of H. annosum and H. parviporum and most likely limited to Europe (Dai et al., 2003; Doĝmus-Lehtijärvi et al., 2006). It occurs predominantly in the southern and central Europe where fir species grow, and extends to Spain in the west, to southern Poland in the north, to the western Caucasus in the east and to southern Greece and Italy in the south (Woodward et al., 1998; Korhonen and Dai, 2005; Sánchez et al., 2005; Capretti et al., 1990; Tsopelas and Korhonen, 1996; Sánchez et al., 2007; Zamponi et al., 2007). Although the distribution areas of the ISGs overlap in Europe, in nature they are genetically isolated and only a few natural intergroup hybrids have been reported (Garbelotto et al., 1996).

The North American H. irregulare attacks mostly trees in the genera Pinus, Juniperus and Libocedrus and shows high degree of compatibility with the European H. annosum s.s. (Chase and Ullrich, 1990a; Stenlid and Karlsson, 1991; Garbelotto et al., 1996). It occurs in the west from British Columbia to Mexico (Eades, 1932; Barrera and Ramirez, 1980) and in the east from Quebec to Florida (Laflamme and Blais, 1995), but is less common in the central parts of the continent.

The North American H. occidentale shows a high partial compatibility with both the European H. annosum s.s. and H. parviporum (Capretti et al., 1990; Stenlid and Karlsson, 1991). Moreover, the H. occidentale shows a wide host range rather than a high degree of host specialization, including trees in genera such as Picea, Abies, Thuja, Pseudotsuga, Tsuga, Sequoiadendron, Pinus and Juniperus (Chase 1985; Korhonen et al., 1998). There are no known avirulent strains of these conifer pathogens and no known host genotype in the Pinaceae family with total resistance (Asiegbu et al., 2005).

The main primary route of infection for H. annosum s.l. is by basidiospores germinating on freshly-cut stump surface or wounds on the roots from where it spreads via root contacts from infected to healthy surrounding trees (Redfern and Stenlid, 1998). The pathogen is capable of infecting conifer roots of all ages (Asiegbu et al., 1993 and 1994; Heneen et al., 1994) and has been reported to remain in the stumps in the forest for periods of up to 62 years (Greig and Pratt, 1976).

1.2 Disease management

1.2.1 Control of Heterobasidion infections

Stump infection and following vegetative spread of the pathogen through root contacts established between stumps and neighboring trees is the primary infection route in managed forests (Stenlid and Redfern, 1998). The control of forest tree diseases is feasible only by interrupting the weak points in the life cycles of the disease agents. The control actions against H. annosum have therefore focused on preventing the infection sources, including basidiospore deposition, germination and growth of the fungus. In practice, several disease control strategies including silvicultural, chemical and biological methods have been used.

1.2.2 Silvicultural methods

As H. annosum complex mainly infects conifer trees rather than deciduous species (Delatour et al., 1998), planting less susceptible species in infected site could help to avoid the root rot problem (Korhonen et al., 1998a; Lygis et al., 2004a, b). In addition, winter cuttings instead of summer cuttings (Möykkynen and Miina, 2000 and 2002) or promotion of mixed stands (Lindén and Vollbrecht, 2002) have been suggested as preventive measures for this disease. Due to industry supply demands, however, these conditions are not always feasible.

1.2.3 Stump removal

Another possibility to eradicate the disease is by removing infected stumps from forest stand (Laflamme, 2010). Infected stump can serve as a source of infection for an extended period as Heterobasidion can survive for 15-60 years on decayed stumps (Greig and Pratt, 1976; Piri, 1996). Extracting stumps with fine roots can ensure removal of all the potential infection inoculum as well as prevent the disease spreading to the next generation (Stenlid, 1987; Laflamme, 2010). Many studies have proven that stump removal, although expensive, is one of the most effective method for control and eradication of Heterobasidion, Armillaria and Phellinus root rot on infested forest sites (Greig and McNabb, 1976; Shaw and Roth, 1978 and 1980; Greig, 1980; Thies, 1984; Pas and Hood, 1984; Morrison et al., 1991; Thies and Sturrock, 1995; Sturrock, 2000; Greig et al., 2001; Gibbs et al., 2002). Due to the demand for biofuel, large interest has focused on stump removal. In Finland and Sweden, biomass from forest has been one of the main sources for renewable fuel, in which the stumps offer biomass resource equally large or larger than the logging residues at harvested forest sites (Hofsten 2006; Egnell et al., 2007). The number of stands, where conifer stumps are lifted and harvested for fuel use in energy production, has increased rapidly over the last years. In Finland, 0.8 million m3 of stumps were used as an energy source in 2009 (Ylitalo, 2010).

Despite the efficacy of stump removal to control the disease, possible site disturbance due to stump removal should be taken into account (Thies 1987; Smith and Wass, 1991; Sturrock et al. 1994; Wass and 11

Senyk, 1999; Sturrock, 2000). A change in microbial diversity has been reported (Warren and English, 2003). The authors showed that some fungi, which were seldom found in natural stands, become more frequent in stands after stump removal. The environmental consequences of stump removal, which might be both negative and positive, should be assessed and evaluated.

1.2.4 Stump treatments with chemicals

Stump treatment has usually been achieved by spraying the stump surface immediately after tree felling with either chemicals such as urea or borate (Pratt et al., 1998; Holdenrieder and Greig, 1998). Chemical fungicides are commonly used to control plant pathogens and to reduce the economical losses from fungal diseases. A large number of commercial products have been tested as control protectants against Heterobasidion infection (Pratt et al., 1998). Of these, urea is the substance currently used as well as borates (Pratt and Lloyd, 1996). Both chemicals have low toxicity and are in common use as agricultural fertilizers. The potential mechanism of urea in the control of H. annosum is through hydrolysis process by urease enzymes in fresh wood, whereby the urea is converted to ammonia raising the pH about 2 units to alkaline level in the surface of the stump, thereby making it toxic for growth of H. annosum (Johansson et al., 2002). Borate compounds prevent the germination of Heterobasidion spores on newly cut stumps; however, the mechanism for this effect is unknown (Pratt, 1996).

It is known, however, that application of chemical substances can markedly influence fungal community structure in both conifer and deciduous stumps (Lipponen, 1991; Meredith, 1959; Rayner, 1977; Varese et al., 1999 and 2003) and also damage the vegetation (Westlund and Nohrstedt, 2000). Moreover, the development of resistance to chemical fungicides is a common phenomenon among plant pathogens (Gisi et al., 2002; Hufbauer and Roderick, 2005; Leroux and Gredt, 1997). Wide spread use of a particular chemical agent tends to pose a strong selection pressure that drives the mutation for fungicide resistance towards fixation in the pathogen gene pool (Samils et al., 2008). The selection imposed by natural enemies is unlikely to be as strong as that imposed by fungicides.

1.2.5 Biological control

Due to possible unfavorable environmental side effects of chemical control method, biological control has been proposed as an alternative to chemical control. Numerous studies have focused on searching for effective competitors and antagonists of H. annosum, such as Phlebiopsis gigantea, Fomitopsis pinicola, Bjerkandera adusta, Resinicium bicolor, Trichoderma spp. (Holdenrieder and Greig, 1998; Holmer and Stenlid, 1996, 1997; Kallio and Hallaksella, 1979; Nicolotti and Varese, 1996; Nicolotti et al., 1999). Among them, only P. gigantea is currently used as an efficient stump treatment agent. Three distinct biological control products based on P. gigantea have been developed: PG Suspension® in the UK, PG IBL® in Poland and Rotstop® in Finland (Pratt et al., 2000).

1.3 Phlebiopsis gigantea as a biological control agent

1.3.1 Biology of P. gigantea

P. gigantea is a corticiaceous basidiomycete and one of the most common fungal species found in dead coniferous wood in the Boreal region, especially in managed forests (Käärik and Rennerfelt 1958; Meredith 1959). It has very large population size due to its easy sporulation (Kallio, 1965; Eriksson et al., 1981). Both morphological characters and molecular evidence proved that P.gigantea is a single species throughout Europe (Eriksson et al., 1981; Korhonen and Kauppila, 1998; Korhonen, 1997; Vainio et al., 1998).

1.3.2 History of P. gigantea as a biological control agent

P. gigantea was first proposed as a biological control agent to prevent primary stump infections by Heterobasidion spp. in England (Rishbeth, 1952 and 1963). It is a common saprotrophic wood decay basidiomycete, which, like Heterobasidion spp. can invade living tissues of freshly cut conifer stumps (Holdenrieder and Greig, 1998). It competes against H. annosum in the woody tissues by rapid colonization of the stumps (Korhonen et al., 1994; Holdenrieder and Greig, 1998; Bailey et al., 2003). Findings by Rishbeth (1951) on the biology of the disease have made it possible to prevent infection through stump treatment. Currently, stumps in over 200,000 ha of forest are being treated with P. gigantea in Europe each year and over 62,000 ha of P. abies stands in Fennoscandia (Thor, 2003; Finnish statistical year book of forestry, 2009).

1.3.3 The mechanism of P. gigantea as a biocontrol agent against H. annosum root rot

Biological control may act in different ways (Postma et al., 1996). The mode of action of biological control agents can be for instance as antibiosis, competition for nutrients, parasitism or induction of host resistance (Baker et al., 1968). During antibiosis process, one or more compounds secreted by microbes with antibiotic or enzymatic activity could suppress the other target organism (Thomashow et al., 2002). The competition occurs between microorganisms for the available nutrients, such as carbon, nitrogen and iron. Limiting the growth of one organism by another is the most common way or outcome for the competition. Competition between pathogens and non-pathogens for nutrient resources is important for limiting disease incidence and severity (Pal and McSpadden Gardener, 2006). The parasitism of one organism by another is defined as destructive mycoparasitism through secretion of degrading enzymes or production of antibiotics during contact.

P. gigantea has been for long time known as a strong competitor against H. annosum (Rishbeth, 1952); however, almost nothing is known about the physiological and molecular basis of the interspecific interaction between these two fungi. So far, there is no evidence of either antibiotics or toxins being 13

secreted by P. gigantea (Holdenrieder and Greig, 1998; Capretti and Mugnai, 1988). Instead, the mode of action of P.gigantea appears to be competitive exclusion of Heterobasidion. The control effect against Heterbasidion is partly based on hyphal interference (Rishbeth 1952; Ikediugwu and Webster, 1970; Ikediugwu, 1976; Tubby et al., 2008). Due to rapid colonization of the stumps, P.gigantea is able to out- compete the pathogen in wood infected by both organisms (Bailey et al., 2003; Holdenrieder and Greig, 1998; Korhonen et al., 1994). Recently, Adomas et al. (2006) reported that a diverse range of genes encoding proteins important for nutrient acquisition were shown to be preferentially expressed during the interaction. This indicates that the mechanism is probably more reliant on rapid colonization of stump wood by P. gigantea (Holdenrieder and Greig, 1998; Asiegbu et al., 2005; Adomas et al., 2006).

1.3.4 Induced resistance as a mechanism for the biological control

Plants actively respond to a variety of environmental stimuli, including biotic and abiotic factors as well as chemical stimuli produced by soil- and plant-associated microbes. The later stimuli can either induce or condition the plant host defenses (Pal and McSpadden Gardener, 2006). These changes enhance resistance against subsequent infection by a variety of pathogens (Pal and McSpadden Gardener, 2006). Induced resistances have been well studied in many horticultural and agricultural systems (Vallad and Goodman, 2004; Walters and Fountaine, 2009). It has previously been shown that induced resistance could be a mechanism of biological control triggered by living bacterial or fungal agents (Cantone and Dunkle, 1990; Steiner and Schönbeck, 1995; Van Loon et al., 1998; Bargabus et al., 2002; Kilic-Ekici and Yuen, 2003). In forest trees, few reports are available on inducible chemical defences (e.g. secondary metabolites) (Keeling and Bohlmann, 2006), but no information is available on the induced resistance being a mechanism of biological control.

Both P. gigantea and Heterobasidion spp. could invade living tissues (Holdenried and Greig, 1998). It is also known that in freshly cut stumps, the resistance mechanism of the parent trees are initially intact and continue to function for a few months (Redfern and Stenlid, 1998; Vasiliauskas et al., 2004). Much research effort has focused on searching for antimicrobials secreted by the fungus (Rishbeth, 1975; Adomas et al., 2006) but very little on induced resistance barriers or host metabolites accumulated due to prior growth of P. gigantea on fresh woody stumps. Therefore, besides acting as competitors to H. annosum, P. gigantea may also induce resistance on fresh woody stumps, which could be part of the mechanism for its biocontrol action. However, this has not been investigated as a potential mechanism for the biocontrol efficacy.

1.3.5 Properties of P. gigantea related to biocontrol efficacy

Although the mode of action of P. gigantea against the establishment of H. annosum on fresh stump surfaces is largely based on higher nutrient acquisition capability by P. gigantea (Asiegbu et al., 2005;

Adomas et al., 2006), three other properties of P. gigantea considered as important factors related to effective biocontrol: 1) ability for rapid colonization of stump wood, 2) competitive ability against H. annosum and 3) ability to produce a sufficient number of asexual spores (oidia) in culture. It is likely that the properties of P. gigantea related to biocontrol also differ between isolates. Much research efforts have been focused on the effectiveness of treament of P. gigantea against H. annosum infection (Korhonen et al., 1993 and 2003; Pratt et al., 2000; Berglund and Rönnberg, 2004; Berglund et al., 2005; Thor, 2003; Adomas et al., 2006; Samils et al., 2008), but little on the variation in properties among P. gigantea isolates. Isolates of P. gigantea that have rapid growth, produce abundant aerial oidia and have high capability to compete with the target pathogen H. annosum can readily be selected from wild population. However, there are no standard protocols for selecting isolates for use in biocontrol products. Different screening processes have been used in the development of the three commercial products of P. gigantea used in Europe (Pratt et al., 2000). Moreover, isolates of P. gigantea are likely to vary in their behavior on different host species, for example, P. gigantea grows on both pine and spruce but appears particularly well adapted to pine compared with Norway spruce in terms of growth speed (Rishbeth, 1951 and 1963; Kallio, 1971).

1.3.6 Mating system of P. gigantea and potential for use of breeding approach

P. gigantea is a heterothallic fungus with bipolar (monofactorial) mating system. The pure cultures can fruit and can be easily identified on the basis of the oidial chains, encrusted upright aerial hyphae, and multiple clamp connections (Stalpers 1978; Eriksson et al., 1981). Pairing experiments (Korhonen and Kauppila, 1998; Korhonen et al., 1997; Grillo et al., 2005) and DNA fingerprinting analysis (Vainio et al., 1998; Vainio and Hantula, 2000) showed that P. gigantea strains in Europe are freely interbreeding. Although the European and North American strains of P. gigantea show some differentiation, they are still regarded as belonging to the same biological species (Grillo et al., 2005). These two populations are interfertile.

Biological control by fungal organisms is commonly used in agriculture and horticulture. Most of the biocontrol agents are selected from natural microbial populations followed by numerous commercial products, which are in extensive use. Despite long history and successful applications, the request for more effective isolates selection is still ongoing, including the use of genetic engineering method to improve the efficiency of biocontrol agent (Baek and Kenerley, 1998; Rey et al., 2001; Lübeck et al., 2002). Samils et al. (2008) has reported that there is a theoretical risk for resistance build-up in the H. annosum population towards P. gigantea. Moreover, two novel double-stranded RNA elements (mycovirus) have been isolated from P. gigantea strains, which have been used as effective biocontrol agent in UK, but lost efficacy against H. annosum later on (Kozlakidis et al., 2009). Mycovirus infection is generally asymptomatic (Ghabrial, 1998), but some of them are known to reduce the virulence of phytopathogenic

fungi and cause obvious metabolic changes in the fungus (Pearson et al., 2009). This could possibly explain the loss of P. gigantea efficacy against H. annosum.

In the case of Rotstop preparation, the same heterokaryotic isolate has been used for stump treatment since 1991 in Finland (Berglund and Rönnberg, 2004; Korhonen et al., 1993). It is necessary to change isolates from time to time to prevent degeneration of the commercial isolate following repeated subculturing. In the long term, this might also safeguard the diversity of wild population of P. gigantea (Pratt et al., 1999). P. gigantea also showed high variability in biocontrol properties (I). The conventional breeding method, where individuals with desired traits are crossed, has been commonly used in crops and domestic animals probably thousands of years. The breeding approach may provide the possibility to improve the performance of P. gigantea in controlling Heterobasidion infection and to obtain potential strains with high control efficacy that can be used in commercial forestry (II).

1.3.7 Risk assessment on the potential for P. gigantea to acquire necrotrophic habit

It has been hypothesized that the lifestyle of a pathogen may have evolved from saprotrophy via acquisition of virulence factors, as saprotrophic and pathogenic fungi share important metabolic routes (Staats et al., 2005). Recent research has suggested that there might be some partial functional and taxonomic overlap among fungi with different primary trophic strategies between communities of endophytes, pathogens and saprotrophs (Vasiliauskas et al., 2007; Fröhlich and Hyde, 1999; Arnold, 2007). P. gigantea is known to be a saprotroph and shows no capacity to act as a pathogen of living trees, even under favorable experimental conditions (Kallio, 1973; Asiegbu et al., 1996). However, recent research provided evidence of the ability of P. gigantea to colonise fine living roots of conifer seedlings (Asiegbu et al., 1996 and 1999; Vasaitis et al., 2007). Both P. gigantea and H. annosum are basidiomycetes and partially share the same ecological niches on the fresh wood surface. In addition, P. gigantea has been intensively applied on stump surfaces in Nordic countries (Thor, 2003). The main concern and a potential risk are the adaptation of the saprotrophic P. gigantea to living wood tissues could facilitate its acquisition of necrotrophic ability. Despite the great ecological importance of P. gigantea, almost nothing has been reported on the interaction between P. gigantea and mature living bark/wood as well as the potential impact on other stump microbiota.

1.4 Impact of P. gigantea treatment on microbial diversity in the stump

At present, the Rotstop strain of P. gigantea has been intensively applied (more than 62 500 ha annually) on stump surfaces in Fennoscandia (Thor, 2003; Finnish Statistical Year book of Forestry, 2009) and over 200 000 ha of forest are being covered for stump treatment in Europe annually (Thor, 2003). Compared to chemical treatments, biological control agents are usually considered environmentally-friendly and often provoke smaller changes in the target organisms (Holt and Hochberg, 1997). However, large-scale

application of a biological control agent in forest ecosystems inevitably brings the issue of its impact on non-target organisms (Enserink, 1999). In the forest ecosystem, the root system of a tree is surrounded by living organisms such as mycorrhizae, bacteria, insects and fungal pathogens (Laflamme, 2010). The lifespan of a tree is calculated in terms of decades instead of weeks for most crops, and all biotic and abiotic factors are important over the course of a tree's lifespan (Laflamme, 2010).

The stumps and the roots of a tree comprise a major proportion of the total biomass (Lundmark, 1988) , which are known to be the key habitat for wood-inhabiting fungi for biodiversity in managed forest areas (Harmon et al., 1986; Samuelsson et al., 1994). In addition, some other organisms inhabiting stumps, which are not responsible for significant decay on the stump, may play an important role in promoting and accelerating stump degradation needed for forest maintenance, and/or oppose decay fungi through direct antagonism and trophic competition (Rayner and Boddy, 1988; Dix and Webster, 1994). Meanwhile, bacteria are probably the most common wood-inhabiting microorganism and the initial colonizer of wood, which play an important role in the overall decay process (Clausen, 1996). Nitrogen-fixing bacteria species associated with wood decay have been reported (Spano et al., 1982; Jurgensen et al., 1984). They contribute significantly to nitrogen estimates of wood during decay (King et al., 1980) and are the primary catalysts for maintaining the nitrogen balance (Barron, 2003; Hutchison and Barron, 1996). Wood-decay basidiomycetes may benefit from the presence of bacteria, in particular with respect to nitrogen supply and detoxification of mycotoxic compounds (De Boer and Van der Wal, 2008). The occurrence of bacteria and a high bacterial diversity in gymnosperm stumps and in natural decaying wood in forest have been demonstrated (Hallaksela, 1977; Woods, 1996; Murrary and Woodward, 2007; Zhang et al., 2008). Bacterial species could also coexist with white-rot basidiomycetes Hypholoma fasciculare in decaying wood (Valášková et al., 2009). Moreover, Folman et al. (2008) have reported that the white-rot fungi Hypholoma fasciculare and Resinicium bicolor had a strong negative effect on the species richness of wood-inhabiting bacteria on decaying beech wood.

Knowledge of the structure and diversity of the microbial community in the stumps could lead to a better understanding of their specific ecological roles in microbe-plant and microbe-microbe interactions. It has been reported that the application of mycelial suspensions of various wood-inhabiting fungi to Picea abies stumps might affect the occurrence of non-target fungal species (Lipponen, 1991; Varese et al., 1999 and 2003). However, it is not yet known how long the commercial strains of P. gigantea could persist in treated stumps and to what extent the microbial diversity is affected over long time. Much research effort has focused on the antagonistic action of P. gigantea against H. annosum infection (Korhonen et al., 1993; Adomas et al., 2006; Samils et al., 2008; Sun et al., 2009a and2009b; Mgbeahuruike et al., 2011), whereas the effect of these treatments on non-target organisms has received little attention. It is possible that stump treatment by P. gigantea can lead to an unnatural and undesirable shift in the biodiversity of forest ecosystems (Varese et al., 2003). More specifically, P. gigantea treatment might present an 17

ecological threat in the form of decreasing the diversity of natural communities of microbe in the stump (Vainio et al., 2005). A number of studies have addressed the impact of Rotstop P. gigantea on fungal communities of conifer stumps by culture-dependent and DNA fingerprinting-based method (Vasiliauskas et al., 2004 and 2005; Vainio et al., 2005) as well as the effects on ground vegetation (Varese et al., 1999 and 2003; Westlund and Nohrstedt, 2000). None, however, has examined the influence of Rotstop P. gigantea on bacterial community in conifer stumps.

The culture- and DNA fingerprinting-based methods have underestimated the total richness of microbial communities (Jumpponen and Jone, 2005; Schadt et al., 2003; Jeewon and Hyde, 2007). For instance, only about 1% of the total number of microbes present in soil is culturable (Schoenborn et al., 2004). This hinders analysis of microbial diversity using culture-based methods. In addition, several studies have shown high genetic diversity and low geographic differentiation in P. gigantea populations (Samils et al., 2009; Vainio et al., 2000). In this case, there might be a risk of loss in intraspecific genetic diversity within the treated areas if single Rotstop strain of P. gigantea is continually used on a large scale and for a long period of time.

1.5 Next generation sequence (454 pyrosequencing)

The use of transcriptome approach offers the possibility for global analysis of gene expression to unravel the molecular basis of the distinction made by host plant tissues during interaction with diverse fungal species. Massive parallel pyrosequencing (454 pyrosequencing) has become a feasible method for high- throughput sequencing (Margulies et al., 2005; Moore et al., 2006; Wicker et al., 2006; Huse et al., 2007; Weber et al., 2007). This has proven to be well adapted for analyzing the transcriptome of both model and non-model species (Morozova et al., 2008).

Furthermore, fungal and bacterial diversity in soil has been investigated using traditional method (Atkins and Clark, 2004; Kowalchuk et al., 1997; Marshall et al., 2003; Öpik et al., 2003). Comparisons between classical culture-dependent and molecular methods have revealed huge gaps and biases in our appreciation of microbial diversity (Hugenholt et al., 1998; Roesch et al., 2007; Jeewon and Hyde, 2007; Woodward, 2010). The application of pyrosequencing has proved to be suitable for analyzing taxonomic diversity within extant microbial communities (Sogin et al., 2006; Roesch et al., 2007; Acosta- Martinez et al., 2008). Partial ribosomal amplification has been commonly used to describe precisely microbial communities in environmental samples (Búee et al., 2009; Woodward, 2010; Jumpponen and Jones, 2009; Kim et al., 2008; Uroz et al., 2010; Lim et al., 2010; Jones, 2009). Short 16S rDNA and internal transcript spacer (ITS) region pyrosequencing reads have been verified to be useful for bacterial and fungal community analysis, respectively (Liu et al., 2007). These have been widely applied in studying microbial diversity in soils (Búee et al., 2009; Jones et al., 2009).

2. AIMS OF THE STUDY

P. gigantea has for a long time been used as biological control agent. The commercial preparation registered as Rotstop (Korhonen et al., 1993) has been intensively applied annually on stump surface in Fennoscandia. Different selection processes have been applied to P. gigantea strains isolated from nature in the development of the three products marketed in the UK, Poland and Finland (Pratt et al., 2000). It would however be of interest to evaluate the properties of P. gigantea important to the control efficacy. An efficient and rapid way for screening and improving the control efficacy is necessary, as there are clear advantages in having a number of isolates available for use in the products (Pratt et al., 1999). Besides acting as a competitor, P. gigantea might induce resistance on the host against Heterobasidion infection. This could partly contribute to the biological control mechanism. On the other hand, there also exists potential for P. gigantea to adapt to living tissues and acquire necrotrophic capability. In addition, long- term application of P.gigantea could affect the overall resident microbial community in the treated forest stands. A better understanding of the successional changes on the microbial community would help to facilitate the evaluation of the environmental effects of using P. gigantea as a biological control agent.

The specific objectives described in this thesis were:

1) To investigate the properties of P. gigantea important for biocontrol against Heterobasidion infection in Norway spruce stumps (I).

2) Use of a breeding approach to improve the biocontrol properties of P. gigantea (II).

3) To evaluate the necrotrophic potential in P. gigantea and to investigate if induced resistance barriers formed due to prior growth of P. gigantea could be one of the mechanisms to restrict invasive growth and necrosis formation by H. annosum (III).

4) To investigate the effect of P. gigantea treatment on bacterial community in conifer stump (IV).

3. MATERIALS AND METHODS

Materials and methods used in this study are summarized in Table 1. Detailed descriptions can be found in the publications I – IV.

Table 1 The materials and methods used in the study. Materials Publication P. gigantea isolates I, II, III, IV H. annosum s.s. isolates I, II H. parviporum isolates I, II, III P. sylvestris seedlings III

Stump samples P. abies, Rotstop-treated or untreated 1-year-old (Liesjärvi, Finland) IV P. abies, Rotstop-treated or untreated 6-year-old (Karja lohja, Finland) IV P. abies, Rotstop-treated or untreated 13-year-old (Saimaa, Finland) IV

Methods Publication fungal strain crossing II RNA isolation III cDNA synthesis III 454 cDNA library construction (III)* Real-time PCR III Bar-coded PCR IV DNA isolation IV 454 pyrosequencing (III, IV)* Sequence analysis and bioinformatics III, IV Genetic heritability analysis II Fluorescence microscopy III *The publications in parenthesis indicate the methods were conducted by the co-authors.

4. RESULTS AND DISCUSSION

4.1 Variation in the biocontrol properties of P. gigantea against Heterobasidion infection (I)

A total of 64 P. gigantea isolates, used for the experiment were collected from southern Finland and a few from other parts of Finland, Sweden, Latvia and Lithuania. The isolates were investigated for spore production, growth rate on agar medium and in spruce wood, competitive ability against Heterobasidion on agar medium and control efficacy against spore infection by Heterobasdion in stem pieces of spruce. The results revealed high variation in traits between the different P. gigantea isolates. The efficacy of control against Heterobasidion spp. and the growth rate in spruce wood were closely related to each other and showed positive correlation (r=0.727, p<0.001). This indicates that the growth rate of a P. gigantea isolate in spruce wood is the most important characteristic predicting its efficiency in controlling the infection and spread of Heterobasidion spp. Among the tested properties, the spore production ability showed the highest variation and had also negative correlation to the growth in spruce wood (p = 0.001).

Besides a sufficient number of P. gigantea spores on the surface of the stump, a very important factor in achieving control of the pathogen seems to be the number of Heterobasidion spores and the ratio between the control agent and the pathogen on the cutting surface (Berglund and Rönnberg, 2004). In our experiment, the proportion H ⁄ P (H.a/P.g) was ca. 1 ⁄ 2.5 (ca. 200 ⁄ 500 spores ⁄ cm2 on the cutting surface), which was higher than normally used ca. 1 ⁄ 10 (ca. 50 ⁄ 500 spores ⁄ cm2 on the cutting surface) (Möykkynen and Kontiokari, 2001; Korhonen, 2003). Compared with the results obtained in earlier experiments (Korhonen, 2003; Berglund and Rönnberg, 2004; Berglund et al., 2005), the control efficacy of the P. gigantea isolates in our study was exceptionally low. This is probably due to the fact that the proportion of H/P used is higher than what usually occurs in treated stumps in forests. This suggests that, in order to obtain satisfactory control of Heterobasidion in spruce stumps, a-ten-fold excess of P. gigantea spores is required.

The sampling depth may affect the observed control efficacy in billets and stumps. Berglund and Rönnberg (2004) noted that higher figures of efficacy were obtained from sample discs cut from the upper parts than from the lower parts of the (relatively long) stumps. Similar results were observed in our study. The growth rate of Heterobasidion in spruce wood was generally faster than that of P. gigantea (Holdenrieder and Greig, 1998), this could indicate that Heterobasidion hyphae has passed through the stump surface and the infections expand spatially in the lower parts of the stump (Redfern et al., 2001).

The efficacy test against Heterobasidion spp. in stem pieces of spruce showed no correlation between spore production capacity and the control efficacy. However, abundant sporulation is a characteristic of many P. gigantea strains (Rönnberg et al., 2006), which makes it easier to produce a satisfactory quantity

to be used in stump treatments, although it is not a good indicator of control efficacy. It is obviously necessary when preparing a spore-based control agent.

It is worthy to note that the control efficacy of both Rotstop (39.5%) and Rotstop S (36.6%) was lower than the mean efficacy of the tested isolates (46.8%). These results were rather different from that obtained in numerous earlier monitoring tests, where Rotstop isolates have performed well (Korhonen, 1993 and 2003; Berglund and Rönnberg, 2004). Generally, a relatively high variation is typical for the results obtained in different stump treatment experiments as the results are affected by many factors, such as moisture content on the stump surface, incubation period after inoculation and the sampling depth of the stumps. It is difficult, for example, to retain the same moisture content in different billets or stumps, and this may considerably affect the result from one experiment to another. Moreover, the number of tested isolates in our study was higher than in earlier experiments. This may also partly explain the low performance of Rotstop. Both the growth rate and the controll effect of P. gigantea isolates against Heterobasidion spp. were strongly dependent on the individual tree. There were significant differences (p < 0.05) between spruce individuals as a woody substrate for both P. gigantea and Heterobasidion.

4.2 Use of a breeding approach to improve the biocontrol properties of P. gigantea (II)

4.2.1 Fruiting of progeny isolates

By crossing the homokaryotic isolates from 10 parental P. gigantea strains, more than 95% of the P. gigantea progeny isolates (172 out of 180) could fruit after 2-3 months incubation and were distinctly heterokaryotic. No fruit bodies were observed in single-basidiospore cultures originating from parental heterokaryotic isolates used for the breeding. It is crucial that the progeny strains produced in pairings between single-basidiospore isolates were heterokaryons as the control efficacy of homokaryons is not known, and the homokaryons also degenerate easily (Garbelotto et al., 1996). The most useful indicator of heterokaryotic cultures is their ready fruiting after 4-6 weeks (Korhonen and Kauppila, 1988; Grillo et al., 2005). In our study, the progeny strains were identified as heterokaryons mainly on the basis of ready and abundant fruiting.

4.2.2 Properties of the progeny and parental isolates of P. gigantea

The same traits of P. gigantea isolates (mentioned in paper I) were investigated for both parental and progeny strains. Compared with parental isolates, the highest values in all the tested traits were observed in the progeny heterokaryons and the differences were particularly substantial in spore production ability and growth rate in spruce wood (see Table 1; Fig. 3). Almost half of the progeny strains (20 out of 50 tested isolates) had faster growth rates than that of the best parental isolate in spruce wood. The growth rate in spruce wood had positive correlation with the control efficacy of P. gigantea. This is consistent 22

with the results obtained in the pervious study (I): growth rate in spruce wood was the most important characteristic indicator trait for control efficacy against Heterobasidion spp.

Among the other traits investigated (spore production, growth rate on agar medium and growth rate over Heterobasidion colonies on agar medium), the progeny also showed better performance than the parental isolates. However, these traits did not directly correlate to control efficacy against spore infection by Heterobasidion, and they were to a lesser degree used as a basis for strain selection.

The results of tested traits for Rotstop were consistent with the results obtained in the earlier study (I), where Rotstop showed moderate performance and Rotstop S had poor growth rate (1.1 mm day-1) in spruce wood but with high spore production ability. The growth rate of the fastest progeny (3.6 mm day-1) was twice as fast as that of commercial Rotstop F (1.8 mm day-1) and 64% faster than the fastest of the parental strain (2.2 mm day-1).

The genetic variation appeared to represent the major part of the phenotypic variation for growth on agar medium alone (61%) and against Heterobasidion (69%) and spore production ability (45%). However, low heritability of growth rate in wood was observed and only about 20% variance was attributed to the growth in wood due to high variability of spruce wood tissue (II; Table 2).

4.2.3 Biocontrol efficacy of the progeny and parental isolates of P. gigantea

The progeny isolates showed better control efficacy than parental isolates in general, especially at higher Heterobasidion spore concentration (10 000 spores mL-1). Significant difference was observed at Heterobasidon spore concentration of 2000 spores mL-1. The control efficacy decreased with increasing Heterobasidion spore load for both progeny and parental isolates.

In the efficacy test, the number of P. gigantea spores in different treatment suspensions (between 3.9 and 9.3 million spores L-1) was in all cases well above the minimum amount that should be applied to spruce stumps [ca. 2 million spores L-1 (Korhonen and Kauppila, 1988)]. However, the concentration of Heterobasidion spores in three treatments was higher, with 300, 2000 and 10 000 sporesmL-1 (corresponding to ca. 30, 200 and 1000 spores cm-2). Under such high infection pressure, the control efficacy of P. gigantea was relatively low. This is consistent with the result obtained in the former investigation (I) and the assumption of Berglund and Rönnberg (2004) that the poor control effect of Rotstop in their experiments was due to the exceptionally high spore load of Heterobasidion.

So far, there is no report on fungal biocontrol agent selection using conventional breeding methods despite long history and successful applications of using fungi for biological control in agriculture and horticulture (Baek and Kenerley, 1998; Rey et al., 2001; Lübeck et al., 2002; Nobre et al., 2005). In our 23

study, the progenies of P. gigantea obtained through breeding showed better control efficacy than parental strains at the different Heterobasidion spore loads applied. Several progeny strains showed relatively high control efficacy even under high spore concentration of Heterobasidion. These could be promising strains that might later replace the current commercial Rotstop isolates of P. gigantea in the future. Moreover, analysis of the component of variation within progeny showed that genetic component accounts for the major part of the phenotypic variation in most of the tested traits of P. gigantea. The breeding method used in this study is an alternative approach for enhancing the efficacy of the biocontrol agent.

4.3 The potential of P. gigantea to acquire necrotrophic capability (III)

By using a combination of histochemical, molecular and transcript profiling (454 sequencing), we investigated the responses of conifer tree to the saprotrophic P. gigantea. The results were compared with those of the pathogenic H. annosum in order to evaluate the potential of P. gigantea to acquire necrotrophic capability through adaptation to living wood tissues. The results revealed that P. gigantea provoked a very strong necrotic reaction in both phloem and xylem on the host, as much as the pathogenic H. annosum at the initial stages of infection. For unknown reasons, however, the necrosis caused by P. gigantea shrank after initial colonization and did not enlarge further. This may indicate that necrotrophs and saprotrophs present different signals to the living cells of the bark and xylem (Asiegbu et al., 1999; Adomas et al., 2008). This further suggests that P. gigantea has very limited potential to acquire necrotrophic capability.

The cell death assay confirmed that H. annosum killed more cells and displayed a typical necrotrophic habit during colonization. However, the necrotic dead cells formed due to challenge with P. gigantea as well as with wounding ceased to expand. This was supported by emergence of new sets of living cells around the necrotic regions (III; Fig. 5). This also suggests possible differences in the structure and perception of the secreted proteins (or effectors) from these two closely related fungi by the tree host.

Several genes involved in cell death regulation were highly transcribed in P. gigantea treatment as well as in wounded tissues (e.g. ATP-binding cassette transport protein, elongation factor-1 alpha and cysteine proteinase) (III; Table 4). Of these, the gene encoding cysteine proteinase is known to be involved in programmed cell death in multi-cellular organisms and plays a critical regulatory role in cell death regulation (Talapatra et al., 2002; Torres et al., 2005; Trobacher et al., 2006; Kobae et al., 2006; Kim et al., 2008). It has also been reported in wound-induced proteins triggered by insect attack (Howe and Jander, 2008). Additionally, the high abundance of transcripts involved in late embryogenesis in both P. gigantea and wounding treatments (III; Table 4) may indicate a participatory role in the cell regeneration process. The common unifying trait for the presence of these proteins is their association with biotic and

abiotic stress. Their actual function at the molecular level is still not fully understood (Hundertmark and Hincha, 2008; Hunault and Jaspard, 2010).

4.4 Induced resistance as a mechanism of P. gigantea control against Heterobasidion root rot (III)

Pre-treatment with P. gigantea or wounding followed by inoculation with H. annosum restricted further necrosis formation (III; Fig. 4a). By contrast, necrotic lesions significantly increased after Heterobasidion inoculation in tissues pre-treated with the pathogen or in fresh wounds (III; Fig. 4a). Elevated transcript levels of genes important for cell lignification, cell death regulation and jasmonic acid (JA) signaling were detected in host tissues pre-treated with P. gigantea. The results suggest that induced localized resistance caused by prior inoculation with P. gigantea on freshly-cut stumps is a contributory factor for the biocontrol efficacy of P. gigantea. The pre-treatment of tissues with P. gigantea delayed the penetration of H. annosum compared to tissues with fresh wounds or pre-treated with H. annosum (Fig. 4). Host tissues pre-treated with P. gigantea had slightly greater numbers of lignified cells (Table 1) and subsequent inoculation with H. annosum on host tissues pre-treated with P. gigantea led to further increases in lignified cells (III; Table 1). The formation of lignified bark necrosis due to pre-challenge with P. gigantea was able to protect the living conifer tissues from further invasive hyphal growth. In contrast, the necrotrophs induced a very rapid necrosis, but did not allow the synthesis of lignin in amounts necessary to restrict invasive hyphal growth. Notably, logging creates similar, major defense priming on the cells that are alive in the stumps. It is possible that the inoculation of P. gigantea on the fresh stump further boosts this effect. All these induced defenses take place in sapwood and phloem as these are the only tissues with living parenchyma, heartwood being completely devoid of living cells and therefore unable to mount induced responses. So the effects of P. gigantea would be restricted to sapwood in this context.

Induced resistance has been well studied in many horticultural and agricultural systems (Vallad and Goodman, 2004; Walters, 2009), but only few reports are available on inducible chemical defences (e.g. secondary metabolites) on forest tree (Keeling and Bohlmann, 2006). Inducible protein-based defences (e.g. pathogenesis-related proteins (PR protein), anti-microbial peptide (AMP), thaumatin and peroxidase) have been shown to be rapidly induced both locally and systemically (Sels et al., 2008) and inducible anatomical defences including cell wall modification have also been documented (Huckelhoven, 2007; Krokene et al., 2008). Lignin precursors such as phenolic compounds are a major class of inducible defences in many woody species, including conifers (Franceschi et al., 2005; Blodgett et al., 2007; Witzell and Martin, 2008). Many studies have demonstrated that the accumulation of certain soluble phenolic compounds and enhanced lignin deposition occurs in induced resistance (Boldgett et al., 2007; Wallis et al., 2008). In P. gigantea-inoculated tissues, several of the induced genes were involved in secondary metabolism, particularly genes encoding enzymes active in lignin biosynthesis (DAHP and SAM synthase) 25

(III; Table 4). Induced lignification and suberization of cell walls are known to contribute to cell wall strengthening (Eyles et al., 2003) and have been reported during pathogen attack (Bonello and Blodgett, 2003). The high abundance of the transcript for DAHP and SAM synthase genes as well as the high number of lignified cells in P. gigantea-treated tissues probably suggests important roles in preventing invasive growth of H. annosum.

A higher transcript abundance for genes (ATP-binding cassette transport gene and pleiothropic drug resistance gene) encoding proteins involved in the jasmonic acid (JA) signaling pathway (III; Table 4) in P. gigantea treated tissues than H. annosum treated tissue further support the hypothesis of rapid signal perception with consequent restriction of hyphal growth due to host defences. It is possible that the necrotrophic H. annosum continuously killed the host cells in advance of the hyphal front, whereas toxic host metabolites induced due to challenge with the saprotroph restricted its invasive growth. JA is one of the key mediators of the regulatory pathway in defence signalling networks (Balbi and Devoto, 2008). It can induce enhanced localized resistance to biotic agents (Heijari et al., 2005; Krokene et al., 2008) or the formation of traumatic resin ducts (TRDs) in conifers (Miller et al., 2005; Erbilgin et al., 2006; Zeneli et al., 2006). In addition, methyl jasmonate (MeJA) could also induce the accumulation of phloem phenolic compounds on ash trees (Eyles et al., 2010). The pleiotropic drug resistance gene in this pathway with high transcript levels is known to induce the secretion of secondary metabolites that could inhibit the growth of invading organisms. Another gene encoding the ATP-binding cassette transport protein was also observed, and has been reported in both constitutive and jasmonic acid-dependent induced defences (Stukkens et al., 2005). Together, these results suggest that rapid recognition coupled with signal perception led to active and effective host defences against wounding and P. gigantea infection.

The results of the RT-PCR analysis revealed that the genes involved in host defense (e.g. thaumatin, PR- 10 and AMP) were upregulated within the first week after inoculation with P. gigantea, but this increase was attenuated after a prolonged infection of eight weeks. This result is consistent with the findings of a previous study (Adomas et al., 2008) on the attenuation of host transcript responses after prolonged incubation with non-pathogens. The induction of defence genes (SAMS, DAHP, cysterine proteinase, pathogen-related protein) by secreted substances from the hyphae without physical contact with host tissues further confirms that P. gigantea is able to release compounds resulting in a very rapid activation of genes for enzymes involved in plant defences as well as in lignin biosynthesis.

4.5 The impacts of treatment with biocontrol fungus (Phlebiopsis gigantea) on bacterial biota of Norway spruce stumps (IV)

4.5.1 Bacterial community richness and diversity in stumps of P. abies

After trimming and quality control of the sequences, a total of 123 562 sequences (83% of the total 154 453 raw sequences) and 4386 – 8902 sequences per sample (mean = 6939 ± 663) were obtained (IV; Table S1). By pooling the sequences from all the three replicates in each site, we obtained about 20 000 sequences per treatment in each site (ranged from 17 801 – 23 397) and used for comparative analysis between treated and control samples as well as for the successional changes over time (IV; Table S1).

We used both an OTUs-based (Schloss et al., 2009) and a naïve Bayesian approach (Wang et al., 2007) to determine the bacterial richness in the stump of P. abies after treatment with P. gigantea. All of the sequence reads (123 562) with the exception of two could be assigned to the Bacteria Domain with a confidence threshold of 80%. The observed OTUs at genetic distance of 3% sequence dissimilarity in each treatment ranged from 2 285 to 4 431 (IV; Table 1). However, the sequencing effort was not large enough to capture the complete diversity of these communities, as the rarefaction curves do not reach the plateau phase (IV; Fig 1). Only 42.4 to 52.0% and 27.0 to 36.2% of the estimated richness were recovered by the sequencing effort for Chao 1 and ACE, respectively (IV; Table 1).

The analysis of the observed OTUs and non-parametric estimators of richness (Chao 1 and ACE) showed that less number of OTUs was obtained in treated stumps compared to control stumps in site 1 (1-year after treatment) and site 3 (13-year after treatment). However, significant difference was observed only in site 1and not in site 3. This suggests that P. gigantea treatment had negative impact on the bacterial richness at the initial decay stage.

At a genetic distance of 3%, Shannon index showed high bacterial diversity across all the samples and ranged from 5.1 to 6.9 (IV; Table S1). The highest bacterial diversity was found in site 3, followed by site 2 and site 1. Within site 1 and 3, the bacterial diversity in treated stumps was relatively lower than that in control stumps. However, there is no significant difference in diversity between the control and treated samples within each site. This probably suggests that P. gigantea treatment has no negative effects on the overall diversity of bacteria in the stump of P. abies.

4.5.2 P. gigantea treatment and bacterial diversity at different stages of stump decay

The number of unique OTUs in treated stump and shared OTUs with control stumps increased over time after P. gigantea treatment (IV; Figures 2 and 3). This suggests that the bacterial community recovered from P. gigantea treatment and the effect of P. gigantea gradually attenuated over time. Previous studies have shown that P. gigantea could persist in the stump for 6 years as it was hardly detected afterwards 27

(Vainio et al., 2001). Vasiliauskas et al. (2004 and 2005) reported that species richness of fungi was lower in P. gigantea-treated stumps after 1-, 4- and 6-year treatment and the occurrence of other fungi increased overtime. On the other hand, the bacterial richness in the control stumps without P. gigantea treatment in our study was found to increase over time. This may further indicate that the overall bacteria communities in the stumps increased following the decay processes of the stump (initial-, intermediate- and advanced-stage of stump decay). Moreover, Shannon index showed the diversity of bacteria in the stump of P. abies continuously increased at advanced stages of stump decay.

4.5.3 Interaction between site, age of stump and P. gigantea treatment on the bacterial biota

The 123 562 sequences classified below domain level, in which more than 92% of total sequences were classified into bacterial phyla, were affiliated to 12 bacterial phyla, 24 classes and 160 genera (IV; Table S3, S4 and S5). The most abundant phyla across all samples were Proteobacteria and Acidobacteria followed by Bacteroidetes, Cyanobacteria and Actinobacteria. The members of rare phyla (≤ 0.5% of all classified sequences) contained Planctomycetes, Verrucomicrobia, OP10, TM7, Firmicutes, Gemmatimonadetes and Bacteria_incertae_sedis (IV; Figure 4 and Table S3). However, the high proportion of unclassified sequences at genus level (39-60%) suggests the large diversity of bacteria in the stumps still remains unknown.

The relative abundance of each bacteria phylum varied between treated and control stumps within each site (IV; Figure 4 and Table S3). The abundance of Proteobacteria in treated stumps was lower compared to the control stumps within site 1 and 3, but was higher in site 2 (IV; Figure 4 and Table S3). In contrast, the abundances of Acidobacteria in treated stump showed the opposite pattern to Proteobacteria in each site (Figure 4 and Table S3). The analysis of the abundance of the bacteria groups showed Proteobacteria and Acidobacteria were the two most dominant phyla in all samples, accounting for more than 40% and 20% of the total sequences in P. gigantea treated samples respectively (IV; Figure 4 and Table S3). The result was consistent with the study done by Valášková et al. (2009), which showed Protecobacteria and Acidobacteria were the most abundant groups coexisting with the fungus Hypholoma fasciculare in decaying wood.

Bacteroidetes appeared equally in treated and control stumps within each site, respectively (IV; Figure 4 and Table S3). This may indicate that this group was not affected by P. gigantea treatment. Compared to the control stump, the relative abundance of Cyanobacteria in treated stump was higher in site 1 and 3, but was lower in site 2. The Actinobacteria had relatively higher abundance (>4%) in both control and treated stumps in site 2 and 3, compared to site 1 (<1%) (IV; Figure 4 and Table S3). The rest of the bacteria groups presented in all samples remained at low abundance (<0.5%). Moreover, No significant

differences in abundance between the treated and control stumps were observed within each site, except Acidobacteria in site 1 (P =0.04).

At genus level, the most abundant genus (>1%) was Acidobacteria-Gp1, followed by Pseudomonas, Burkholderia, Sphingomonas, Dyella, Mucilaginibacter, Duganella, Methylobacterium, Acidisoma and Janthinobacterium (IV; Figure S4 and Table S5). Most of the genera mentioned above, except Acidobacteria-Gp1and Acidisoma, had relatively high abundance in site 1 and low abundance in site 2 and 3. However, significantly higher abundance was only observed for Acidobacteria-Gp1 (p =0.05) in site 1 in treated stumps compared to control stumps and no significant differences were observed for the rest of genera within each site. Genus Burkholderia was one of the most abundant bacteria in all samples at different sites (IV; Figure 7). Burkholderia is known to be efficient mineral weathering and nitrogen- fixing bacteria (Uroz et al., 2007 and 2009; Leveau et al., 2010). It has been isolated from the white-rot fungus, Phanerochaete chrysosporium (Lim et al., 2003). It was also reported to be a very efficient degrader of aromatic compounds derived from lignin degradation by white-rot fungi (Seigle-Murandi et al., 1996). It was also found to be associated with the root of hybrid aspen (Yrjälä et al., 2010) and more related to rizhosphere than in the soil (Uroz et al., 2010). No significant differences in the abundance for this genus between the treated and control stumps were observed. This suggests that Burkholderia might play important role in the decay process in the stump and that P. gigantea treatment had no effect on this particular group.

Genus Pseudomonas was found with relatively high abundance (>8.9%) in stumps after 1-year, but with a very low level abundance after 6- and 13-year (IV; Figure 7 and Figure S4). It is possible that Pseudomonas was more sensitive to changes in the substrate structure and nutrient availability at advanced stage of decay, which probably led to a decrease in the abundance of this group. The well-known ability of Pseudomonas to degrade phenols and aromatics has been reported (Kamath et al., 2004). Initial decay of the woody-tissue in the stump produces more phenolic compounds, which could favour more Pseudomonas bacteria.

4.5.4 Successional changes in bacterial community composition during stump decay

The successional pattern of the bacterial group differs from each other in the stumps over time. In treated stumps, Proteobacterial population declined with the progress of stump decay whereas Acidobacteria increased at advanced stages of the decay (IV; Figure 5 and Table S3). High abundance of Proteobacteria has previously been associated with high availability of carbon (McCaig et al., 1999; Fazi et al., 2005; Fierer et al., 2007). On the stump surface, woody-tissue harboured many microbes and served as nutrient sources. Consequently, the stumps are subjected to different stage of decay over time, which causes the change in nutrient composition as well as in microbial biota. Both density and moisture content of the stump wood are likely to influence bacteria community. Density of stump wood decreased with age, 29

whereas moisture content (fresh moisture or relative moisture) have been reported to increase (Murray and Woodward, 2007). Fewer nutrients are available in the stumps at advanced stages of the decay process. The decrease in the availability of C in the stump over time might have contributed to the change in relative abundance of the Proteobacteria.

The Acidobacteria are also commonly found in soils (Neufeld and Mohn, 2005; Roesch et al., 2007) and are known to grow under oligotrophic conditions (Dion, 2008). Previous study has shown that the abundance of Acidobacteria had negative correlation with carbon availability (Fierer et al., 2007).The abundance of Acidobacteria over time may reflect the low levels of the available nutrients and perhaps acidic conditions on the stump due to decay caused by wood-decay fungi. Moreover, higher abundance of this group were observed in treated stumps after 1-year and 13-year treatment, but significant differences in abundance between treated and control stumps existed only after 1-year. This suggests that P. gigantea treatment has positive effect on this group at initial stage of decay and the population of Acidiobacteria significantly increased after P. gigantea treatment.

Interestingly, Actinobacteria had lowest abundance (0.5%) after 1-year treatment, but increased to 11.2% during intermediate stages of stump decay and declined to 4.3% at advanced stage of decay. Bacteroidete and Cyanobacteria had relatively high abundance (>4.0%) at initial decay stage, but decreased at intermediate stages of decay, which later increased in highly decayed stumps in the 13-year treated stumps. The rest of the bacteria groups remained at low abundance (<0.5%) across the different time points. Moreover, the shift in population of each bacterial group also occurred at different stages of decay (IV; Figure 5). For example, the abundances of the bacterial group at initial stage of decay was as follows: Proteobacteria > Acidobacteria > Bacteroidetes > Cyanobacteria > Actinobacteria, intermediate stage of decay: Proteobacteria > Acidobacteria > Actinobacteria > Cyanobacteria > Bacteroidetes and advanced stage of decay: Acidobacteria > Proteobacteria > Actinobacteria > Cyanobacteria > Bacteroidete. In addition, the redundancy analysis of relative abundances of bacterial phyla with respect to different stages of decay revealed significant differences between initial and intermediate stage of decay for Actinobacteria, between initial and advanced stage of decay for Proteobacteria, Acidobacteria and Actinobacteria (IV; Figure 6 and Table S3). The same results were supported by non-parametric multidimensional scaling analysis (data were not shown).

The population of Bacteroidetes shifts between different stages of decay in the stump and decreased after initial stage of decay. Members of Bacteroidetes have been implicated in the degradation of lignocellulosic litter due to their ability to degrade complex biopolymers (Martínez-Alonso et al., 2010). On the other hand, Murray and Woodward (2007) found that the abilities of bacteria to degrade different substrate did not correlate strongly with age of the stump from which they were isolated. Besides changes in the nutrient content in the stump along with advanced decay process, other factors may also affect the 30

population shift of this group over time. The fluctuation in the abundance of Cyanobacteria in the stumps was also observed (IV; Figure 4 and Table S3). As Cyanobacteria were considered to play a fundamental role in soil nutrient cycling in fluctuated environments (Blume et al., 2002), the different decay process in the stump over time may have contributed to the fluctuation. The aerobic Actinobacteria were the most common initial colonizers in decaying wood (Lynd et al., 2002). Interestingly, in our study, Actinobacteria had relatively higher abundance at intermediate and advanced stages of decay in the stump, but with low population at the initial stage of stump decay.

At genus level, Acidobacterial GP1 was the most abundant genus at different stages of decay and the population increased over time in treated stump (IV; Figure 7). However, significantly higher abundance was only observed after 1-year treatment in treated stumps. This indicates P. gigantea treatment had strong promoting effect on this group at initial stage of decay. On the other hand, not much is known about the physiology and metabolic functions of Acidobacterial GP1. In addition, many genera had relatively high abundance in the stumps at initial stage of decay, but the abundance decreased to a very low level at intermediate and advanced stage of decay, such as Sphingonomas, Dyella, Mucilaginibacter and Dugnella (IV; Figure 7). Thus, the shifts in stump bacterial community composition may correlate with a change of decay process, in which wood-decay fungi were involved. The negative effect of white-rot fungi on bacteria in the stump wood might prevent bacteria from competing for easily utilizable wood nutrients and low-molecular-weight compounds released by fungal extracellular enzymes. Previous studies have demonstrated that the selective effects of the white-rot basidiomycete on the bacterial community were stronger during initial decay than at later phases (Folman et al., 2008; Valášková et al., 2009). This may explain the abundance as well as the decrease of certain bacteria groups. Notably, the stump harbours a complex set of microbes, such as wood-decay fungi and is subject to varying abiotic environment. The change in fungi diversity in the stump might also indirectly influence the bacteria community. Allison and Martiny (2008) also showed that changes in the composition of microbial communities are often associated with changes in the process rates of ecosystems. Competition between bacteria and wood-decay fungi may also influence establishment and subsequent growth of the major decay–causing agent in the stump (Murray and Woodward, 2003). Therefore, to fully understand how the bacterial communities in the stumps are influenced by P. gigantea, it is also necessary to draw a bigger picture, in which all the factors involved in the decay process are taken into account.

5. CONCLUSIONS

In this thesis, the efficacy of P. gigantea against Heterobasidion root rot and its impacts on conifer tree (P. sylvestris) defences and bacterial biota on the stumps of P. abies were studied. The P. gigantea traits (asexual spore production, growth rate, competitive ability) which are potentially important for biocontrol efficacy against H. annosum infection were evaluated. There was a positive correlation between the growth rate of P. gigantea isolates in wood and their biocontrol efficacy against H. annosum (paper I). Therefore the determination of the growth rate of P. gigantea in wood was considered a very useful trait in screening of isolates for relative biocontrol efficacy.

In a follow-up article (paper II), homokaryotic isolates of P. gigantea were paried and the resulting heterokaryotic progeny were compared with the parental isolates for biocontrol related traits. The progeny of P. gigantea isolates obtained through breeding approach had generally better properties and control efficacy against Heterobasidion infection compared to the parental isolates. The genetic effects accounted for a part of the variation between progeny and parental strains. Breeding approach was therefore considered as an optional tool for the study and development of biological control agents in general.

In paper III, the necrotrophic capability of P. gigantea and induced localized resistance as a mechanism for its biocontrol action were investigated. The results revealed that P. gigantea comparatively appears to have a very limited necrotrophic capability, despite its intensive use in managed forests, and that induced localized resistance is a contributory factor for the biocontrol efficacy of P.gigantea.

The fourth article (paper IV) evaluated impacts of P. gigantea treatment on bacteria biota of conifer stumps. Although the treatment of P. gigantea significantly decreased the bacterial richness in the spruce stumps at the initial stage of decay, over time, the bacterial community gradually recovered and the negative effects of P. gigantea was attenuated. Overall the results were considered relevant and provide additional insight on the risk assessment as well as environmental impact on the long-term use of P. gigantea in the control of Heterobasidion root rot in conifer forests.

6. FUTURE PERSPECTIVES

P. gigantea has for a long time been known as a strong competitor against H. annosum (Rishbeth, 1952); however, very little is known about the physiological and molecular basis of the interspecific interaction between these two fungi. Recently, Adomas et al. (2006) reported that a diverse range of genes encoding proteins important for nutrient acquisition were preferentially expressed during the interaction between P. gigantea and H. annosum. Isolate-specific differences in gene expression could be used to identify candidate genes important for the efficacy of P. gigantea used for biocontrol of H. annosum. The

development of DNA transformation system for P. gigantea will facilitate functional knockout assays that can be used to validate the importance of such candidate genes.

The use of the transcriptome approach offers the possibility for global analysis of gene expression to unravel the molecular basis of interaction between diverse fungal species. High-throughput sequencing (454 pyrosequencing) has proven to be well adapted for analyzing the transcriptome of both model and non-model species (Morozova and Marra 2008; Parchman et al., 2010). This provides the possibility to investigate transcriptomic profile of P. gigantea isolates with different biocontrol efficacy from that of the wild type and progeny isolates and to identify candidate genes contributing to the control efficacy as well as genes that are affected by the breeding process. Identifying the genes affected by the breeding process should be a high research priority. The availability of whole genome sequences of P. gigantea could also help to facilitate a large-scale transcriptomic study. Moreover, to scan potential genes contributing to the control efficacy of P. gigantea against Heterobasidion, it would be necessary to differentially screen considerably higher number of isolates of P. gigantea. Additionally, whole genome sequencing of P. gigantea will enable comparative genome analysis of progeny isolates with good biocontrol ability against non-effective isolates.

Large-scale applications of a biological control agent in forest ecosystems inevitably involve potential risk as well as impact on non-target organisms. Besides bacteria, wood-inhabiting fungi are another important community on the stump surface. This should be equally taken into account in terms of the mycobiota affected by P. gigantea.

ACKNOWLEDGEMENTS

It has been a long process to write this dissertation and it would not have been possible without the guidance, help and support from numerous people. Thus, I owe my gratitude to all those who have made this dissertation possible over the last few years.

This work was carried out during the year 2006 to 2011 at the Department of Forest Sciences, University of Helsinki and at the Vantaa Research Unit, Finnish Forest Research Institute (Metla). I wish to thank Prof. Jarkko Hantula at Metla and Prof. Fred. O. Asiegbu at the University of Helsinki for providing excellent research facilities.

My deepest gratitude is to my supervisor Prof. Fred. O. Asiegbu for excellent guidance. Not only was he readily available for me, as he so generously is for all of the students, but he always answer the questions, read and responded to the writing of my work more quickly than I could have hoped. He has taught me the way of scientific thinking and how to question thoughts and express ideas. Dr. Risto Kasanen, my co- supervisor, has always been there to listen and give advice to me. I am deeply grateful to him for helping me financially starting with the project. I am also thankful to him for believing in me and gave me the freedom to explore on my own. Dr. Kari Korhonen is one of the best researchers that I have had in my life. I am indebted to him for teaching me how to do research and most importantly, how to hold right attitude to life.

I am grateful to the pre-reviewers, Prof. Richard Hamelin and Dr. Ari Hietala, for their valuable suggestions to improve this thesis. I thank to Docent Frederick Stoddard, for the linguistic revision of the thesis. I also wish to thank other co-authors of the papers, Dr. Edward Alatalo, Dr. Lars Paulin, Eeva Terhonen, Kaisa Koskinen, for their invaluable expertise and contributions to the papers.

It would have been a lonely lab without so many people helping me in the laboratory. I am thankful to the former and current members of the Forest Pathology group to create such enjoyable atmosphere in and out of the lab. It has been honour and pleasure to work with all of you. Particularly, I am indebted to Anna-Maija, Susanna, Tommaso and Tony for improving the thesis with valuable suggestions; to Lu, Chen, Chaowen, Eeva, Susanna, Nicolas, Monica, Nevena and Jiayao for sharing ideas and life over lunch time; to Susanna, I will definitely remember the ´Dynamic network´; to Eeva for being such a nice working and office mate. Many people in other research groups assisted and encouraged me in various ways. I am especially grateful to Marjo, Katrin, Linda, Tuuli, Mikko, Isa, Tian, Sidona, Xianbao, Shahid and Anssi in Plant Pathology group. Especially, I owe gratitude to Marjo for her friendship and encouragement as well as always having time for discussions on everything. I am also thankful for having received much assistance from Marjut in the lab. In addition, I also would like to thank Jukka Lippu at Department of Forest Sciences for administrative and practical help.

Many friends have helped me stay sane through this difficult process. Their support and care helped me overcome setbacks and stay focused on the study. My studies would not have been the same without them. I deeply appreciate the support and welcome I have received over these years from Rana Sinha and Seija Sinha. Thanks for taking me as a family member and always believing in me that I can make it. I am also grateful to Tanja and Ben for bringing so much energy and dragging me out of the work and being such good friends. I am particularly thankful to Susanna, Jan and Anton for understanding and always supportive. I also would like to thank Teija and Esa for their friendship and I still need to continue practicing the Karaoke.

Finally, none of this would have been possible without the love and patience of my family. I would like to express my gratitude to my parents and elder sisters in China for their love and support. I also owe my deepest gratitude to my Finnish family. I am truly indebted and thankful to Olli, Anneli, Marko, Marika and Raija for love, patience, strength and understanding.

Helsinki, November 2011

Hui

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