the effect of fungicide treatment on the non-target foliar
TRANSCRIPT
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
Faculty of Agriculture and Forestry Laitos Institution Department
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
Master‟s Thesis
Aika Datum Month and year
May 2012
Sivumäärä Sidoantal Number of
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
Maatalous-metsätieteellinen tiedekunta Laitos Institution Department
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
Maisterin tutkielma
Aika Datum Month and year
Toukokuu 2012
Sivumäärä Sidoantal Number of
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
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
2
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
3
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
4
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
5
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
6
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
7
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.
8
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
9
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-
10
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
11
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
12
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
13
(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).
14
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
15
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).
16
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.
17
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.
18
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.
19
Figure 1. Experimental seedling rack in June.
Figure 2. Experimental Scots pine seedling in June.
20
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.
21
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
22
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).
23
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
24
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.
25
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 %
26
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).
Figure 3. The frequencies of endophytic isolates in control and fungicide treated P.
sylvestris needles between June and October.
Figure 4. The number of endophytic OTUs in control and fungicide treated P.
sylvestris needles between June and October.
0 1 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
June July August September October
freq
uen
cies
Control Fungicide treatment
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
June July August September October
freq
uen
cies
Control Fungicide treatment
27
Table 3. Endophytic fungal taxa isolated from P. sylvestris needles
Isolate
group
number
GenBank
accession no.
of best
matches
Mi(%) /
Qc(%)a)
Description of
best matches
Our suggestion
of isolate name
Freqd) Classb)
Phylumc)
10 AF473558 100/100 Lophodermium
pinastri
Lophodermium
pinastri
8 L(A)
8 AY373923
100/98 Penicillium
melinii
Penicillium
sp.
8 E(A)
7
DQ093676
98/100 Gibberella
avenacea
Gibberella
sp.
5 S(A)
9 AF473556
100/100 Lophodermium
pinastri
Lophodermium
sp.
4
L(A)
26 FN868459
100/99 Phoma
herbarum
Phoma
sp.
4F) D(A)
14 FN868459
100/99 Phoma
herbarum
Phoma
sp.
68 D(A)
49 FR837918
99/99 Lophodermium
piceae
Lophodermium
sp.
2C) L(A)
20 AF473556
100/100 Lophodermium
pinastri
Lophodermium
sp.
1F) L(A)
53 AF201717
100/99 Hypoxylon
multiforme
Hypoxylon
sp.
1C) P(A)
58 DQ093676
100/100 Gibberella
avenacea
Gibberella
sp.
1F) S(A)
39 FN868459 100/98 Phoma
herbarum
Phoma
sp.
2C) D(A)
24 DQ093653
100/94 Sistotrema
brinkmannii
Sistotrema
sp.
8 A(B)
5 AF473558
100/100 Lophodermium
pinastri
Lophodermium
sp.
5 L(A)
28
3 FN868459 100/99 Phoma
herbarum
Phoma
sp.
2 D(A)
12 HM036611
100/99 Phoma
macrostroma
Phoma
macrostroma
3 D(A)
55 DQ093728
95/98 Mucor
hiemalis
Mucor
sp.
1C) Z(Z)
15 FN868459 100/99 Phoma
herbarum
Phoma
sp.
7 D(A)
21 AF201717
100/99 Hypoxylon
multiforme
Hypoxylon
sp.
1C) P(A)
45 HQ533788
100/100 Cladosporium
sp.
Cladosporium
sp.
1C) D(A)
11 FR686560
100/98 Hypholoma
fasciculare
Hypholoma
sp.
2 A(B)
36 FN868459 100/98 Phoma
herbarum
Phoma
sp.
5C) D(A)
33 FN868459 99/100 Phoma
herbarum
Phoma
sp.
4 D(A)
35 DQ093699
100/100 Phoma
glomerata
Phoma
glomerata
12 D(A)
27 AY465468
98/99 Phomopsis
sp.
Phomopsis
sp.
1F) S(A)
4 DQ093668
100/100 Epicoccum
nigrum
Epicoccum
nigrum
6 D(A)
54 EF197084
100/98 Podospora
pleiospora
Podospora
sp.
1C) S(A)
2 FN868459 100/99 Phoma
herbarum
Phoma
herbarum
8 D(A)
23 AY561198
98/94 Foliar
endophyte
of Picea
glauca
Unidentified
fungal
endophyte
4
29
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
40 GQ153112
100/99 Dothideo-
mycetes sp.
Unclassified
Dothideo-
mycetes
1F) D(A)
1 GU062252
82/86 Lecythophora
sp.
Unidentified
Sordario-
mycetes
1C) S(A)
30
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).
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
0
1
2
3
4
5
6
7
freq
uen
cies
Control Fungicide treatment
31
(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).
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
0 1 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18 19 20 21
freq
uen
cies
Control Fungicide treatment
32
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.
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
0 1 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18 19
freq
uen
cies
Control Fungicide treatment
33
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.
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
0 1 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18
Fre
qu
enci
es
Control Fungicide treatment
34
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).
35
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.
0
1
2
3
4
5
6
7
8
9
10
11
Fre
qu
enci
es
Control Fungicide treatment
36
Table 5. Common and exclusive fungal OTUs for control and fungicide treatments
between June and October.
June July August September October
Control
exclusive
4 6 6 7 8
Fungicide tr.
exclusive
5 2 5 6 7
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).
37
Table 6. Shannon‟s diversity index for isolated endophytic fungi in control and
fungicide treated needles of P. sylvestris from June to October.
Treatment/
month
Species
richness
Abundance Shannon‟s (H)
Control/
June
9 15 2,06
Control/
July
10 16 1,92
Control/
August
10 22 1,92
Control/
September
12 18 2,13
Control/
October
13 18 2,51
Fungicide
treatment/
June
10 13 2,24
Fungicide
treatment/
July
6 18 1,04
Fungicide
treatment/
August
9 20 1,77
Fungicide
treatment/
September
11 23 1,95
Fungicide
treatment/
October
14 23 2,1
38
Table 7. Sorenson‟s similarity index for isolated endophytic fungi between
control and fungicide treated needles of P. sylvestris from June to October.
Comparison
month/treatment
Shared
species
Sorenson
index
June/Control June/Fungicide
treatment
5 0,53
July/Control
July/Fungicide
treatment
4 0,5
August/Control August/Fungicide
treatment
4 0,42
September/Control September/Fungicide
treatment
5 0,43
October/Control October/Fungicide
treatment
5 0,4
Table 8. Mann-Whitney test results for the frequencies of isolated endophytic
fungi between control and fungicide treated needles of P. sylvestris.
Comparison
month/treatment Asymp. Sig. (2-tailed)
June /Control
June/Fungicide
treatment 0,813
July/Control
July/Fungicide
treatment 0,738
August/Control
August/Fungicide
treatment 1,000
September/Control
September/Fungicide
treatment 0,663
October/Control
October/Fungicide
treatment
0,231
39
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.
40
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
month/treatment Asymp. Sig. (2-tailed)
September/Control September/Fungicide
treatment 0,034
October/Control October/Fungicide
treatment 0,843
0 1 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
September c September t October c October t
freq
uen
cies
Endophyte Epiphyte
41
Table 10. Mann-Whitney tests for epi- and endomycota frequencies.
Month Treatment Comparison Asymp. Sig.
(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
42
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).
Figure 11. Average heights of Scots pine seedlings in control and fungicide treated P.
sylvestris seedlings.
0 5
10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
100 105 110
May June July August September
Hei
gh
ts o
f th
e se
ed
lin
gs,
mm
Control Fungicide treatment
43
Table 11. Paired t-test values for the heights of the P. sylvestris seedlings/treatment
in June to September.
Month Mean
difference
Std.
error df Sig. a
Lower
bound
Upper
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
44
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
45
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
46
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
47
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
48
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
49
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
50
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).
51
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
52
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
53
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
54
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.
55
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).
56
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
57
are very close to each other, so this may lead to misidentification of the fungi (Ko et
al. 2011).
58
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
59
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
60
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.
61
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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
76
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.