The Effect of Fungicide Treatment on the Non-Target Foliar Mycobiota of Pinus Sylvestris- Seedlings in Finnish Forest Nursery

The effect of fungicide treatment on the non-target foliar mycobiota of Pinus sylvestris- seedlings in Finnish forest nursery

Master‟s thesis

Ninni Klingberg

University of Helsinki

Department of Forest Sciences

Forest Ecology

May 2012

Acknowledgements

I would like to thank my supervisors Professor Fred Asiegbu, Docent Risto Kasanen and Researcher Marja Poteri from Metla for providing me this interesting thesis topic. I am grateful for all the constructive comments concerning the manuscript and for the patience towards the “silent times” when other matters were taking my focus and time.

Especially I would like to thank Marja Poteri. I want to thank her for helping me with the establishment of the field experiment and all the guidance and support provided during this long journey. Also I want to express my gratitude with the guidance in statistical analysis and for answering all of my many questions.

I want to thank the Forest Pathology group and especially Eeva, Hui, Ximena and Tommaso. Your help and practical guidance in the research lab made my molecular work phase a little bit more tolerable despite of all the misfortune I faced with the processing of the samples. I would like to give special thanks to Eeva for the persistence in encouraging me at the moment of despair.

I would like to thank the Faculty of Agriculture and Forestry for providing the chance to write in the “thesis clinic”, graduklinikka. That was the place where my thoughts were quite clear and written text was born.

I would like to thank my family and friends for all the support and peace that they have giving me during this thesis “journey”. Above all I can‟t thank enough Miika, who kept me together during my most difficult times and pushed me forward to complete this thesis.

HELSINGIN YLIOPISTO  HELSINGFORS UNIVERSITET  UNIVERSITY OF HELSINKI

Tiedekunta/Osasto  Fakultet/Sektion  Faculty Laitos  Institution  Department Faculty of Agriculture and Forestry Department of Forest Sciences

Tekijä  Författare  Author Ninni Marjaana Klingberg

Työn nimi  Arbetets titel  Title The effect of fungicide treatment on the non-target foliar mycobiota of Pinus sylvestris- seedlings in Finnish forest nursery

Oppiaine Läroämne  Subject Forest Ecology

Työn laji  Arbetets art  Level Aika  Datum  Month and year Sivumäärä  Sidoantal  Number of Master‟s Thesis May 2012 pages 80 + appendices

Tiivistelmä  Referat  Abstract

Fungi are the major causal agents of several plant diseases. Fungicides are regularly used in forest tree nurseries to protect and eradicate fungal pathogens. However, the use of fungicides can create problems such as the alteration of natural fungal communities in the upper and lower part of the seedling, and fungicide resistance. These factors may lead to new disease problems in the nursery. Excessive use of fungicides is harmful to environment and might prevent the emergence of novel beneficial fungal species. Some foliar endo- and epimycota are known to suppress fungal diseases and protect the host from herbivoria and abiotic stress. The aim of this study was to investigate if routinely used fungicide (Tilt 250 EC propiconazole as an active ingredient) against Scleroderris canker (Gremmeniella abietina) has side-effects on the non-target foliar mycobiota as well as on the height growth of Scots pine (Pinus sylvestris) seedlings in a Finnish forest nursery. The experiment was conducted in a Finnish forest tree nursery during one growing period. Altogether 100 needles were sampled which resulted in a total of 186 fungal endophytic isolates, and 40 needles sampled resulted in a total of 86 epiphytic isolates. Endophytic isolates were further analysed and assigned to 37 operational taxonomic units (OTUs). Phoma spp. were the most frequently isolated OTUs in both treatments. There were no statistically significant differences between mycota isolated from fungicide treated and control seedlings (except between epiphytes in September), however there were quantitative and qualitative differences which was mainly seen in the higher number of exclusive fungi in control seedlings. There were no statistically significant differences between the growth of fungicide treated and control seedlings but fungicide treated seedlings grew faster at the end of the growing season. These results suggests that fungicide treatment has side-effects on the non-target foliar mycobiota and the growth of Scots pine seedlings.

Avainsanat  Nyckelord  Keywords Foliar mycobiota, diversity, Pinus sylvestris, forest tree nursery, fungicide, propiconazole

Säilytyspaikka  Förvaringsställe  Where deposited University of Helsinki: Department of Forest Sciences and Viikki Science Library

Muita tietoja  Övriga uppgifter  Further information Supervisor: Professor Frederick Asiegbu. Assistant supervisors: Docent Risto Kasanen, Researcher Marja Poteri (Finnish Forest Research Institute) and M. Sc. Eeva Terhonen

HELSINGIN YLIOPISTO  HELSINGFORS UNIVERSITET  UNIVERSITY OF HELSINKI

Tiedekunta/Osasto  Fakultet/Sektion  Faculty Laitos  Institution  Department Maatalous-metsätieteellinen tiedekunta Metsätieteiden laitos

Tekijä  Författare  Author Ninni Marjaana Klingberg

Työn nimi  Arbetets titel  Title Fungisidin käytön vaikutukset männyn (Pinus sylvestris) taimen neulasen sieniin suomalaisella metsäpuutaimitarhalla

Oppiaine Läroämne  Subject Metsäekologia

Työn laji  Arbetets art  Level Aika  Datum  Month and year Sivumäärä  Sidoantal  Number of Maisterin tutkielma Toukokuu 2012 pages 80 + liitteet

Tiivistelmä  Referat  Abstract

Sienet ovat pahimpia kasvien ja puiden taudinaiheuttajia. Metsäpuiden taimitarhoilla käytetään fungisideja estämään ja hävittämään taimien patogeenejä. Fungisidien käyttö voi kuitenkin kehittää ongelmia, kuten luonnollisen sieniyhteisön muuttumisen taimen maanpäällisissä osissa sekä juuristossa, ja patogeenien resistanssin käytetyille fungisideille. Nämä tekijät voivat kehittää uusia tautiongelmia taimitarhalla. Fungisidien liiallinen käyttö on haitallista ympäristölle, sekä sienimyrkkyjen rutiininomainen käyttö voi estää uusien hyödyllistem sienilajien ilmaantumisen. Puiden neulasten ja lehtien endo- ja epifyyttisienten tiedetään torjuvan sienitauteja ja tuhohyönteisiä sekä edistävän isännän abioottisen stressin sietokykyä. Tämän tutkimuksen tarkoitus oli selvittää onko metsäpuiden taimitarhoilla rutiininomaisesti käytetyllä fungisidilla (Tilt 250 EC, propikonatsoli vaikuttava aine) sivuvaikutuksia männyn (Pinus sylvestris) taimen neulasen endo- ja epifyyttisieniin sekä taimen pituuskasvuun. Kenttäkoe suoritettiin FinForelian taimitarhalla Nurmijärvellä yhden kasvukauden aikana. Sadasta analysoidusta neulasesta eristettiin yhteensä 186 endofyyttisientä ja 40 neulasesta eristettiin 86 epifyyttisientä. Endofyyttisienet analysoitiin molekulaarisin menetelmin ja tuloksena saatiin 37 eri lajia/sukua (OTU, Operational Taxonomic Unit). Yleisimmät ja runsaimmat endofyyttisienet fungisidillä käsitellyissä sekä kontrollitaimissa olivat Phoma- lajit. Endo- ja epifyyttisienien frekvensseissä sekä lajeissa ei ollut tilastollisesti merkitsevää eroa käsittelyjen välillä (paitsi epifyyttifrekvensseissä syyskuussa) mutta määrällisiä ja laadullisia (eksklusiiviset lajit) eroja oli havaittavissa. Myöskään taimien pituuksissa käsittelyjen välillä ei ollut tilastollisesti merkitsevää eroa mutta fungisidillä käsitellyt taimet kasvoivat paremmin kuin kontrollitaimet kasvukauden loppupuolella. Tulokset antavat viitteitä siitä, että fungisidin käytöllä on sivuvaikutuksia männyn taimen neulasen sieniyhteisöön sekä taimen pituuskasvuun.

Avainsanat  Nyckelord  Keywords Endofyyttisieni, epifyyttisieni, neulanen, monimuotoisuus, Pinus sylvestris, metsäpuiden taimitarha, fungisidi, propikonatsoli

Säilytyspaikka  Förvaringsställe  Where deposited Helsingin Yliopisto: Metsätieteiden laitos, Viikin tiedekirjasto

Muita tietoja  Övriga uppgifter  Further information Ohjaaja: Professori Frederick Asiegbu. Avustavat ohjaajat: Dosentti Risto Kasanen, tutkija Marja Poteri (Metsäntutkimuslaitos) ja MMM Eeva Terhonen.

Table of contents

1 Introduction ...... 1 1.1 The ecological significance of mycobiota ...... 1 1.2 Endophytes ...... 2 1.2.1 Definition ...... 2 1.2.2 Diversity and abundance ...... 3 1.2.3 Ecological significance ...... 4 1.3 Pesticides ...... 8 1.3.1 Fungicides ...... 9 1.3.2 Use of pesticides in Finland ...... 11 1.3.3 Side-effects of propiconazole on non-target organisms – previous studies ...... 14 1.4 Aims of the study ...... 16 2 Materials and methods ...... 17 2.1 The forest tree nursery and the experimental design ...... 17 2.2 The fungicide treatment ...... 20 2.3 Measurement of the growth height of the seedlings ...... 20 2.4 Sample collection ...... 20 2.5 Isolation of the mycobiota ...... 21 2.6 DNA extraction ...... 22 2.7 PCR (polymerase chain reaction) amplification...... 23 2.8 Sequencing ...... 23 2.9 Phylogenetic analysis ...... 23 2.10 Statistical and diversity analyses ...... 24 3 Results ...... 25 3.1 Endophytic fungal isolates between control and fungicide treatments ...... 25 3.2 Endophytic fungal population – seasonal differences between treatments ...... 29 3.3 The impact of fungicide treatment to species richness and community structure ...... 36 3.4 The impact of fungicide treatment to isolated epimycota ...... 39 3.5 The impact of fungicide treatment to growth heights of the Scots pine seedlings ...... 42

4 Discussion ...... 44 4.1 Specific aims of the present study ...... 44 4.2 The impact of fungicide treatment and some seasonal differences on frequencies of fungal endophyte isolates ...... 44 4.3 The impact of fungicide treatment to endophytic species (OTU) richness ...... 45 4.3.1 Dothideomycetes ...... 47 4.3.2 Leotiomycetes ...... 49 4.3.3 Sordariomycetes...... 50 4.3.4 Pyrenomycetes ...... 51 4.3.5 Eurotiomycetes ...... 51 4.3.6 Agaricomycetes ...... 51 4.3.7 Zygomycetes ...... 52 4.4 The impact of fungicide treatment to community structure of endophyte population ...... 52 4.5 The impact of fungicide treatment on fungal epiphytes ...... 53 4.6 The impact of fungicide treatment on the growth height of Scots pine seedlings ...... 55 4.7 Technical considerations ...... 56 5 Conclusions ...... 58 References ...... 61 Appendices ...... 75

1 Introduction

1.1 The ecological significance of mycobiota

Fungi (termed mycobiota when restricted to specific habitat/area) have many functional roles in the dynamic biosphere. Fungi play an important role in the forest ecosystem as a form of mycorrhiza and lichen symbionts, and as the main decomposer of organic matter. Some fungal species overcome the host‟s defence mechanisms and continue living inside of the host tissues as harmless endophytes. Some endophytes have been reported to play a role in host‟s defence against herbivores (Sumarah & Miller 2009) and diseases (Li et al. 2012), and even abiotic stress (Kane 2011).

Every interior and exterior parts of plants has abundant mycobiota, epi- and endomycota, which colonizes the external and internal tissues of the host, respectively. Normally these fungi are harmless but their lifestyle can turn pathogenic, if the host is weakened by abiotic factors, or/and environmental conditions become favourable for plant disease development. Fungi are the major causal agents of plant diseases all over the world.

Three major terrestrial ecological roles for fungi are: saprobes, parasites (including plant pathogens) and mutualistic symbioses (Kendrick 2001). However, the boundaries of these roles are vague and they can overlap each other. One example of a very common fungal genus of conifers which is reported to have all of these roles is Lophodermium spp. (Diwani & Millar 1987).

Saprotrophic fungi are dominant decomposers of plant debris and wood. They use dead organic matter as a nutrition source and this is essential in the recycle of nutrients. Especially in boreal forest, where nutrient mineralization is slow, decomposing fungi are crucial for plant growth. Unlike saprotrophs, fungal parasite needs a living host. Fungus can live as a harmless parasite inside a host‟s tissue but pathogenic activity may occur if there are some alterations in the condition of the host. If the parasite causes visible physiological symptoms in the host then it is a pathogen, a causal agent of a plant disease. All pathogenic fungi have latent phase (i.e. they don‟t cause any visible symptoms in the host) in their lifecycle, and

1 possible disease development depends on many biotic and abiotic factors. A mutualistic symbiosis on the other hand, is the association between two (or more) organisms which is beneficial to both (or all) partners (Saffo 2001). Mutualistic symbioses are everywhere and they have important role in ecology. The most common mutualistic symbioses are the association between a mycorrhizal fungus and a tree. Typically the mycorrhizal fungus absorbs mineral nutrients from the soil and directs these to the tree, while the tree provides the fungus with sugars. Fungal endophytes also form symbiotic associations with grasses and woody plants. In this case, endophytes receive nutrition and protection from the host, and host may benefit from enhanced competitive abilities and increased resistance to herbivores, pathogens and abiotic stress. There is a thin line between parasitism and mutualistic symbioses. This means that a beneficial relationship between symbiotic partners can turn harmful if the balance between them is disturbed by abiotic or biotic stress factors, or a decrease in plant resistance or/and increase in fungal virulence (Schulz et al. 1999; Eaton et al. 2011).

1.2 Endophytes

1.2.1 Definition

Fungal endophytes live asymptomatically and internally (intercellularly or intracellularly) all or spend at least significant part of their life cycle within host plant tissues (Wilson 1995). These fungi grow hyphae between the spaces of plant cells, within the cell walls or inside the cells. The term endophyte is derived from the greek words éndon (inside) and phytón (plant) and it was first applied by De Bary 1866. Since then the term by De Bary, which only described the place where the microorganism was, has evolved together with more intense microbial studies and more complex definitions by researchers (Hyde & Soytong 2008).

The definition by Wilson (1995) is probably the most relevant because it is the most common definition in use today in scientific papers (Müller 2003). The term includes fungi and bacteria, and also pathogenic fungi which have a latent phase in their life cycle. It excludes mycorrhizal fungi, root nodule bacteria and mistletoes. The term endophyte is used to indicate what type of connection internally living fungi or

2 bacteria forms with the host. In the interaction endophytic fungi or bacteria do not elicit symptoms of disease. This is common to all endophytes irrespective of their taxonomy or infection cycle (Wilson 1995). In practice, fungi isolated from surface sterilised healthy-looking plant tissues are considered endophytes, even if there is a possibility that some of these isolated fungi may be epiphytic survivors of surface- sterilization (Müller 2003). In addition, it may be difficult to determine if the fungus is endo- or epiphyte because some species may grow in both habitats during part of their life cycle (Osono & Mori 2005, Osono 2006).

1.2.2 Diversity and abundance

Tropical forests are known to be biodiversity hot spots and they harbour large numbers of endophytic fungi (Blackwell 2011 and references in it) but also boreal forests have been reported to harbour numerous amounts of fungal species (Higgins et al. 2007, Taylor et al. 2010). Fungal diversity is enormous in trees, especially in conifers but also in the soil (Faeth & Fagan 2002). Untouched, mature, dense, mix- tree stands have the highest fungal diversity (Helander et al. 2006).

Fungal endophytes have been found in every investigated plant around the globe (Yuan et al. 2010). They usually occur in above-ground plant tissues and occasionally in roots (Kernaghan 2011). Endophytes are diverse group and they have associations with various plant families, especially in grasses and woody plants (Saikkonen 2007). The most studied fungal endophytes of grasses are the ascomycetes. Neotyphodium sp. is probably the most studied grass endophyte genus because of the production of toxic alkaloids which cause toxic symptoms and even death to herbivores and grazing livestock (Qawasmeh et al. 2012). Grass endophytes are usually seed-borne and termed systemic fungi. This means that seed-borne endophytes are present also in following generations of the host plant.

Fungal endophytes in woody plants usually belong to ascomycetes (Hyde & Soytong 2008). Also endophytes belonging to basidiomycetes, oomycetes, and zygomycetes have been found from trees (Petrini 1986, Sinclair & Cerkauskas 1996; ref. Saikkonen, et al. 1998; Giordano et al. 2009). Endophytes in woody plants are

3 thought to be more diverse and complex than in grasses because of the wider taxonomic host-range (Rodriquez et al. 2008; ref. Yuan et al. 2010). However, some studies suggest that tree-endophytes are highly specific to host-species and also host- neutral species occurs, but these are exceptions (Carroll 1988; Higgins et al. 2007). Usually the host-tree has few dominant endophyte species, and at low frequencies high number of other species (Sieber 1988, Espinosa-Garcia & Langenheim 1990, Fisher & Petrini 1990, Petrini et al. 1992, Kowalski & Krygier 1996, Petrini 1996; ref. Müller 2003). The most dominant species on Scots pine needles have been reported to be Lophodermium pinastri (Schrad.) Chevall, Hormonema dematioides Lagerb. & Melin and Cyclaneusma minus (Butin) DiCosmo, Peredo & Minter (Kowalski 1993; Helander 1994; Terhonen et al. 2011). Giardano et al. (2009) investigated endophytes in the sapwood of Scots pine (Pinus sylvestris L.) and the most dominant species detected were Penicillium spp. and Alternaria alternata (Fr.) Keissl. Tree-endophytes are generally dispersed horizontally by spores and termed non-systemic endophytic fungi. Endophytes can also occasionally be disseminated by insects (Monk & Samuels 1990; Devarajan & Suryanarayanan 2006).

Tree-endophytes are found in all parts of conifer trees. The most endophyte- harbouring and endophyte diverse part is foliage, and needle endophytes are the most studied endophytes of conifers (Müller 2003). Older, mature needles have usually the highest infection frequencies (Kowalski 1993; Helander et al. 1994; Hata et al. 1998, Guo et al. 2008). Infection frequencies correlate negatively with elevation to canopy and positively with annual precipitation (Carroll 1988, Arnold & Lutzoni 2007). Also growing season, source of inoculum, moisture, temperature and latitude affect on the abundance of endophytic fungi (Kowalski 1993, Terhonen et al. 2011).

1.2.3 Ecological significance

Endophyte-host relations are presently intensively studied because of the complex nature of their interaction. Fungal endophyte lives within host tissues and utilizes leaking nutrients without causing any visible damage. The host regulates the growth of the fungus and this makes living mutually possible. Endophytic hyphae usually grow very slowly and it is restricted to certain areas (Müller 2003). Some facultative

4 saprobic endophyte species become active only when the senescence of the tissue begins (Müller et al. 2001; Promputtha et al. 2007; Oses et al. 2008). Changes in the biotic or abiotic environment can push the endophyte towards pathogenic, sabrobic or mutualistic life style. It has been reported that a mutation in a single locus of fungal genome (e.g. avirulence to virulence), changes in fungal community in foliage and changes in host plant (e.g. altered nutrient uptake, damage or senescence) can be one of the factors determining further interaction between the endophyte and the host (Minter 1981, Freeman & Rodriguez 1993, Faeth & Hammon 1997; ref. Saikkonen 2007).

Endophytes can overcome the defence mechanism of the host or at least can tolerate it. Probably the same mechanism is involved in the infection as is in the formation of mycorrhizal symbioses between a tree and a fungus. Endophyte-host relation is characterized as a mutualistic symbiosis, and this probably explains why fungal endophytes are able to infect host without provoking any major resistance response. Plants allow in some level these associations because of the benefits obtained from the presence of the endophytes (Eaton et al. 2011).

Endophytes are able to increase host resistance in many ways. Firstly, the endophyte infection enhances the defence mechanisms of the host (Ganley at al. 2008). This is beneficial against foliar pathogens and herbivores. Secondly, the assemblages of endophytes in the host-plant can diminish the presence of foliar pathogens via direct competition (Minter 1981; Mejía et al. 2008). Thirdly, direct antagonism against microbial pathogens and herbivores, which is commonly found in grasses (Diehl 1950, Clay et al 1993; ref. Blackwell 2011). This occurs through the production of toxic secondary metabolites by endophytes. Also coniferous needle endophytes are reported to produce toxins and act as antagonists against foliar pathogens (Mejiá et al. 2008) and herbivores (Carroll 1988; Clark et al. 1989; Calhoun et al. 1992).

Example of endophyte-pathogen antagonism is the interaction between Lophodermium species. Lophodermium species are commonly found in Scots pine needles. Lophodermium seditiosum Minter, Staley & Millar is a severe needle pathogen in young Scots pine stands, especially found in Scandinavian tree seedling nurseries. L. pinastri is reported to act as an endophyte and become active after the senescence of the needle (Kowalski 1993). L. conigenum (Brunaud) Hilitzer is also

5 categorized as a needle endophyte and a primary decomposer of falling needles. However, this fungus is reported to prevent the infection of L. seditiosum when needles are still attached in fallen branches. If the endophyte is present the pathogen will often avoid these needles (Minter 1981; ref. Diwani & Millar, 1987). This helps to prevent the source of sporulation of the pathogen.

Induced resistance response was reported from Ganley et al. (2008) in their research focusing on endophyte-mediated resistance against white pine blister rust in Pinus monticola Douglas ex. D. Don. They demonstrated that fungal endophytes were effective at increasing survival in host plant against the pathogen. This induced resistance was found to be effective over time, indicating persistence. Authors suggested, that fungal endophytes may play a significant role in the establishment of fungal community, and could have a potential in biological control of pests and diseases.

Mejía et al. (2008) reported endophytic fungi of Theobroma cacao L. to act as antagonistic against major pathogen of cacao. The antagonism mechanism was mainly competition for substrate, which was seen among fast-growing good colonizers. Toxic chemicals were produced for antibiosis reaction and this mechanism was seen among slow-growing weak colonizer fungi.

One example of herbivore resistance by fungal endophytes is the interaction between elm bark beetles and endophyte Phomopsis oblonga (Desm.) Traverso. P. oblonga often invades the inner bark of elm. The attack of elm bark beetles to the same host- tree is disturbed by this fungus. It is seen as a disruption of the breeding of bark beetles and leading to a decline of elm bark beetle populations (Webber 1981). This endophyte probably plays a major role in preventing the infection of Ceratocystis ulmi (Buisman) C. Moreau, the causal agent of Dutch elm disease, to elms. Bark beetles act as vectors of this fungal pathogen and the presence of P. oblonga prevents the attack of the beetles on the hosts (Webber & Gibbs 1984).

Carroll found in his studies evidence of endophyte-mediated resistance in woody plants between Douglas fir (Pseudotsuga menziesii (Mirb). Franco) and Contarinia- midges. Common needle endophyte of Douglas fir, Rhabdocline parkeri Sherwood, J. K. Stone & G. C. Carroll, invades the galls of Contarinia-midges and cause death of larvae. Results showed higher mortality in the endophyte-infected samples than in

6 the uninfected. R. parkeri does not invade the living larvae so the death of larvae may be caused by fungal toxins (Carroll 1988).

Clark et al. (1989) studied the secondary metabolites of endophytes of balsam fir (Abies balsamea L. Mill.) and red spruce (Picea rubens Sarg.) and their toxicity to spruce budworm. Their data suggested that some of these endophytic metabolites were toxic and it manifested as mortality and retarded larvae development. Calhoun et al. (1992) found in their studies that balsam fir endophyte Hormonema dematioides produced toxic metabolites which reduced growth rate of the spruce budworm and increased the mortality of larvae. This endophyte is also found in Scots pine buds (Pirttilä et al. 2003).

Sumarah et al. (2008) reported that Phialocephala scopiformis Kowalski & Kehr, an endophyte of Picea glauca (Moench) Voss and P. rubens, reduced the growth of spruce budworm Choristoneura fumiferana Clemens which is the most economically important pest in North America. This endophyte was investigated to produce anti- insect toxins against the insect-pest, and hence reduced its development and growth.

Fungal endophytes have also been reported to protect its host from abiotic stress factors (Redman et al. 2002; Kane 2011). Novel endophyte Curvularia sp. isolated from thermotolerant plant Dichanthelium lanuginosum raised the host heat tolerance. The plant host and the endophyte could survive only together at high temperatures (Redman et al. 2002). Kane (2011) investigated if endophyte infection had effects on the drought stress tolerance of Lolium perenne L. in the Mediterranean regions. Endophyte-free plants tolerated drought less than plants with natural endophyte communities. This indicates that endophytes may enhance the host ability to tolerate abiotic stress.

Foliar endophytes are very sensitive to changes in the surrounding biotic and abiotic environment. This can be seen as changes in the endophyte species community and isolate infection frequencies. Environmental factors, such as humidity and elevation (Carroll & Carroll 1978; Lehtijärvi & Barklund 1999), density of the trees (Helander et al. 1994), geography (Higgins et al. 2007), nutrient availability of the host (Ranta et al. 1995; Lehtijärvi & Barklund 1999), air pollution (Ranta et al. 1995) and chemical applications (Riesen & Close 1987; Mohandoss & Suryanarayanan 2009) have impact on the infection rate.

1.3 Pesticides

Pesticides (i.e. plant protection products) are chemicals intended for preventing, destroying, repelling or mitigating any pest on cultivated plants. Target pests can include insects, plant pathogens, weeds, birds, mammals, nematodes and microbes. Herbicides are used to eliminate vascular weeds, insecticides are used to kill harmful insects and fungicides are used against pathogenic fungi. Fungicides are categorised according to their mode of action (e.g. protectant or eradicant) or the purpose for which they are used. Protectant protects the plant from infections of plant pathogens and eradicant kills the pathogen which already has colonized the plant.

Pesticide can be specific when it has a selective toxicity against one species or a group, or it can have a wide spectrum whereby it is toxic to many species or groups. The selective toxicity of a pesticide depends on the target sites (foliage, soil) and the resistance mechanisms of the organism (Laatikainen 2006).

Formulated pesticide product contains one or more active ingredients. Active ingredient can be a naturally occurring compound, like antibiotics, or synthetic inorganic or organic compound, or mixture of compounds or ligands. Inactive ingredients are also added, like solvents, diluents, acidicity control chemicals, pigments or binding agents. The purpose of using inactive ingredients is often to increase the effectiveness of the active ingredient or make the product easier to use (Laatikainen 2006). Organic compounds are the most widely used active ingredients in modern pesticides. Many inorganic and heavymetal containing products are presently no more in use, like DDT, because of the environmental risks (Laatikainen 2006).

Pesticides is a heterogeneous group of chemicals, and their risks to human health and environment are thoroughly investigated and assessed before they are allowed into commercial production (“Plant protection products”. Internet-site of Finnish Safety and Chemicals Agency, Tukes. < http://www.tukes.fi/ >. 11.5.2012). Toxicity, dose and biodegrability are often the factors that determine the environmental risks of a pesticide. Pesticides are applied in the fields, especially in agriculture and forestry, when there is a possibility that other organisms are affected too than the protected plants. Harmful side effects to the non-target organisms have raised concern and

8 modern pesticides are under continuous development (Laatikainen 2006). Nowadays pesticides are developed to be more effective, more target-specific and more biodegradable than in the past (“What are pesticides?” Internet-site of European Crop Protection Association, ECPA. < www.ecpa.eu >. 11.5.2012).

1.3.1 Fungicides

Fungicides are used for protecting cultivated plants for the infection or for eradication of the pathogenic fungi. Fungicides can be grouped in many ways such as the role in protection of plants, chemical group, mode of action, resistance risk, breadth of activity, and mobility in the plant (“FRAC-code-list 2011”. Internet-site of Fungicide Resistance Action Committee, FRAC. < www.frac.info >. 11.5.2012; “Fungicides terminology”. Internet-site of Integrated Crop Management, by Darren Mueller, Department of Plant Pathology, University of Iowa State < www.ipm.iastate.edu >. 11.5.2012).

Fungicides can be grouped according to their role in plant protection. This includes preventative-, early-infection-, eradicative- and antisporulant-activity. Some fungicides can be categorized simultaneously in more than one group. Fungicides can be grouped according to their chemical group. This means that the fungicide is based on a chemical group which share a biochemical mode of action and in some cases a similar chemical structure (“Fungicides terminology”. Internet-site of Integrated Crop Management, by Darren Mueller, Department of Plant Pathology, University of Iowa State < www.ipm.iastate.edu >. 11.5.2012).

Fungicides are grouped according to their mode of action in the target fungi. This includes nucleic acid synthesis, mitosis and cell division, respiration, amino acids and protein synthesis, signal transduction, lipids and membrane synthesis, sterol biosynthesis in membranes, cell wall biosynthesis, melanin biosynthesis in cell wall, and host plant defence induction (“FRAC-code-list 2011”. Internet-site of Fungicide Resistance Action Committee, FRAC. < www.frac.info >. 11.5.2012). Resistance risk is given almost to all commercial fungicides. It reports detailed information of molecular mechanism of resistance development and the resistance risk (“FRAC-

9 code-list 2011”. Internet-site of Fungicide Resistance Action Committee, FRAC. < www.frac.info >. 11.5.2012). A breadth of activity reports reveals if fungicide has one or more target in the metabolic sites of the fungus. Finally, fungicides can be grouped according to their mobility in the plant. This is categorized as contact fungicides and systemic fungicides (“Fungicides terminology”. Internet-site of Integrated Crop Management, by Darren Mueller, Department of Plant Pathology, University of Iowa State < www.ipm.iastate.edu >. 11.5.2012).

Inorganic fungicides were the first chemicals to be used to prevent plant pathogens. It started in 1846 with elemental sulphur, 1882 with copper salts and 1920 with mercury (extremely toxic to living organisms). Sulphur is a complete natural product and it is the only fungicide that organic growers can use in their crops. Copper salts have wide antifungal and antibacterial range. They have been used to control many leaf and fruit diseases, and copper salts based fungicides have been proved to control successfully powdery mildew in grapevines. Copper salts disrupt many basic metabolic processes, so the fungal pathogens do not develop resistance to them easily. However, copper salts are possible phytotoxic compounds (Deacon 1997). Nowadays copper fungicides are used against fungal diseases for example in wine- growing areas (Vitanovic et al. 2010).

Organic contact fungicides, termed also as protectant fungicides, were developed in the 1930s. They act only near the site where they are applied. They need to be applied over the whole plant surface and re-applied to protect new growth. These fungicides interrupt the basic metabolic processes of fungi such that resistance is not easily developed towards them. Organic contact fungicides are long-lasting and many of them are in use today (Deacon 1997).

Systemic fungicides were developed in the 1960s. These fungicides are absorbed by plants and spread internally to the foliage, stem and roots. Systemic fungicides can act as curative to already existing infections, and prevent new infections. They are used widely but the mode of action of these fungicides can be highly specific, such as binding to a specific component of a fungal cell. Most fungi can easily develop resistance towards such fungicides. Usually the resistance in a fungus can be derived from a single-gene mutation. If resistance is developed, the fungus often has cross- resistance to all other members of that fungicide group. That is why systemic

10 fungicides are needed to combine with fungicides with different modes of action or use the combination of systemic and broad-range contact fungicides (Deacon 1997). Only few fungicides are truly systemic (i.e. move freely inside the plant): some are upwardly systemic (i.e. move only upward through the xylem tissue), and some are locally systemic (i.e. move into treated foliage and redistribute to some degree within the treated portion of the plant (“Fungicides terminology”. Internet-site of Integrated Crop Management, by Darren Mueller, Department of Plant Pathology, University of Iowa State < www.ipm.iastate.edu >. 11.5.2012).

Sterol biosynthesis inhibitors (SBIs) are systemic fungicides which inhibits the sterol synthesis in fungal cell membrane. This implies that the fungus will be unable to synthesize ergosterol, which is the main fungal sterol (Laatikainen 2006). These SBIs are toxic to fungi but non-toxic to several species of mitosporic fungi because of their lack of ergosterol (Weete 1973). Changes of sterol composition in plasma membrane may alter fluidity of the cell membrane, thickness of the molecules, mitochondria and membrane structure. Also it can have effect on chitin synthethase activation and free fatty acid accumulation, both of which affect the growth and morphology of fungi. One important example of SBIs is propiconazole (triazole) which cause leakage of cell membrane and ultimately leads to cell death (Laatikainen 2006). This fungicide is used routinely in agriculture and forestry in Scandinavia (Juntunen 2002).

1.3.2 Use of pesticides in Finland

Use of pesticides is a routine part of the Finnish agriculture and forest tree seedling production. Although Finland is located in the cold north there are also several plant pathogens, harmful insects and harmful plants. In Finland Tukes (Finnish Safety and Chemicals Agency) is the agency that makes the decisions concerning the authorisation and terms of use of plant protection products. The active ingredients are assessed under a joint EU procedure and the approved plant protection products are listed in the Plant Protection Products Directive. This list is also named as the “positive list”. Products are authorised at national level in each member country and only those accepted and registered products can be placed on the market and can be

11 used (“Plant protection products”. Internet-site of Finnish Safety and Chemicals Agency, Tukes. < http://www.tukes.fi/ >. 11.5.2012).

There are approximately 350 pesticides including 150 active ingredients registered for use in plant protection in agriculture and forestry in Finland. Yearly sales of these products have been followed since 1953 and the amount of sold pesticides has been increasing since then. In year 2010, most part of the yearly sold plant protection products (based on active ingredient) were used in agriculture, and approximately 1/3 in forestry. This fraction in forestry has been increasing because of the intensive use of stump treatment with Rotstop and Urea against Heterobasidion root rot. Overall, herbicides are the most used pesticides followed by fungicides and then insecticides (“Plant protection products”. Internet-site of Finnish Safety and Chemicals Agency, Tukes. < http://www.tukes.fi/ >. 11.5.2012).

Commercial tree seedling production is a part of forestry and it takes place at forest tree nurseries. In Finland the main commercial tree species are Norway spruce (Picea abies L. Karst.), Scots pine and silver birch (Betula pendula Roth). In 2011, the number of all domestic seedlings delivered for planting was approximately 142 million. The majority of seedlings were Norway spruce (67 %), Scots pine (30 %) and silver birch (2 %) (“Statistics from forest seed and seedling production”. Internet-site of Finnish Food Safety Authority, Evira. < www.evira.fi >. 19.5.2012).

99 % of the seedlings are grown in containers and the rest as bareroot seedlings. During growing season container trays are placed on a rack and lifted 20 cm above the ground to get good aeration for root system. Containers are filled with medium- texture, low-humified Sphagnum peat. Usually dolomite lime and a base fertilizer are added. First seedlings are grown in plastic-covered greenhouses and later outdoors when the night frost risk is over. Density of seedlings depends on the age and size of the seedlings but mainly seedlings are cultured in densities of 400-900/m2 (conifers) and 150-400/m2 (hardwood) (Lilja et al. 2010). Irrigation and fertilization is automatic. Greenhouses are ventilated, and have regulation of temperature and relative humidity. Seedlings are transferred outdoors in June-July, depending on the tree species and age. No shelter is used but in the case of too early night frost seedlings are either transported into plastic house or protected by water irrigation (Oral communication; Researcher Marja Poteri, Finnish Forest Research Institute

(Metla), 12.5.2011). About 40-50 % of all the seedlings are removed in the autumn and packed in cardboard boxes. Boxes are stored at -1 to -4oC for 6-7 months. The rest of the seedling containers are placed on ground surface and they are let to overwinter under the snow (Lilja et al. 2010). If snow is lacking, snow canoons are used to make artificial snow to protect the seedlings.

Forest nursery is a good environment for pathogens and biotic diseases. High density of monocultured seedlings, pathogen-free growth medium, fertigation (irrigation with fertilizers), outdoor growing and abiotic stress are all factors that can help especially fungal pathogens spread and cause infections. Occurence of pests always causes economical losses for the nursery. Spore dispersal of pathogenic fungi from neighbouring forests creates a high risk of disease especially to Scots pine seedlings. Finnish forest nurseries have developed good cultural practises and chemical protection to prevent possible epidemics. However, pesticides can also eliminate other living organisms and have an impact on the environment.

In Finland the improvements in commercial tree seedling nurseries have decreased the amount of used pesticides. One main reason for the decrease in pesticide use from 1980s has been the decrease in seedling production. Also the novel pesticide products are more developed than before so the required doses are lower (Juntunen 2002). Due to the higher density of seedlings and shorter growing time the same number of container seedlings can be produced in a much smaller growing area than former bareroot seedlings which also decreases the used amount of pesticides per growing area (Oral communication; Researcher Marja Poteri, Metla, 12.5.2011).

The biggest number of pesticide products is used in Scots pine production. Also the number of applications is largest and the duration of chemical prevention is longest in Scots pine seedling production. There is, however, variation between Finnish nurseries in the use of pesticides (Juntunen 2002).

Nowadays, Finnish forest nurseries use herbicides only around the nursery and planting areas without seedlings, because of the phytotoxicity of most herbicides to forest tree seedlings (Oral communication; Researcher Arja Lilja, Metla, 14.5.2012). Insecticides are mainly used in forest tree nurseries to pre-protect Scots pine seedlings from the attack of large pine weevil (Hylobius abietis L.) in forestation sites (Juntunen 2002).

Seedlings overwinter under the snow and this facilitates easy spreading of fungal pathogens like snow blights of conifers (Phacidium infestans P. Karst., Herpotrichia juniperi (Duby) Petr.) (Lilja et al. 1997). Fungicide treatment against snow blights is a necessary routine practice in Finnish nurseries and practically there is no Scots pine seedling production without it (Juntunen 2001).

In Finland propiconazole is used as an active ingredient in a routine treatment against specific fungal pathogens in forest nurseries and in agriculture. In forest nurseries two products, one having propiconazole and another prochloraz and propiconazole as active ingredients, are sprayed on pine seedlings to prevent infection of snow blights and Scleroderris canker (Gremmeniella abietina (Lagerb.) M. Morelet). A mixture of strifloxystrobin and propiconazole is used to control rust infections on birch seedlings (Oral communication; Researcher Arja Lilja, Metla, 14.5.2012). In agriculture propiconazole is sprayed on cereals, especially wheat, to prevent rust fungi, spot diseases and powdery mildew (Mäki-Valkama 2005). Propiconazole treatment is also used in golf courses to prevent spot diseases of grass. The application rate of propiconazole as an active ingredient is 125 g ha-1. It is less than other routinely used fungicides (Juntunen & Kitunen 2003).

Currently there are less than fifteen fungicides registered for use at the forest tree nurseries in Finland (“Plant protection products”. Internet-site of Finnish Safety and Chemicals Agency, Tukes. < http://www.tukes.fi/ >. 11.5.2012). However, this number changes every year.

1.3.3 Side-effects of propiconazole on non-target organisms – previous studies

Propiconazole (Tilt 250 EC), 1-[2,4-dichlorophenyl)-4-propyl-1,3-dioxolan- 2ylmethyl]-1H-1,2,4-triazole (IUPAC, International Union of Pure and Applied Chemistry), is a SBI foliar fungicide. It has a broad range of activity, and it can be used as a protectant or an eradicant treatment. It is a systemic fungicide and it is usually used as a foliar application. The needles of the seedling absorb the fungicide and after some time it is translocated into to the stem and later into the roots in small amounts (Laatikainen 2006; Blaedow et al. 2010). Propiconazole is used routinely in

14 forest tree seedling nurseries in Scandinavia to control Scleroderris canker and snow blights during overwinter cold storage (Laatikainen 2006). It is very effective against these pathogens but it is also shown to have some impacts on seedlings and other non-target organisms (Elmholt 1991; Peltonen and Karjalainen 1992; Ylimartimo & Haansuu 1993; Manninen et al. 1998; Laatikainen & Heinonen-Tanski 2002; Laatikainen 2006).

Manninen et al. (1998) investigated if propiconazole application had effects on fine root and mycorrhiza condition of Scots pine. In their 2-year field experiment they found that propiconazole application reduced mycorrhizal infection and killed selectively ascomycete symbionts (ectendomycorrhizas). Also soil respiration was reduced which implies additional side-effects on soil organisms. At the ultrastructural level, propiconazole caused increased transparency and gradual granulation and degeneration of cytoplasm in the fungal part of mycorrhiza. Results suggested that propiconazole has severe effects on mycorrhizas in Scots pine and on non-target soil fungi.

Laatikainen and Heinonen-Tanski (2002) studied the effects of pesticides to ectomycorrhizal fungi of boreal forest trees in vitro. Propiconazole was one of the two most harmful pesticides to ectomycorrhizal fungi. It inhibited strongly the growth of mycorrhizal fungi.

Elmholt (1991) investigated the side–effects of propiconazole application to non- target soil fungi in a field experiment in winter wheat. Significant inhibitory effects to filamentous fungi, especially Cladosporium, were detected.

Propiconazole has been reported to stimulate plant growth. Peltonen and Karjalainen (1992) investigated in a field trial the effects of propiconazole sprayings to spring wheat. There were genotypic differences between cultivars but in some cultivars propiconazole application did increase plant growth and quality with increasing efficient nitrogen uptake Laatikainen (2006) has reported in their study an improvement in nitrogen uptake resulting in better plant growth of Scots pine seedlings. Enhanced nitrogen uptake followed by imbalanced nutrient ratios in the needles may decrease the resistance of the seedlings and increase their susceptibility to plant pathogens such as Scleroderris canker (Ylimartimo & Haansuu 1993).

1.4 Aims of the study

The aim of this study was to investigate if propiconazole (Tilt 250 EC®) fungicide application has effects on the non-target foliar mycobiota of Scots pine seedlings. Also the effect of fungicide treatment to the height growth of Scots pine seedlings was investigated.

Our specific aims and hypotheses were:

a) To evaluate the effect of fungicide treatment on the non-target foliar mycobiota abundance of Scots pine seedlings.

H1: Fungicide treatment will have effect on the non-target foliar mycobiota abundance of Scots pine seedlings.

b) To evaluate the effect of fungicide treatment on the non-target foliar fungal endophyte structure of Scots pine seedlings.

H1: Fungicide treatment will have effect on the non-target foliar fungal endophyte structure of Scots pine seedlings.

c) To evaluate the effect of fungicide treatment on the non-target foliar fungal endophyte diversity of Scots pine seedlings.

H1: Fungicide treatment will have effect on the non-target foliar fungal endophyte diversity of Scots pine seedlings.

d) To evaluate the effect of fungicide treatment on the height growth of Scots pine seedlings.

H1: Fungicide treatment will have effect on the height growth of Scots pine seedlings.

To the author‟s knowledge, there are yet no published studies about propiconazole application and its side-effects on the non-target foliar mycobiota of Scots pine. The purpose of this study was to create new information about toxicity of fungicides to non-target mycobiota.

2 Materials and methods

2.1 The forest tree nursery and the experimental design

The field trial was established in the beginning of June 2008 in Fin Forelia forest tree nursery which is located in Nurmijärvi, South Finland. The seedlings were 2-months- old Scots pine (Pinus sylvestris) containerised seedlings (Lannen PL81© containers). Each container accomodates 81 seedlings and there was a total of 30 containers (2430 seedlings) which were placed on a lifted rack (20 cm above ground) (Figure 1). The seedlings were sown in the end of March and grown first in the plastichouse. In the beginning of May the seedlings were transferred into open field. Experimental rack was located at the same field with the other commercial seedlings growing in similar racks. It was separated from each other by approximately 5 meters to prevent fungicide contamination when commercial seedlings were sprayed by the nursery staff. The rack was sealed from the sides with foam to prevent the drying effect of the wind. Experimental seedlings had the other normal nursery routines such as irrigation and fertilization (Figure 2).

Initially the condition of the seedlings was evaluated and the containers were paired. The idea was that the control and experimental fungicide treated container had similar looking seedlings (size, condition). The rack containers were arranged into 6 columns and 5 rows (Table 1). In columns 1, 3 and 5 all containers were used as controls and columns 2, 4 and 6 were selected for fungicide treatment, giving a total of 15 containers per a treatment. Containers as well as the racks were marked.

Table 1. The experimental design for the fungicide treated and the control seedlings in the rack.

25 C 26 T 27 C 28 T 29 C 30 T

19 C 20 T 21 C 22 T 23 C 24 T

13 C 14 T 15 C 16 T 17 C 18 T

7 C 8 T 9 C 10 T 11 C 12 T

1 C 2 T 3 C 4 T 5 C 6 T

T=treatment, C= control; numbers refer to container numbers.

Table 2. Identification of the seedlings in a container.

9 9 9 9 9 9 9 9 9 8 8 8 8 8 8 8 8 8 7 7 7 7 7 7 7 7 7 6 6 6 6 6 6 6 6 6 5 5 5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 2 3 4 5 6 7 8 9

Each container had a marking tape in the left front corner (red circle). In this position row numbers run from left to right 1-9. In each column seedling numbers run from front to backwards 1-9.

Figure 1. Experimental seedling rack in June.

Figure 2. Experimental Scots pine seedling in June.

2.2 The fungicide treatment

The fungicide (commercial name Tilt 250EC®) used had propiconazole as an active ingredient (250g/l). The spraying was targeted against Scleroderris canker (Gremmeniella abietina) following the guidelines of plant protection in the Finnish forest tree nursery. The first spraying was in the beginning of June (2.6.) and the last one in the end of August (30.8.). Within this period the seedlings were treated with fungicide twice a month, every second week. When fungicide spraying was performed, control seedling containers were moved 10 meters apart paying attention on the wind direction to prevent fungicide contamination. Control seedlings were sprayed at the same time with same amount of water than fungicide treatment. The fungicide concentration was 0,125% (recommendation to apply 0,5 l/ha and use 400 l water/ha) which corresponded to 31,25 g/ha of active ingredient. The spraying was performed with 1L Birchmeyer spraying bottles and the volume used was 8-10 ml/container (container area 0,16 m2).

2.3 Measurement of the growth height of the seedlings

300 seedlings were chosen systematically and 10 seedlings from each container were used for the growth measurements. Each month during sample collection the height of the same seedlings were measured and their condition were observed and documented. Height was measured systematically from the edge of the container to the tip of the main shoot. The first measurement was in the beginning of June (2.6) and it is referred to as May in the Results section.

2.4 Sample collection

The samples were collected once a month starting from the end of June till the end of October. A total of 20 healthy looking Scots pine seedlings were collected per sampling, altogether 100 seedlings. Ten fungicide treated and ten control seedlings were cut from the base of the stem from containers next to each other to form pairs.

Criteria for sample seedlings were that seedlings looked healthy and looked similar to each other in both treatments. Seedlings in the edge of the rack were ignored because of the “edge effect”. Whole seedling were taken and stored in sterile tube. Tubes were marked with an identification number of the seedling. The identification number refers to the exact place where the samples were taken from the rack. For example 1.1.1, the first number “1.” is the first container (control), the second number “.1.” is the first column of that container and the last number “.1” is the first seedling of that column (Table 2). The tubes were transported to laboratory for further investigation within 24 hours from the sampling time.

2.5 Isolation of the mycobiota

One needle was taken from the seedling. The criteria were that the needle was a fully developed older needle and looked healthy. The needle was surface sterilised to eliminate epiphytic organisms. First the needle was dipped into 4 % sodiumhydroxide (NaOH) for 30 seconds, then 70 % ethanol for 30 seconds and finally into autoclaved sterile Milli Q-water for one minute. The needle was allowed to air dry for a few seconds and there after cut in a 5 few millimetres long fragments. These fragments were placed in a 1,5 % malt extract agar (MEA) Petri dishes (agar plate). Incubation was at room temperature in a dark cabinet. Newly emerging fungal hyphae growing out from the needle fragments were subcultured into a new MEA agar plate to obtain a pure culture.

Pure cultures were classified visually into many different morphological groups. Classification was mainly based on the outlook of fungal pure culture, such as colour and shape of the mycelia. Also the growth speed and the structure of mycelia were used. One pure culture from each morphotypes was selected to be the representative one based on the similar outlook to its group members. The representatives were transferred to an autoclaved membrane on new MEA agar plates.

In September and in October epiphytic fungi were isolated. The same batch of sample seedlings was also used for isolation of endomycota. One needle was taken from the sample seedling in both treatments, a total of 40 needles (September and

October). The needle was transferred directly into the 1,5 % MEA agar plate and after fungal growth subcultured to obtain pure cultures.

2.6 DNA extraction

DNA was extracted with a few modifications using protocol described by Asiegbu et al. (2004). Mycelia was harvested from the top of the cellophane membrane and placed in a 1,5 ml eppendorf tube. 100 μl CTAB 2 % + buffer were added to each tube (see appendix 1 for preparation of the solutions). Little bit of crushed sterilised sand were added to these tubes and the samples were homogenised with a micro- pestle. 500 μl CTAB 2 % + buffer were added and samples were heated in the waterbath at 65oC for 1,5 hours. Samples were mixed a few times during this period.

One volume of chloroform IAA (24:1) was added to the tubes and quickly vortexed. Samples were centrifuged for 8 minutes at 13 300 rpm (maximum speed). Upper phase was transferred in to a new 1,5 ml eppendorf tube. DNA was precipitated by adding 2 volumes of cold isopropanol-MIX and vortexed quickly. Samples were left to sit in the ice for 30 minutes.

Samples were centrifuged at 4oC for 30 minutes at 13 500 rpm (maximum speed). The supernatant was poured out carefully. The pellet was washed by adding 200 μl 70 % cold ethanol. Samples were centrifuged at 4oC for 5 minutes at 6 500 rpm (half speed). The supernatant was poured out and the pellet was air dried for 5 minutes in a sterile laminar flow. The pellet was re-suspended in 50 μl TE buffer. The DNA samples were stored in a freezer at -20oC.

The concentration and purity of genomic DNA in each sample were measured by using NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, USA).

2.7 PCR (polymerase chain reaction) amplification

PCR was used to amplify ITS regions for the sequencing. ITS 1 and ITS 4 were used as primers. DNA was diluted in autoclaved MQ water (1/10), based on the results of NanoDrop. Negative control was used to check for contaminations. PCR was performed using a Biohit XP Cycler according to protocol explained in Appendix 2. PCR samples were stored at -20oC.

PCR samples were run on 1 % agarose gel with ethidium bromide to assess the quality of DNA amplification. 5 μl were taken directly from the PCR product and mixed with 2 μl of loading buffer. The samples were run in the gel 25 min. at 120 V. After the gel was run, picture was taken with gel image system (Molecular Imager Gel Doc XR+ System).

2.8 Sequencing

PCR products were sent to Haartman Institute, Helsinki Finland and sequenced with the ITS1 primer. The obtained sequences (52) were sent back as a chromatogram files (quality files) and text files. Chromatograms were manually analysed with a chromatogram viewer, Finch TV (http://www.geospiza.com/products/finchtv.shtml) and text files were cleaned according to the quality files. After this procedure, the Fungal ITS extractor software (http://www.emerencia.org/FungalITSexctractor.html) was used to extract the ITS1 and ITS2 subregions from the sequences.

2.9 Phylogenetic analysis

The cleaned sequences were used for BLAST searches against GenBank/NCBI to establish the taxonomic identification of the fungal isolates. The sequences with similarity of over 94 % and the query coverage over 95 % were used to determine species, taxon or order. Phylogenetic analyses of the sequences were done with using Clustal X (Larkin et al. 2007) and MEGA 5 (Tamura et al. 2001). The sequences were aligned using multiple sequence alignment with Clustal X and

23 phylogenetic tree was drawn with the use of MEGA5. Finally, the sequences were assigned to operational taxonomic units (OTUs) (Arnold & Lutzoni 2007) according to closest BLAST matches, morphological descriptions and phylogenetic analysis. This decreased the number of morphological groups from 52 to 37.

2.10 Statistical and diversity analyses

Differences in endo- and epimycota isolate frequencies between the two treatments were tested with a Mann-Whitney test. The impact of fungicide treatment to the height growth of the Scots pine seedlings were tested with a paired t-test. For statistical analyses SPSS version 19 (Chicago, IL, USA) was used.

Similarity in fungal community structure between treatments was estimated with Classical Sorenson index. The species richness between treatments was calculated using Shannon‟s diversity index. Both indices were calculated using Microsoft Excel 2010.

3 Results

3.1 Endophytic fungal isolates between control and fungicide treatments

Altogether 186 endophytic fungal isolates grew out from the 100 needles analysed. Isolates were grouped into 37 OTUs based on mycelia morphotyping, molecular identification and phylogenetic analyses (Table 3). These units consisted of both identified and unidentified distinct fungal isolates. Twenty-seven (27) OTUs were identified at least to genus level. OTUs were divided into Ascomycota (34), Basidiomycota (2) and Zygomycota (1). Fungal isolates were found in 5 classes within Ascomycota: Dothideomycetes (13 taxa), Leotiomycetes (5 taxa), Sordariomycetes (5 taxa), Pyrenomycetes (2 taxa) and Eurotiomycetes (1 taxa). Fungal isolates in Basidiomycota were from the class Agaricomycetes and an isolate from Zygomycota was identified belonging to class Zygomycetes. Ascomycota and Basidiomycota were found equally in both treatments. The only Zygomycota isolated was from the control seedling.

The highest frequency of endophytic isolates was found in needles of the fungicide treated Scots pine seedlings, altogether 97 isolates. Control seedlings had a lower frequency of isolates, with a total of 89. Fungal frequencies increased from spring to autumn in fungicide treated seedlings. Similar trend was shown in control seedlings until September when isolate frequencies decreased and stayed at the same level in October (Figure 3).

Control seedlings harboured more endophytic OTUs than fungicide treated seedlings, with a total of 28 and 26, respectively (Figure 4). Altogether 37 OTUs were found, 16 of which were common for both treatments. 9 species were found only in fungicide treated seedlings and 12 species were found only in control (Table 3). The number of endophytic OTUs in control seedlings slightly increased from June to October, being highest in October. Similar trend was not shown in fungicide treated seedlings. The highest number of endophytic OTUs including both treatments were in October (42) and lowest in July (16).

One OTU was overwhelmingly common and it was the only OTU present in both treatments at every sampling time (group 14 identified as Phoma sp. covering 36,6 %

25 of all isolates). Other common OTUs were Phoma glomerata (Corda) Wollenw. & Hochapfel (6,5 %), Lophodermium pinastri (4,3 %), Sistotrema sp. (4,3 %), Phoma herbarum Cooke (4,3 %) and Penicillium sp. (4,3 %) (Table 4).

Control Fungicide treatment 25 24 23 22 21 20 19 18 17

16 15 14 13 12 11

frequencies 10 9 8 7 6 5 4 3 2 1 0 June July August September October

Figure 3. The frequencies of endophytic isolates in control and fungicide treated P. sylvestris needles between June and October.

Control Fungicide treatment 15 14 13 12 11

9 8 7

frequencies 6 5 4 3 2 1 0 June July August September October

Figure 4. The number of endophytic OTUs in control and fungicide treated P. sylvestris needles between June and October.

Table 3. Endophytic fungal taxa isolated from P. sylvestris needles

Isolate GenBank Mi(%) / Description of Our suggestion Freqd) Classb) group accession no. Qc(%)a) best matches of isolate name Phylumc) number of best matches 10 AF473558 100/100 Lophodermium Lophodermium 8 L(A) pinastri pinastri 8 AY373923 100/98 Penicillium Penicillium 8 E(A)

melinii sp. 7 DQ093676 98/100 Gibberella Gibberella 5 S(A)

avenacea sp. 9 AF473556 100/100 Lophodermium Lophodermium 4 L(A)

pinastri sp. 26 FN868459 100/99 Phoma Phoma 4F) D(A)

herbarum sp. 14 FN868459 100/99 Phoma Phoma 68 D(A)

herbarum sp. 49 FR837918 99/99 Lophodermium Lophodermium 2C) L(A)

piceae sp. 20 AF473556 100/100 Lophodermium Lophodermium 1F) L(A)

pinastri sp. 53 AF201717 100/99 Hypoxylon Hypoxylon 1C) P(A)

multiforme sp. 58 DQ093676 100/100 Gibberella Gibberella 1F) S(A)

avenacea sp. 39 FN868459 100/98 Phoma Phoma 2C) D(A) herbarum sp. 24 DQ093653 100/94 Sistotrema Sistotrema 8 A(B)

brinkmannii sp. 5 AF473558 100/100 Lophodermium Lophodermium 5 L(A)

pinastri sp.

3 FN868459 100/99 Phoma Phoma 2 D(A) herbarum sp. 12 HM036611 100/99 Phoma Phoma 3 D(A)

macrostroma macrostroma 55 DQ093728 95/98 Mucor Mucor 1C) Z(Z)

hiemalis sp. 15 FN868459 100/99 Phoma Phoma 7 D(A) herbarum sp. 21 AF201717 100/99 Hypoxylon Hypoxylon 1C) P(A)

multiforme sp. 45 HQ533788 100/100 Cladosporium Cladosporium 1C) D(A)

sp. sp. 11 FR686560 100/98 Hypholoma Hypholoma 2 A(B)

fasciculare sp. 36 FN868459 100/98 Phoma Phoma 5C) D(A) herbarum sp. 33 FN868459 99/100 Phoma Phoma 4 D(A) herbarum sp. 35 DQ093699 100/100 Phoma Phoma 12 D(A)

glomerata glomerata 27 AY465468 98/99 Phomopsis Phomopsis 1F) S(A)

sp. sp. 4 DQ093668 100/100 Epicoccum Epicoccum 6 D(A)

nigrum nigrum 54 EF197084 100/98 Podospora Podospora 1C) S(A)

pleiospora sp. 2 FN868459 100/99 Phoma Phoma 8 D(A) herbarum herbarum 23 AY561198 98/94 Foliar Unidentified 4

endophyte fungal of Picea endophyte glauca

40 GQ153112 100/99 Dothideo- Unclassified 1F) D(A)

mycetes sp. Dothideo- mycetes 1 GU062252 82/86 Lecythophora Unidentified 1C) S(A)

sp. Sordario- mycetes a) Mi, Max identity; Qc, Query coverage b) D, Dothideomycetes; S, Sordariomycetes; L, Leotiomycetes; P, Pyrenomycetes; A, Agaricomycetes; Z, Zygomycetes c) A, Ascomycota; B, Basidiomycota d) F, only observed in fungicide treated seedlings; C, only observed in control seedlings

Table 4. Most common OTUs with their frequencies in control and fungicide treatments at different sampling times.

Control Total Fungicide tr. Total OTUs Ju Ju Au Se Oc Ju Ju Au Se Oc

Phoma sp. (14) 2 7 9 7 2 27 1 13 9 10 8 41 Phoma glomerata (35) 1 1 3 1 0 6 1 1 3 1 0 6 Lophodermium pinastri (10) 4 1 0 0 1 6 2 0 0 0 0 2 Sistotrema sp. (24) 1 0 2 0 2 5 0 1 0 1 1 3 Phoma herbarum(2) 0 1 1 1 1 4 0 1 0 1 2 4 Penicillium sp. (8) 1 1 1 1 0 4 1 1 1 0 1 4

3.2 Endophytic fungal population – seasonal differences between treatments

The first sampling had the lowest frequencies of endophytes in the whole sampling period (Figure 3). Fungicide treated seedlings harboured less isolates than control seedlings. More OTUs were detected in fungicide treated seedlings (Figure 4). The most dominant class was Leotiomycetes, 46,6 % in control and 38,5 % in fungicide

29 treated seedlings, whereas the second most dominant class was Dothideomycetes, 33,3 % in control seedlings and 30,8 % in fungicide treated seedlings.

The most common OTU was Lophodermium pinastri (isolate group number 10) (Figure 5). It was observed in both treatments, however it was more abundant in control seedlings. The second most common OTU was group 14 Phoma sp. It was observed in both treatments. Lophodermium sp. (nr. 5), Hypholoma sp. (nr. 11), Lophodermium sp. (nr. 20), Phoma sp. (nr. 26) and unidentified fungal endophyte (nr. 50) were only detected in fungicide treated seedlings. Similar numbers of exclusive OTUs were observed in control seedlings including Sistotrema sp. (nr. 24), Phoma sp. (nr. 36), Lophodermium sp. (nr. 49) and unidentified fungal endophyte (nr. 57).

Control Fungicide treatment 7

3 frequencies 2

Figure 5. Endomycota frequencies observed in June in control and fungicide treated needles of P. sylvestris.

In July, the observed fungal frequencies were higher than in June. Fungicide treated seedlings harboured more isolates than control seedlings which was the opposite situation in June (Figure 3). However, more OTUs were detected in control seedlings

(Figure 4). The most dominant class was Dothideomycetes (75 %) and the second most common class was Leotiomycetes (12,5 %) in control seedlings. In fungicide treated seedlings the most dominant class was Dothideomycetes (83,3 %).

Fungicide treated seedlings harboured only six OTUs while control seedlings had 10 distinct OTUs. The most common OTU was Phoma sp. (nr. 14) (Figure 6). It was observed in both treatments, more in fungicide treatment. Two OTUs were observed only in fungicide treatment; Gibberella sp. (nr. 7) and Sistotrema sp. (nr. 24). Lophodermium sp. (nr. 5), Lophodermium pinastri (nr. 10), Phoma sp. (nr. 36), Phoma sp. (nr. 39), Cladosporium sp. (nr. 45) and Hypoxylon sp. (nr. 53) were exclusive OTUs in control seedlings. The observed frequencies of different OTUs were only one or two with the exception of Phoma sp. (nr. 14) (freq. 20).

Control Fungicide treatment 21 20 19 18 17 16 15 14 13 12 11 10 9 8 frequencies 7 6 5 4 3 2 1 0

Figure 6. Endomycota frequencies observed in July in control and fungicide treated needles of P. sylvestris.

Fungal isolate frequencies increased in both treatments in August compared to frequencies observed in July. Also the highest population of endophytes were observed in August compared to other sample collection times. More isolates were

31 observed in control seedlings (Figure 3). Control seedlings also harboured more OTUs than fungicide treated seedlings (Figure 4). The most dominant class was Dothideomycetes (77,3 %) and the second most common class was Agaricomycetes (9,1 %) in control seedlings. In fungicide treated seedlings the most dominant class was Dothideomycetes (85 %).

The most common OTUs were Phoma sp. (nr. 14) and Phoma glomerata (nr. 35) (Figure 7). Both OTUs were observed equally in both treatments. Five OTUs were exclusive in fungicide treatment, including Epicoccum nigrum Link (nr. 4), unidentified fungal endophyte (nr. 19), Phoma sp .(nr. 26), Phoma sp. (nr. 33) and Gibberella sp. (nr. 58). Phoma herbarum (nr. 2), Lophodermium sp. (nr. 5), Phoma macrostroma (nr. 12), Sistotrema sp. (nr. 24), Phoma sp. (nr. 36) and Mucor sp. (nr. 55) were exclusive OTUs in control seedlings.

Control Fungicide treatment 19 18 17 16 15 14 13 12 11 10 9 8 7 frequencies 6 5 4 3 2 1 0

Figure 7. Endomycota frequencies observed in August in control and fungicide treated needles of P.sylvestris.

In September, fungal isolate frequencies decreased in control seedlings (Figure 3). In fungicide treated seedlings frequencies increased from August. The number of OTUs

32 increased in both treatments (Figure 4). Control seedlings harboured more OTUs than fungicide treated seedlings. In September the most dominant class was Dothideomycetes (55,5 %) and the second most common class was Sordariomycetes (16,7 %) in control seedlings. In fungicide treated seedlings the most common class was Dothideomycetes (82,6 %).

The most common OTU was Phoma sp. (nr. 14) (Figure 8). It was detected in both treatments, more in fungicide treatment. Lophodermium sp. (nr. 9), Phoma macrostroma (nr. 12), Phoma sp. (nr. 15), Sistotrema sp. (nr. 24), Phoma sp. (nr. 33) and unclassified Dothideomycetes (nr. 40) were only detected in fungicide treated seedlings. Control seedlings harboured more exclusive OTUs including unidentified Sordariomycetes (nr. 1), Lophodermium sp. (nr. 5), Penicillium sp. (nr. 8), Hypoxylon sp. (nr. 21), unidentified fungal endophyte (nr. 23), unidentified fungal endophyte (nr. 51) and Podospora sp. (nr. 54). Also in this sample collection other OTU frequencies than Phoma sp. (nr. 14) (freq. 17) were one to three.

Control Fungicide treatment 18 17 16 15 14 13 12 11 10 9 8 7 6 Frequencies 5 4 3 2 1 0

Figure 8. Endomycota frequencies observed in September in control and fungicide treated needles of P. sylvestris.

Observed fungal frequencies were exactly the same in October as they were in September (Figure 3). Fungicide treated seedlings harboured more fungal isolates

33 than control seedlings. The number of OTUs increased in both treatments (Figure 4) being the highest in the whole sampling period. More OTUs were detected in fungicide treated seedlings. In October the most dominant class was Dothideomycetes (55,5 %) and the second most common class was Agaricomycetes 16,6 % in control seedlings. In treated seedlings the most common class was Dothideomycetes (60,9 %) and the second most common class was Sordariomycetes (8,7 %).

Still the most common OTU was Phoma sp. (nr. 14) (Figure 9). However, its observed frequencies started to decrease after the peak in July and other OTUs frequencies were slightly higher in October, including Phoma sp (nr. 15), unidentified fungal endophyte (nr. 23) and Sistotrema sp. (nr. 24). All common OTUs were detected in both treatments. This time Phoma sp. (nr. 14) was clearly more abundant in fungicide treatment. There were seven exclusive OTUs in fungicide treated seedlings: Epicoccum nigrum (nr. 4), Gibberella sp. (nr. 7), Penicillium sp. (nr. 8), Phoma sp. (nr. 26), Phomopsis sp. (nr. 27), unidentified fungal endophyte (nr. 50) and unidentified fungal endophyte (nr. 56). Control seedlings had more exclusive OTUs including Phoma herbarum (nr. 2), Lophodermium sp. (nr. 9), Lophodermium pinastri (nr. 10), Hypholoma sp. (nr. 11), Phoma macrostroma (nr. 12), Phoma sp. (nr. 33), Phoma sp. (nr. 39) and unidentified fungal endophyte (nr. 42).

Control Fungicide treatment 11 10 9

7 6 5

4 Frequencies 3 2 1 0

Figure 9. Endomycota frequencies observed in October in control and fungicide treated needles of P. sylvestris.

As a summary, in June the most abundant OTU was Lophodermiun pinastri (nr. 10) a month after the first fungicide treatment was sprayed. This species was observed in both treatments, clearly more in control seedlings. After June it was only observed once in July and in October and it was isolated only from control seedlings. Phoma sp. (nr. 14) was overall the most abundant OTU in both treatments. In June, it was the second most common OTU and after that it was the most isolated OTU at every sampling time. The highest number of observed frequencies of Phoma sp. (nr. 14) was in July and the lowest number was in October. At the same time the number of other species increased. In June, the number of OTUs was higher than in July when the detected number dropped. After this the number of OTUs started to increase slowly, being highest in October. Only in June and in October the fungicide treated seedlings harboured more endophytic OTUs than control seedlings. In July, August and September the control seedlings had more species than fungicide treated seedlings.

Table 5. Common and exclusive fungal OTUs for control and fungicide treatments between June and October.

June July August September October

Control 4 6 6 7 8 exclusive Fungicide tr. 5 2 5 6 7 exclusive Common 5 4 4 5 5

3.3 The impact of fungicide treatment to species richness and community structure

Diversity for observed endophytic fungal OTUs in fungicide treated and control seedlings were calculated with Shannon‟s diversity index. Shannon‟s diversity index varies between 1,5 to 3,5 where 3,5 indicates the highest diversity. In this study, the index ranged between 1,04 and 2,51 (Table 6). This suggests that there were differences in fungal diversity between treatments over time. The highest diversity was in October in control seedlings and the lowest diversity was in July in fungicide treated seedlings.

Differences in community structure between treatments over time were estimated with Sorensen similarity index (Table 7). The most similar fungal isolates were observed in July and the least similar were observed in October. The number of common and exclusive fungal OTUs is shown in Table 5.

No significant differences were found in Mann-Whitney test for isolate frequencies between the two treatments over time in the 95 % confidence level (Table 8).

Table 6. Shannon‟s diversity index for isolated endophytic fungi in control and fungicide treated needles of P. sylvestris from June to October.

Treatment/ Species Abundance Shannon‟s (H) month richness

Control/ 9 15 2,06 June

Control/ 10 16 1,92 July

Control/ 10 22 1,92 August

Control/ 12 18 2,13 September

Control/ 13 18 2,51 October

Fungicide 10 13 2,24 treatment/ June

Fungicide 6 18 1,04 treatment/ July

Fungicide 9 20 1,77 treatment/ August

Fungicide 11 23 1,95 treatment/ September

Fungicide 14 23 2,1 treatment/ October

Table 7. Sorenson‟s similarity index for isolated endophytic fungi between control and fungicide treated needles of P. sylvestris from June to October.

Comparison Shared Sorenson month/treatment species index June/Control June/Fungicide 5 0,53 treatment

July/Control July/Fungicide 4 0,5 treatment

August/Control August/Fungicide 4 0,42 treatment

September/Control September/Fungicide 5 0,43 treatment

October/Control October/Fungicide 5 0,4 treatment

Table 8. Mann-Whitney test results for the frequencies of isolated endophytic fungi between control and fungicide treated needles of P. sylvestris.

Comparison Asymp. Sig. (2-tailed) month/treatment

June /Control June/Fungicide 0,813 treatment

July/Control July/Fungicide 0,738 treatment

August/Control August/Fungicide 1,000 treatment

September/Control September/Fungicide 0,663 treatment

October/Control October/Fungicide 0,231 treatment

3.4 The impact of fungicide treatment to isolated epimycota

Epiphytic fungi were isolated in September and October from both treatments. A total of 86 fungal isolates grew out from the 40 needles analysed. In September, the epiphytic frequencies were higher in control seedlings but found to be higher in October in fungicide treated seedlings (Figure 10). When the endo- and epimycota were compared in both treatments it was shown that epiphytic frequencies were higher in September in control seedlings and endophytic frequencies were higher in fungicide treated seedlings. In October epiphytic frequencies were higher in both treatments.

Mann-Whitney tests for epiphytic fungi in both treatments indicated that there was a significant difference between treatments in epiphytic fungi in September but not in October (Table 9). Mann-Whitney tests for endo- and epimycota individually, indicated that isolation frequencies did not differ significantly between the treatments (Table 10).

The most frequently isolated epiphytic fungus was Epicoccum nigrum and the second most isolated fungus was Sistotrema brinkmannii (Bres.) J. Erikss. Both species were observed in both treatments. Further analyses could not be done because only the representative epiphytic isolates from the most isolated two groups were sent for sequencing.

Endophyte Epiphyte 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11

frequencies 10 9 8 7 6 5 4 3 2 1 0 September c September t October c October t

Figure 10. Observed endo- and epimycota frequencies in September and in October in control and fungicide treated needles of P. sylvestris. c=control, t=fungicide treatment.

Table 9. Mann-Whitney test results for epimycota frequencies between treatments.

Comparison Asymp. Sig. (2-tailed) month/treatment

September/Fungicide September/Control 0,034 treatment

October/Fungicide October/Control 0,843 treatment

Table 10. Mann-Whitney tests for epi- and endomycota frequencies.

Asymp. Sig. Month Treatment Comparison (2-tailed)

September Control Epiphytes Endophytes 0,137

September Fungicide Epiphytes Endophytes 0,606

October Control Epiphytes Endophytes 0,810

October Fungicide Epiphytes Endophytes 0,782

3.5 The impact of fungicide treatment to growth heights of the Scots pine seedlings

There was a clear trend shown between the average heights of the fungicide treated and control seedlings (Figure 11). The seedlings grew fairly similar in June and July but in August fungicide treated seedlings started to grow faster than control seedlings. However, in paired t-test between treatments, there was no significant difference between mean heights of the seedlings (Table 11).

Control Fungicide treatment 110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30

Heights of theofHeights seedlings, mm 25 20 15 10 5 0 May June July August September

Figure 11. Average heights of Scots pine seedlings in control and fungicide treated P. sylvestris seedlings.

Table 11. Paired t-test values for the heights of the P. sylvestris seedlings/treatment in June to September.

Mean Std. Lower Upper Month df Sig. a difference error bound bound

June -,1867 1,3916 14 0,895 -3,1713 2,7980

July -3,5267 2,0269 14 0,104 -7,8740 0,8207

August -4,5333 2,4633 14 0,087 -9,8166 ,7499

September -4,1400 2,4691 14 0,116 -9,4358 1,1558

4 Discussion

4.1 Specific aims of the present study

The aim of this study was to investigate if routinely used fungicide propiconazole (Tilt 250 EC®) application has side-effects on the non-target foliar mycobiota of Scots pine seedlings in Finnish forest nursery. The primary objective of the investigation was to determine if there were differences in endomycota abundance, diversity and community structure in current year needles of Scots pine between fungicide treated and control seedlings. Differences between the abundance of epiphytic fungi were also investigated later during the growing season. The impact of propiconazole application on the growth height of Scots pine seedlings was equally monitored.

4.2 The impact of fungicide treatment and some seasonal differences on frequencies of fungal endophyte isolates

Observed fungal frequencies during the growing season followed the same pattern as shown in earlier studies concerning the changes in abundance of endophytes in Scots pine needles. Helander et al. (1994) found that endophyte frequencies increased in young needles during the summer. In the present study, the lowest and the highest endophytic frequencies were observed in fungicide treated seedlings, being lowest in June and highest in September/October. Similar results were obtained in the study of Guo et al. (2008). Foliar endophyte frequencies isolated from Pinus tabulaeformis Carr. were higher in August than in May but they did not detect any endophyte infection in 1-year-old needles. In our study, the youngest Scots pine seedlings were 3-month-old. Other authors have noted that changes in the physiology of needles with ageing could be a factor enhancing the endophyte isolate frequencies (Hata et al. 1998, Lehtijärvi & Barklund 1999). Hata et al. (1998) suggested that possible explanations for increased frequencies could be a decrease of antifungal compounds in the needle or improved physical condition of the needle. Lehtijärvi & Barklund (1999) stated that the colonization frequencies of endophytic fungus Lophodermium

44 piceae (Fuckel) Höhn. in the needles of Norway spruce increased after the tree growth had stopped in autumn.

The results of the present study suggested that fungicide treatment had effect on the abundance of foliar endophytic fungi on Scots pine seedlings as shown in Figure 3. Arnold et al. (2003) reported that natural plants growing close together have the same kind of fungal community. In this study, only the effect of fungicide treatment could possibly explain these differences in the abundance. Gamboa et al. (2005) reported in their study that propiconazole application decreased the number of isolated endophytic fungi compared to the control plant Guarea guidonia (L.) Sleumer. Also Mohandoss & Suryanarayanan (2009) found in their study that a systemic fungicide, hexaconazole, reduced the colonization frequency of foliar fungal endophytes compared to the colonization frequency of the control plant Mangifera indica L.

4.3 The impact of fungicide treatment to endophytic species (OTU) richness

The number of species was different in both treatments in the whole sampling time as seen in Figure 4. In control seedlings the number of OTUs increased from June till October. In fungicide treated seedlings the pattern was different. This could be due to the starting and finishing date for the fungicide application which was in the beginning of June and in the end of August. Tuomainen et al. (1999) found in their study that propiconazole had disappeared from the needles of Scots pine within 8 days after fungicide application.

The diversity of fungal endophytes was highest in October in control seedlings and lowest in July in fungicide treated seedlings according to Shannon‟s diversity indices (Table 6). Martín-Pinto et al. (2004) studied the seasonal influences on the endophytic fungal community in needles of Pinus spp. seedlings in nurseries in Spain. They observed that the highest number of species was in the spring. In our study the highest number of species was in October. Terhonen et al. (2011) investigated the seasonal influences on the endophytic fungi of Scots pine needles in

Finland. Their results were similar than ours, species richness was higher in the fall than in the spring.

Mohandoss & Suryanarayanan (2009) studied the effect of fungicide hexaconazole treatment on foliar fungal endophyte diversity in mango (M. indica). Fungicide treatment decreased the total number of detected species compared to control. Recovered species were 4-7 in treated leaves and 10-15 in control leaves. However, the species diversity, according to Shannon‟s diversity index, did not vary significantly. The authors concluded that the fungicide treatment possibly altered the traits of susceptibility of M. indica to fungal endophytes as the established endophyte community structure after treatment was quantitatively and qualitatively different. They speculated that the elimination of fungal species with fungicide, makes it possible for other competing weaker endophytic species to invade the host which would not usually have the ability to do that.

Barklund and Unestam (1988) studied the interaction of Gremmeniella abietina and foliar mycobiota in Norway spruce seedlings. They found that the abundance of endo- and epiphytic fungi decreased in acidic mist treated shoots of Norway spruce seedlings. Simultaneously, the number of artificially inoculated G. abietina isolates increased, and the authors suggested that this happened because of the absence of antagonistic endo- and epiphytic species. However, in further studies conducted by Ranta et al. (1995), they did not find any evidence that endophytic fungal species were able to antagonize G. abietina. These results suggested that environmental changes, like acidic rain or fungicide application, affects the abundance of foliar mycota in needles and hence, makes the environment more suitable for weaker competitors, including pathogens, to invade the host.

The majority of the detected fungal species belonged to Ascomycetes (91,9 %.) and the most dominant class was Dothideomycetes, (38,2 %). Leotiomycetes and Sordariomycetes were the second most dominant classes, 14,7 % each. There have been several earlier studies concerning endophytes of conifers and major part of the identified endophytic fungi isolated from needles belonged to phylum Ascomycota (Müller 2003; Martín-Pinto et al. 2004; Ganley & Newcombe 2006; Kauhanen et al. 2006; Arnold & Lutzoni 2007; Alonso et al. 2011; Terhonen et al. 2011). In our study, Dothideomycetes had the richest species assemblages and highest isolation

46 frequency, 44,8 % and 66,1 %, respectively. The high percentage of isolation frequency can be explained with Phoma sp. (14), which was isolated at every sampling time in both treatments.

Botella and Diez (2011) investigated fungal diversity in Pinus halepensis Miller stands in Spain. Their results were similar to the findings in the present study; Dothideomycetes was the dominant class in species richness and in isolation frequencies. The second most dominant classes were Leotiomycetes and Sordariomycetes. Terhonen et al. (2011) studied the diversity of mycobiota in Scots pine needles in Finland and their results revealed that Leotiomycetes was the most dominant class. The differences in result pattern could be due to the age of the trees and sampling sites. On the other hand, Leotiomycetes was the most dominant class in June at the first sampling in both treatments. After that, isolates from Dothideomycetes became more abundant in both treatments and they were overwhelmingly abundant in fungicide treated seedlings.

In this study, endophytic fungal taxa isolated from the Scots pine needles consisted of few common and many rare taxa. This is a common pattern that has been seen in earlier studies of endophyte assemblages in Pinus spp. needles (Kowalski 1993; Deckert & Peterson 2000; Ganley & Newcombe 2006; Guo et al. 2008; Zamora et al. 2008; Botella & Diez 2011; Terhonen et al. 2011). Hormonema dematioides, Lophodermium spp., Alternaria sp., Epicoccum sp., Cenangium ferruginosum Fr. and Cyclaneusma minus have been observed as common foliar endophytes of Scots pine (Kowalski 1993; Gourbière & Debouzie 2003; Martín-Pinto et al. 2004; Terhonen et al. 2011). Only a few groups of earlier reported endophytes were detected in our study.

4.3.1 Dothideomycetes

The most isolated genus in the present study was Phoma. It was isolated from both treatments over the entire sampling period. However, it was more abundant in fungicide treated seedlings (Table 4; Figure 5-9). The genus Phoma has worldwide distribution and consists of a large group of fungi that are found in many different

47 environments, hosts and tissues. To date, more than 220 species have been recognised but the actual number of species is most likely higher. Taxa within this genus, is problematic for identification because of the asexual (mitosporic) nature of most species (Aveskamp et al. 2008). Phoma has been reported to include functionally different fungi such as, endophytic (Mohandoss & Suryanarayanan 2009), saprotrophic (Kim et al. 2007) and pathogenic (Thomidis et al. 2011) in many different hosts. Also a few results have been reported about the antagonism by P. etheridgei L. J. Hutchison & Y. Hirats and P. glomerata against plant pathogens (Hutchinson et al. 1994; Sullivan & White Jr. 2000).

Phoma has been detected in earlier studies in Scots pine but not with frequencies as high as in this study (Martin-Pinto et al. 2004; Menkis et al. 2006, Terhonen et al. 2011). The Phoma species identified in this study were P. glomerata, P.macrostroma and P. herbarum. The most isolated OTU was morphogroup 14 and it was identified only to genus level. P. glomerata and P. herbarum were the most isolated fungi in CCA-treated Pinus radiata D. Don wood, so they could have an ability to degrade preservatives or have the ability to tolerate chemicals (Kim et al. 2007). Thomidis et al. (2011) studied triazole fungicide tebuconazole and its effectiveness on P. glomerata in vitro. Tebuconazole is a demethylation inhibitor fungicide, like propiconazole. They did find a significant inhibitory effect on P.glomerata, whereas in our study there was no inhibitory effect when compared to the number of Phoma isolates from the control and fungicide treated seedlings. Their study was conducted under a laboratory condition, so it is crucial to test these applications also in the field situation.

Phoma spp. have been reported as a plant pathogens in some temperate and in tropical areas but not in Scandinavia yet. There could be a possibility with climate change, that new pathogens are introduced into new areas with more suitable adaptive capability. Phoma spp. in Scandinavian nurseries should be further investigated because of the pathogenicity of the fungus as well as the great amount of isolation frequencies obtained in this study.

Epicoccum nigrum has been isolated from conifers (Menkis et al. 2006; Botella & Diez 2011; Terhonen et al 2011). It is a possible generalist and opportunistic endophyte with an epiphytic lifestage (Petrini 1991; Botella & Diez 2011). Bakys et

48 al. (2009) investigated fungi in declining common ash (Fraxinus excelsior L.) in Sweden and they isolated E. nigrum from symptomless shoots. In inoculation tests, E. nigrum caused necrotic lesions to bark and cambium. E. nigrum is also studied to be an effective biocontrol agent against brown rot of peach fruit (De Cal et al. 2009).

Cladosporium sp. isolate was observed once in July in control seedlings. This genus is ubiquitous and distributed worldwide in many ecological niches (Zalar et al.2007). Cladosporium spp. have been isolated also from Scots pine (Menkis et al. 2004; Terhonen 2011). This fungus is reported to be very sensitive to pollutants (Magan et al. 1995) and fungicides (Elmholt 1991). Moricca et al. (2001) reported antagonistic activity by Cladosporium tenuissimum Cooke against spore germination of Cronartium flaccidum (Alb. & Schwein.) G. Winter and Peridermium pini (Willd.) Lév., causal agents of pine rust in Europe. Authors concluded that the fungus is an aggressive mycoparasite, and its survival without rusts makes it a promising agent for the biological control of pine rusts. Cladosporium spp. has also been studied to control powdery mildew (Mmbaga et al. 2008). In addition, Cladosporium sp. has been studied in vitro to antagonize Gremmeniella abietina (Santamaria et al. 2007), although the effect was intermediate. Also Alternaria sp., Aureobasidium sp. and Trichoderma sp. (common endophytes of pine) inhibited the growth of G. abietina. The antagonistic activity was mainly the ability to compete against the pathogen. None of these three endophytes were isolated in our study. Propiconazole is routinely used and mainly targeted against G.abietina in the protection of Scots pine seedlings. The fungicide application does not give any opportunity to these possible natural competitors to evolve in the seedlings during growth period.

4.3.2 Leotiomycetes

The second most dominant genus was Lophodermium. Group 10 was identified as Lophodermium pinastri and it was the second most isolated species after Phoma spp. Lophodermium spp. are very common species to find in conifer needles (Stone et al. 2000; Müller 2003; Alonso 2011). The genus contains needlecast pathogens and endophytes with a saprotrophic mode (Minter & Millar 1980). L. pinastri is reported to be a common endophyte of Scots pine (Kowalski 1993; Gourbiére & Debouzie

2003; Terhonen et al. 2011) and other Pinus spp. It is a primary colonizer of symptomless still attached fresh needles (Gourbiére et al. 2003). This fungus has the ability to remain active as saprotroph when the needles fall (Osono & Hirose 2010; Boberg et al. 2011). It is classified as a facultative biotroph, so if the host is weakened, fungus has the ability to become pathogenic. L. pinastri is usually isolated from older needles and also in younger needles with lower frequencies (Stenström & Ihrmark 2005).

Although more than 20 species from genus Lophodermium are known to colonize needles of conifers (Ortiz-García 2003), only L. seditiosum is considered to be pathogenic to Pinus spp. (Diwani & Millar 1987). It is a serious needle pathogen which often infects pine seedlings in nurseries and in plantations. Infection can result in reduced growth with needle loss or even death of young seedlings (Stenström & Ihrmark 2005). The fungus infects green primary and secondary needles during late summer or autumn by ascospores from fallen needles (Minter & Millar 1980; Diwani & Millar 1990). Lophodermium needlecast is routinely controlled by fungicides in Finnish nurseries (Poteri 2008). L. seditiosum was not identified to be present in our study.

L. piceae is a common needle endophyte of Norway spruce. It is a neutral host specific endophyte and it has an active role as a significant decomposer of senesced needles (Müller 2003; Korkama-Rajala et al. 2008). There is a possibility that this species ended in the Scots pine needles by accident of dispersal because L. piceae is a very host specific fungus (Müller 2003).

4.3.3 Sordariomycetes

Gibberella sp. has been detected earlier in decayed roots of conifer seedlings and in symptomless shoots of common ash (Fraxinus excelsior) (Menkis et al. 2005; Bakys et al. 2009). The genus has also species which are severe plant pathogens, like G. circinata Nirenberg & O‟Donnell which causes pitch cankers on Pinus spp. (Wingfield et al. 2008; Gordon et al. 2011).

Phomopsis sp. was isolated once in fungicide treated seedlings during the whole sampling period. This genus has been reported to be an endophyte of Pinus spp. (Guo et al. 2008; Botella & Diez 2011; Kowalski & Drozynska 2011). Phomopsis sp. has been reported to coexist with Gremmeniella abietina in stem lesions of Norway spruce nursery seedlings (Borja et al. 2007). Phomopsis has also been suggested to have a facultative saprotrophic mode of infection as it has been found in the necrotic tissues, which Thekopsora areolata causes, on the shoots of Norway spruce seedlings (Hietala et al. 2008). Phomopsis is occasionally isolated from the lesions on birch leaves and sometimes on nursery seedlings of silver birch (Lilja et al.2010).

Podospora sp. was isolated once in control seedlings in September. In an earlier study it has been isolated from the roots of broadleaved trees (Kwasna et al. 2007).

4.3.4 Pyrenomycetes

Hypoxylon sp. was isolated in control seedlings once in June and in September. This genus has earlier been reported as an endophyte of white spruce (Picea glauca) (Stefani & Berube 2006) and Scots pine (Terhonen et al.2011).

4.3.5 Eurotiomycetes

Penicillium sp. was one of the common OTU isolated in the present study. It was isolated from both treatments. It includes species of soil microfungi (Baar & Stanton 2000; De Santo et al. 2002). This genus has been isolated from Scots pine needles as an endophyte (Terhonen et al. 2011) and from decayed roots of conifers (Lilja et al. 1992; Menkis et al. 2006).

4.3.6 Agaricomycetes

Sistotrema sp. was one of the common OTU isolated in this study. This fungus is a saprotrophic basidiomycete. Species of this genus are often detected in decayed

51 wood of pine (Veerkamp et al. 1997) and in needle litter of Scots pine (Boberg et al. 2011).

Hypholoma sp. was isolated once in June in fungicide treated seedlings and once in October in control seedlings. Terhonen et al. (2011) isolated Hypholoma as an endophyte of Scots pine. However, this genus is reported to be widespread decomposer of dead conifer wood in temperate and boreal forests (Vasiliauskas et al. 2007).

4.3.7 Zygomycetes

One isolate from the class zygomycetes was observed: Mucor sp. It was isolated in August from the control seedlings. In earlier studies, Mucor sp. has been detected in decayed roots of conifers (Menkis et al. 2006) and in soil (De Santo et al. 2002).

4.4 The impact of fungicide treatment to community structure of endophyte population

Overall, all isolated fungal populations in control and fungicide treated seedlings were dominated by one morphological type that had high isolation frequencies (Figure 4-9). In the beginning of sampling (in June) and at the end of sampling (in October) there were few dominant morphotypes. The isolation frequencies of common species varied with season in both treatments (Table 5).

Although there were no statistical differences between population sizes in the fungicide treated and control seedlings, there were qualitative differences in the composition of endophyte OTUs. A total of five OTUs were detected only in fungicide treated seedlings (Table 3). They were: Phoma sp. (group 26), Lophodermium sp. (20), Gibberella sp. (58), Phomopsis sp. (27) and Unclassified Dothideomycetes (40). Control seedlings harboured 9 exclusive OTUs: Lophodermium sp. (49), Hypoxylon (53), Phoma sp. (39), Mucor sp. (55), Hypoxylon (21), Cladosporium sp. (45), Phoma sp. (36), Podospora sp. (54) and unidentified

Sordariomycetes (1). There were also seasonal differences in the presence of common and exclusive OTUs (Table 4-5). This indicates that seedlings in both treatments were colonized by different fungi. Similar kind of results have been obtained in earlier studies concerning foliar mycobiota and fungicide application (Mmbaga & Sauvé 2009; Mohandoss & Suryanarayanan 2009)

According to Sorenson similarity indices, the most similar community structure between treatments was in June and the least similar was in October (Table 7). This indicates that there were differences in fungal community structure between treatments through growing period, hence fungicide treatment had effect on the endophyte community structure of Scots pine needle.

4.5 The impact of fungicide treatment on fungal epiphytes

The number of epiphyte frequencies was higher than isolated endophyte frequencies in control seedlings in both sampling times. Isolated epiphytes from fungicide treated seedlings in September were lower than endophytes. In October the isolated epiphytes increased to the same level as endophytes. According to Mann-Whitney tests results, there were no significant difference between endophytes and epiphytes in both treatments during sampling period. However, in September there was a significant difference (p = 0,034) between epiphyte isolate frequencies from fungicide treated and control seedlings. In October there was no significant difference between treatments. Epicoccum nigrum and Sistotrema brinkmannii were the most common isolated fungi. Phoma was not isolated among the two most common epiphytes. Since the increase in epiphytic isolates in October were clear, it might indicate that the fungicide was removed/degraded from the external part of the treated needles and this enabled new infections of foliar mycobiota occur.

Coppola et al. (2011) investigated the effects of six commonly used fungicide applications in the Mediterranean area on the fungal diversity in grapevine yard soils (compost composed of ligneous pruning residues; a biomixture). Fungicide application did alter the species diversity due to lack of different new species in the fungicide treated soil biomixture compared to the control substrate. However, 70

53 days after the last fungicide application there were no differences in the species assemblages between fungicides treated and control biomixture. The authors suggested that the changes in microbial community induced by fungicides were only temporary and the substrate was able to restore the original fungal assemblages in a short time. Few species were detected at all times in fungicide treated soil residues. Authors suggested that these constant species could possibly degrade the fungicides applied or at least the fungicides did not affect them.

Tu studied (1993) the effects of two fungicides, captanol and chlorothanil, on fungal populations in soil. Application of fungicides decreased fungal populations at first but recovery of the fungi was fast. Elmholt & Smedegaard-Petersen (1988) investigated the effect of two fungicides, propiconazole and captafol, on non-target soil fungi and non-target epiphytes in leaves of spring barley. They found that both fungicides did have a diminishing effect on the colonization frequency of epiphytes and on soil fungi. After 30 days of last fungicide application, there were not anymore statistically significant differences between untreated and treated plots. There were also differences between the fungicide treatments: propiconazole did affect significantly on the species assemblages for a one month, whereas captafol significantly reduced the fungi for more than a month.

Mmbaga & Sauvé (2009) investigated if different fungicide treatments did affect on the epiphytic microbial communities on foliage of flowering dogwoods. Authors concluded that fungicide treatment did not kill all the epiphytes or if they did, there was a rapid re-colonization of new epiphytic fungi. They did not find difference between treatments in microbial diversity but there were differences between microbial community structure. Also they detected several exclusive epiphytic fungi species in control leaves that are known to suppress foliar diseases. Authors suggested that factors, which alter epiphytic fungal populations, could have an impact on the host‟s defence against pathogens by disrupting the natural fungal communities on foliage.

One example of routine fungicide application against a specific pathogen and the increased abundance of another weaker pathogen is the control of dogwood anthracnose caused by Discula destructive Redlin in Tennessee commercial flowering dogwood nurseries in 1990s (Hagan & Mullen 1995; Daughtrey et al.

1996). Shortly after the extensive applications of fungicides began for the control of the main disease, powdery mildew (weaker competitor) became more abundant and hence, routine fungicide application became obligatory for the both diseases in the commercial production of flowering dogwoods.

4.6 The impact of fungicide treatment on the growth height of Scots pine seedlings

There were no statistically significant differences in the mean heights of seedlings between control and fungicide treatments. However, there was a clear difference observed in the seedling growth rate between treatments. Seedlings growth was comparable between May to June but the fungicide treated seedlings grew faster till the end of growth period. According to previous studies, reason for this difference could be that propiconazole induces the growth of Scots pine seedlings by enhancing the uptake of nitrogen and modifying the synthesis of free amino acids when the seedlings are grown at good fertilization levels (Peltonen & Karjalainen 1992; Laatikainen 2006).

On the other hand, endophytes are known to produce plant growth hormones such as gibberellins (MacMillan 2002; Kawaide 2006). The number of endophytes slowly increased in fungicide treated seedlings from June to September. In control seedlings the isolate frequency dropped in September, although, the species composition could play more important role than isolate frequencies in plant growth. Hamayun et al. (2009) studied Phoma herbarum and its potential to promote soybean growth. They showed that P. herbarum did produce gibberellins, and hence, did have a potential in promoting plant growth. Phoma glomerata is also reported to produce gibberellins (Rim et al. 2007). Phoma species were isolated more frequently from fungicide treated seedlings. Penicillium sp. was isolated in the present study, and Penicillium citrinum Thom is also reported to produce gibberellins (Khan et al. 2008).

4.7 Technical considerations

Distinction was made between isolated foliar mycota, as endophytes that grew out from the surface sterilized needle pieces, and epiphytes that grew out from the non- sterilised needles. However, some epiphytic species were observed among the isolated endophytes, such as Cladosporium sp., Epicoccum sp. and Penicillium sp. Petrini suggested (1991) that endophyte assemblages often contains facultative epiphytic species which enter foliar tissues when the senescence of tissue has began. Müller speculated (2003) that some of the epiphytic fungal isolates obtained from the surface sterilized needles might have endedup in the plant by accidental dispersal and survived as spores trapped in surface cavities, stomata or in a vascular system. Arnold speculated (2007) that subcuticular infections may persist through surface sterilization and result as an “endophytic” isolate.

There is a possibility that only fast-growing fungi were detected in our study. Slow- growing fungi or obligate biotrophs may escape detection and hence, were not isolated (Hyde & Soytong 2008). Also many endophytes are fastidious and will not grow on the common artificial media (Guo et al. 2001). In the present study epiphytic fungi were present among endophytic isolates. This could suggest that there were problems with the surface sterilization method. Leaf imprint –method is one of the new methods used for testing whether surface sterilization efficiently eliminates epiphytes. It involves making leaf imprints on the agar surface. If no fungi emerge, then sterilization protocol can be considered to be effective (Schulz et al. 1998; Sánchez-Marquez et al. 2007). In addition, morphogrouping may have been biased because populations of some fungal species are morphologically either homogenous or heterogeneous, therefore it is possible that all species may not have been detected (Müller 2003). Also the amount of fungal isolates and different species might increase with the increase of number of the collected needles (Hyde & Soytong 2008).

Identification of fungi with sequencing and using BLAST searches could be questionable. The sequence data is compared with sequences loaded from public databases e.g. GenBank. The problem is that these databases include a lot of isolates that have been incorrectly named. Also many different species have sequences that

56 are very close to each other, so this may lead to misidentification of the fungi (Ko et al. 2011).

5 Conclusions

The present study indicates that propiconazole treatment does have side-effects on the non-target foliar mycobiota and growth of Scots pine seedlings. The differences were not statistically significant but there were quantitative and qualitative differences in fungal assemblages between fungicide treated and control seedlings.

Propiconazole application is routinely used on Scots pine during growing season in forest nurseries in Finland. It is targeted against Scleroderris canker and snow blight pathogens of Scots pine. Propiconazole is reported to have side-effects on mycorrhizal fungi (Laatikainen 2006), non-target soil fungi (Manninen et al. 1998) and now also in non-target foliar mycobiota. The lack of mycorrhiza can result in slow development and even death of conifer seedlings after out-planting. Also if the non-target soil fungi in the rhizosphere are reduced, it can reduce plant growth, increase the risk of pathogen infection and alter the availability of nutrients. All these factors may reduce the survival of host plant (Allen 1992).

Fungicides have been reported to alter fungal species composition (Mmbaga & Sauvé 2009) and reduce the number of fungal isolates in foliage (Stirling et al. 1999; Mohandoss & Suryanarayanan 2009). Elimination of competitor endo- or epiphytes enables the weaker competitors to colonize the host. Species composition was different between propiconazole treated and control seedlings. Mainly it was seen in the number of exclusive fungi. Also Cladosporium sp. was detected only in control seedlings and it is reported to suppress foliar diseases. Hence, it could be stated that the use of fungicides could reduce the potential of biological biotic disease control.

Routinely used fungicides may result to fungicide resistance by the target organism. Jo et al. (2008) investigated the development of resistance by pathogen Sclerotinia homoeocarpa F. T. Benn. on turfgrass towards systemic fungicides, e.g. propiconazole. They concluded that field populations of the pathogenic fungi continuously changed towards resistant population and adapted to the changing environment.

Any common nursery foliar disease pathogens were not isolated nor detected from control seedlings. However, there were several unidentified fungal isolates, so the possibility of latent pathogens present in the needles could not be excluded. In

58 addition, there were isolates identified to belong in the genus Lophodermium, which contains the species L. seditiosum, a severe needlecast causing pathogen of Scots pine.

Gremmeniella abietina, the causal agent of Scleroderris canker, was not isolated in our study. Epidemics occur commonly in nurseries and forests after mild winter, start of cool and rainy growing season and warm late autumn (Petäistö & Heinonen 2003; Kurkela 1981, Sairanen, 1990, Borja et al.2006; ref. Lilja et al. 2010). G. abietina has a life cycle of 2-3 years. Asexual conidia are formed on pine one year after infection and ascocarps are formed 3 years after infection or later (Petäistö 2008). First year seedlings are most susceptible to spore infection in late summer, and second year seedlings in early summer (Petäistö & Kurkela 1993, Petäistö 1999, Petäistö & Laine 1999, Petäistö, unpublished data; ref. Petäistö & Heinonen 2003). First year seedlings are at risk of pathogen attack but second year seedlings are more at risk because G. abietina spore release extends from the end of May into late summer during the active shoot growth (Petäistö 1999; Petäistö & Heinonen 2003). Propiconazole induced the height growth of Scots pine seedlings in the present study, which may increase the susceptibility of the host to G. abietina (Ylimartimo & Haansuu 1993). Also increased nitrogen levels and incomplete lignificaton due to rapid growth, are factors that could increase host susceptibility to G.abietina (Petäistö & Repo 1988, Ylimartimo 1991, Barklund 1993; ref. Lilja et al. 2010). This could provide a basis for further studies to investigate possible infection of G.abietina in second year seedlings that have not been fungicide treated at all in their lifetime.

Many beneficial plant-microbe interactions are possibly disturbed in agriculture with the use of pesticides (Andrews et al. 2010). Species have even gone into extinction because of the excessive use of toxic chemicals in agriculture (Geiger et al. 2009). The ongoing climate change may increase problems with disease causal agents that benefits from the high precipitation and warm autumn, like rusts (Lilja et al. 2010). More suitable climatic conditions may be seen also as new introduced fungal species, and native species that now have the ability to expand to new geographical areas (Lilja et al. 2010). Seedlings should be healthy, strong and associated with beneficial microbial community in their roots and upper parts, in order that, the survival after out-planting is secured in the best way. Climate change has brought major storms to

Finland and these can be drastic to trees, especially in late fall when frost has not developed. Fallen-trees increase the risk of insect epidemics in the forest, so it is crucial that planted seedlings have natural competitors already in their root, stem and foliar systems.

No drastic changes were observed in the fungal abundance, diversity and community structure of Scots pine seedlings. There were qualitative and quantitative differences in foliar mycobiota between propiconazole treated and control seedlings. However, differences were small and mainly seen as difference in exclusive fungi. Fungicide treatment alters the community structure of foliar mycobiota in needles, so further studies could be conducted to investigate if the resultant mycobiota alters the host susceptibility or resistance to disease. Also studies that includes more growing seasons and includes epiphytic fungi could create more information about fungicides and their side-effects to non-target foliar mycobiota of Scots pine.

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Appendices Appendix 1: DNA exctraction for preparing the solutions

TE buffer (10:1)

1 M Tris HCL 1 ml

0.5 M EDTA pH 8.0 0.2 ml

H2O, adjust to 100 ml

EDTA 0.5 M pH 8.0

EDTA X 2 H2O 186.1 g

H2O 800 ml

Adjust pH with ~ 20 g NaOH, EDTA will not be dissolved until the pH is high enough.

Add H2O to 1000ml.

2 % CTAB + buffer 10 ml

0,2 g CTAB (Hexadecyltrimethyl amonium bromide)

H2O 5,78 ml

Dissolve with heating.

Then Add:

1 M Tris-HCl 1ml

5 M NaCl 2,8 ml

0,5 M EDTA 0,4 ml

-mercaptoethanol 20 l

Appendix 2: PCR protocol

PCR master mix for one sample:

1. autoclaved MilliQ-water 13,5 μl 2. Biotools buffer (10X) 2,5 μl 3. ITS1 (25 μM) 0,5 μl 4. ITS4 (25 μM) 0,5 μl 5. dNTPs (10 μM) 0,5 μl

6. MgCl2 2,0 μl 7. Biotools DNA polymerase (1U/ μl) 0,5 μl

total volume 20 μl

8. DNA directly from the extraction

(diluted 1/10) 5 μl

total 25 μl

Negative control had sterile autoclaved MilliQ-water 5 μl instead of DNA.

PCR program:

1. 4 minutes at 95oC (initial denaturation) 2. 45 sec. at 94oC (denaturation) 3. 45 sec. at 55oC (annealing) 4. 1 min. at 72oC (extending) 5. 10 min. at 72oC (extension) 6. 1 min- 60 min. at 4oC (cooling)

Step 2 to 4 was cycled 30 times.