Effects of cutting height, fungal symbiont and soil
properties on meadow fescue’s growth
Sanna-Maria Keronen
Ecology and Evolutionary Biology
Master’s thesis
Credits: 30 ECTs
Supervisors:
Benjamin Fuchs
Marjo Helander
Kari Saikkonen
5.5.2022
Turku
The originality of this thesis has been checked in accordance with the University of
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Master's thesis
Subject: Ecology and Evolutionary Biology
Author: Sanna-Maria Keronen
Title: Effects of cutting height, fungal symbiont and soil properties on meadow fescue’s growth
Supervisors: Dr. Benjamin Fuchs, Dr. Marjo Helander, Dr. Kari Saikkonen
Number of pages: 43 pages
Date: 5.5.2022
Abstract
Grasses have a beneficial effect on the soil organic matter content, and hence carbon
sequestration of agricultural soils throughout the world. Because grasslands cover one-third of
the land on Earth and meadows and pastures approximately 70% of the world’s agricultural
area, grasses serve as a powerful carbon sink. Meadow fescue is an important perennial
pasture and forage grass in Scandinavia. It has deep roots, rapid growth in spring, high after
growth ability, low straw formation and excellent winter hardiness. It has excellent forage
quality and amount of yield and superior fiber digestibility. Meadow fescue cultivars are often
symbiotic with seed transmitted systemic fungal endophytes (Epichloë uncinata) offering
various benefits for their hosts, for example increased growth and reproduction, and resistance
to herbivores, pathogens and abiotic environmental stresses, and systemic fungal endophytes
thereby enhance the competitive abilities of endophyte-infected plants. In my master’s thesis I
studied the effects of cutting height, residues of glyphosate based herbicides (GHBs) in soil,
sterilized soil and fungal endophyte Epichloë uncinata to aboveground biomass production,
root biomass and chlorophyll content in a greenhouse experiment. To test the importance of
cutting height to grass performance, I assigned grasses to three different cutting treatments:
uncut, cut to the height of 5 cm and cut to the height of 15 cm. Half of the plants were symbiotic
to the fungal endophyte, the other half were endophyte-free. Plants were assigned to three
different soil groups: control, GBH treated and sterilized soil. Cutting height significantly
affected the total aboveground plant biomass, root biomass and chlorophyll content. Uncut
meadow fescues produced the largest total aboveground biomass and root biomass which
were significantly higher compared to total aboveground biomass and root biomass from
grasses cut to 15 cm or 5 cm. The grasses cut to the height of 5 cm had the smallest total
aboveground biomass and root biomass, which were further significantly smaller compared to
grasses cut to 15 cm. Endophyte did not affect the total aboveground biomass, root biomass
or the chlorophyll content of the plants. Meadow fescues produced higher amount of total
aboveground biomass when growing in a sterilized soil compared to control or GBH treated
soil. Root biomass was not significantly affected. Glyphosate residues in the soil decreased
the amounts of total aboveground and root biomass of grasses. Chlorophyll content was
highest in plants growing in sterilized soil. I conclude that to improve the total aboveground
plant biomass and root biomass, the use of GBHs should be avoided and rotational grazing,
in which animals are moved between pastures before they have been eating the grass too low,
should be preferred. This will enable farmers to collect more harvest and sequester more
carbon at the same time.
Keywords: Schedonorus pratensis, Epichloë uncinata, systemic fungal endophyte,
glyphosate, soil sterilization, cutting treatment, grazing, silage
0Contents
1 Introduction ................................................................................................................. 1
1.1 Carbon and soil ............................................................................................................... 1
1.2 Removing biomass from perennial grass fields ........................................................... 2
1.3 Meadow fescue (Schedonorus pratensis) ..................................................................... 4
1.4 Systemic fungal endophyte Epichloë uncinata ............................................................ 4
1.5 Glyphosate based herbicides ........................................................................................ 6
1.6 The aims and hypothesis of this study ......................................................................... 7
2 Material and methods ................................................................................................. 9
2.1 Study design and treatments ......................................................................................... 9
2.2 Biomass and chlorophyll content sampling ............................................................... 12
3 Results ....................................................................................................................... 14
3.1 Effect of cutting height and endophyte status on plant parameters ......................... 14
3.2 Effect of soil treatment and endophyte on plant parameters .................................... 17
4 Discussion ................................................................................................................. 21
4.1 Effect of cutting height, soil treatment and endophyte status on plant parameters 21
4.2 General conclusions ..................................................................................................... 23
4.3 The larger scale ............................................................................................................ 24
5 Acknowledgements ................................................................................................... 26
References ....................................................................................................................... 27
11 Introduction
1.1 Carbon and soil
Soil carbon sequestration is a key mitigation strategy for rising carbon dioxide concentration in the
atmosphere, and important in improving the fertility and quality of soil. Carbon stored in soils
worldwide exceeds the amount of carbon stored in phytomass and the atmosphere (Jobbágy &
Jackson 2000). There is no consensus of the size of global soil organic carbon stocks, but most
studies report a global soil organic carbon estimate to be roughly 1500 petagrams of carbon to a
depth of 1 meter and 2400 Pg of carbon to 2 meter depth (Pg C: 1015 g or billion tons of carbon)
(Scharlemann et al. 2014; Paustian et al. 2019). Globally agricultural land covers approximately 5
billion hectares, or 38 percent of the global land surface. About 33 percent of this is used as cropland
and 67 percent consist of pastures for grazing livestock (FAO 2020). At the moment agricultural
lands have been seriously degraded by widespread, unsustainable management. Soil quality is
directly linked to food production, food security and environmental quality through its effects of
energy use in food production, greenhouse gas emissions and water quality. Soil quality describes
the capacity of soil to function as a provider of key ecosystem services, such as decomposing organic
matter, supplying and cycling of nutrients for optimum plant growth, filtering water passing through
soil to support clean groundwater, receiving rainfall and storing water for root utilization, and storing
organic carbon for nutrient retention and mitigating greenhouse gas emissions (Franzluebbers 2012;
O’Mara 2012).
Soils growing perennial grasses have high organic matter content and they can contribute to an
agricultural future with high soil quality, and therefore can mitigate greenhouse gas emissions
through soil carbon sequestration and improve a multitude of other ecosystem responses, including
controlling water quality, improving water and nutrient cycling, and supporting biological diversity.
Agricultural soils would benefit from the reintroduction of perennial grasses to regain soil organic
matter and strengthen their capacity for long-term productivity and environmental resiliency
(Franzluebbers 2012). Crop production results in a loss of soil organic carbon due to decreasing
carbon inputs in the soils and by causing soil erosion (Bakker et al. 2004; Kirkels et al. 2014; Doetterl
et al. 2016). Soil erosion affects vegetation growth and biomass production by changing physical
and chemical properties of soil related to soil fertility, such as water holding capacity, nutrient status
and soil depth (Bakker et al. 2004). Intensification of agricultural practices, like ploughing and the
application of agrochemicals and artificial fertilizer, are linked to a reduced soil biodiversity (Tsiafouli
et al. 2014).
Land use, for example whether the area is primary forest, plantation, pasture or growing crop, affects
to the soil carbon levels. Soil carbon stocks increase when area is converted from crop to pasture
2(+19%), from forest to pasture (+8%), from crop to plantation (+18%) and from crop to secondary
forest (+53%) (Guo & Gifford 2002). Soil carbon declines when area is converted from pasture to
plantation (-10%), from pasture to crop (-59%), from forest to crop (-42%) and from forest to
plantation (-13%) (Guo & Gifford 2002). Pasture grasses maintain a cover of vegetation on the soil,
add organic matter on both above- and belowground, and reduce soil temperatures (Brown & Lugo
1990). The annual carbon input to soil from crop residues originates from straw, stubble and surface
debris, and root biomass left in the soil at harvest, root turnover, exudates and secretions (Bolinder
et al. 2002). Perennial grasses have a beneficial effect on the soil organic matter content, and hence
carbon sequestration of agricultural soils throughout the world. This is mostly because of the root
biomass production of perennial forage crops and the absence or reduction of tillage compared to
annual crops (Paustian et al. 1997). Well-managed grasslands can maintain and accumulate soil
carbon and hence contribute to climate change mitigation (Leifeld & Fuhrer 2010; Soussana et al.
2014; LaCanne & Lundgren 2018; Poeplau et al. 2018).
The soil is where most terrestrial plants start their growth, and home to a diverse community of
microbes and animals. The soil is the source of most beneficial microbes that colonize the
rhizosphere, and that are key players in plant immunity and overall plant performance (Bulgarelli et
al. 2013). Soil microbes provide plants with key functions, such as enhanced growth via improved
nutrition and suppression of soil pathogens (Pieterse et al. 2016; Raaijmakers & Mazzola 2016). An
important soil service is also the protection of above-ground plant tissues against pests and diseases
(Bardgett & Wardle 2010; Mariotte et al. 2018). Plants provide the organic carbon for the
decomposers and the resources for root-associated organisms, for example root herbivores,
pathogens, and symbiotic mutualists (Wardle et al. 2004). Decomposers break down dead plant
material and thereby regulate plant growth and community composition by determining the supply of
available soil nutrients. Root-associated organisms and their consumers influence the quality,
direction, and flow of nutrients and energy between plants and decomposers (Wardle et al. 2004).
The complexity of soil foodweb is essential to maintain high rates of ecosystem function. Activities
that contribute belowground biodiversity losses, such as loss of taxa and trophic levels, cause a
reduction in foodweb complexity and the capacity of soils to perform ecosystem functions (Wall et
al. 2015).
1.2 Removing biomass from perennial grass fields
Grasses can tolerate repeating disturbance well, because by their evolution they have adapted to it.
For example pastures are continually grazed, and lawns and fields are mowed. In addition grass
breeding has increased for example to the forage yields and regrowth, and improved resistance
against pests and pathogens (Saari et al. 2010). In addition to the fast regrowth, grasses can tolerate
3grazing also because of their underground storage organs, basal meristems and tillering capacity
(Huitu et al. 2014).
Herbivores alter carbon and nitrogen inputs to the soil by changing the quality and quantity of organic
inputs (for example herbivore dung and plant litter), by decreasing biological nitrogen fixation through
the consumption of legumes, and through changes in soil conditions, like temperature and moisture
(Bardgett & Wardle 2003; Pineiro et al. 2010), which in turn have an impact in soil microbial
communities and activity (Bardgett & Wardle 2010). Greater microbial activity increases litter
decomposition rates and carbon respiration and also carbon transfer into slow-cycling forms of
carbon, for example microbial necromass (Lange et al. 2015; Sokol & Bradford 2019), which further
increases the potential for carbon sequestration under grazing. Under eutrophied conditions
herbivores promote soil carbon and nitrogen storage in grasslands (Sitters et al. 2020).
The negative effect of defoliation on growth rate or final biomass is typically less than proportional to
the removal of live biomass. Sometimes it can even be positive (McNaughton et al. 1983). When
defoliated plants can partially or fully compensate for the removal of biomass, the response is known
as compensatory regrowth (Ferraro & Oesterheld 2002). The magnitude how much grasses can
compensate defoliation, are dependent on evolutionary mechanisms, for example coevolutionary
history with big vertebrate grazers (Vail 1992), nutrient levels (Georgiadis et al. 1989; Alward & Joern
1993; Ferraro & Oesterheld 2002), carbon allocation (Briske et al. 1996), recovery conditions
(Oesterheld & McNaughton 1988; Ferraro & Oesterheld 2002) and light environment (McNaughton
1992). In addition defoliation can affect to the root growth and belowground carbohydrate reserves,
decreasing root biomass (Holland et al. 1996; Thornton & Millard 1996) and belowground growth
rate (Oesterheld 1992).
A species of animal, frequency and severity of grazing, method of prehension, treading, excreta
deposited on pastures, and saliva deposited on plants during grazing influence to the plants that are
grazed. These animal factors can cause substantial changes in the persistence, productivity, and
botanical composition of the sward and the regrowth rate of plants following grazing. The extent of
leaf-tissue removal, the accumulation of dead material, canopy structure in relation to light
interception, the microenvironment within the canopy, species composition of the sward and the
general physiological well-being of plants will be altered by the intensity and frequency of grazing
(Matches 1992). Saliva of grazing animals might have an important role for increasing grass growth
(Reardon et al. 1974; Gullap et al. 2011). Grazing intensity of pastures should be regulated to
maintain adequate leaf area for maximum plant growth rates throughout the grazing season
(Matches 1992). Meadow fescue has been shown to have a bigger regrowth if it is cut from the height
of 9 cm instead of 3 cm (Virkajärvi 2003).
41.3 Meadow fescue (Schedonorus pratensis)
Meadow fescue [Schedonorus pratensis (Huds.) P. Beauv syn. Festuca pratensis (Huds.) and
Lolium pratense (Huds.)] is an important forage grass in temperate and cold climates.  It is perennial
and has height of 40-100 cm. It grows as a tuft formation, has rapid growth in spring, high after
growth ability and low straw formation. Meadow fescue has an excellent winter hardiness and forage
quality. Meadow fescue tends to be outcompeted from multispecies grasslands, because its
competitive ability is relatively low. Meadow fescue can cross-breed with tall fescue, perennial
ryegrass (Lolium perenne) and Italian ryegrass (Lolium multiflorum) (Fjellheim & Rognli 2005; Brink
et al. 2010; Saari et al. 2010; Rao & Rognli  2014; Rikkinen 2014). Meadow fescue is a viable
alternative for example to tall fescue and orchardgrass in managed intensive rotational grazing
systems because of its comparable yield and superior fiber digestibility. Meadow fescue has higher
nutritional quality than for example tall fescue (Schedonorus arundinacea) or orchardgrass (Dactylis
glomerata). (Brink et al. 2010). Both cattle and horses prefer meadow fescue in their diet (Allen et
al. 2013).
1.4 Systemic fungal endophyte Epichloë uncinata
Meadow fescue’s fungal endophyte Epichloë uncinata [(W. Gams, Petrini & D. Schmidt) Leuchtm. &
Schardl syn. Neotyphodium uncinatum] subsists entirely on the host’s resources, and the fitness of
an endophytic symbiont depends on the fitness of the host plant. The host, meadow fescue, receives
various benefits, for example increased growth and reproduction, and resistance to herbivores,
pathogens and abiotic environmental stresses, which thereby enhance the competitive abilities of
endophyte-infected plants (Saikkonen et al. 2004; Lehtonen et al. 2005). Epichloë uncinata
endophytes in meadow fescue plants are producing loline alkaloids which are known to have
detrimental effects on invertebrates (Hume et al. 2009; Jensen et al. 2009; Popay et al.  2007; Popay
et al. 2009; Patchett et al. 2011), but have not known effect on grazing mammals (Schardl et al.
2007). However, female voles have been shown to suffer from weight lost because of loline alkaloids
(Huitu et al. 2014). The genotype of meadow fescue affects the amount of alkaloids and fungal mass
in the host (Cagnano et al. 2020). There are several combinations and concentrations of alkaloids,
and their presence is dependent on for example environmental conditions (Spiering et al. 2005).
Systemic fungal endophytes cannot exist without its grass host (except as a cultured mycelia on
artificial media), because they need the host grass for supply for nutrients and water and
dissemination through seeds of the host plant to the next plant generation. Systemic Epichloë
endophytes grow internally and intercellularly throughout the above-ground tissues of the host plant
and into the developing inflorescences and seeds (Saikkonen et al. 2004; Lehtonen et al. 2005). The
coiled hyphae of fungal endophytes do not penetrate the plant cells (Cheplick & Faeth 2009).
5Epichloë uncinata reproduces asexually via the plant seeds. The fitness of the symbiotic endophyte
is totally dependent the fitness of the grass host. Epichloë uncinata provides benefits to its host, such
as competitive advantage against other plants, and higher shoot and root dry weight comparing to
endophyte-free meadow fescues. Extensive root system is a vital characteristic for drought
avoidance, and most likely increases persistence of endophyte-symbiotic meadow fescue under
drought and when competing with other species (Malinowski et al. 1997; Malinowski & Belesky 2000;
Saari et al. 2010). The symbiosis between Epichloë uncinata and meadow fescue can range from
antagonistic to mutualistic depending on the genetic match of the fungal endophyte and the host
grass, and also environmental conditions (Ahlholm et al. 2002; Saikkonen et al. 2004; Saikkonen et
al. 2010). Nutrients are vital for the mutualism between meadow fescue and Epichloë uncinata.
When resources are limited, endophyte-symbiotic meadow fescue produces fewer tillers and seeds,
and has lower total biomass and lower root mass compared to endophyte-free meadow fescue
(Ahlholm et al. 2002). The positive effect from the endophyte to the grass host is strongest in the
environments where nutrients are not a limiting factor, e.g. often in the agroenvironments (Saikkonen
et al. 2006; Cheplick & Faeth 2009; Saikkonen et al. 2013). Endophyte infection affects host’s
morphology, chemistry and physiology. It alters for example leaf rolling, photosynthesis, nutrient
uptake, water-use efficiency, hormonal changes and root morphology (Malinowski & Belesky 2000;
Morse et al. 2007; Cheplick & Faeth 2009). Study with perennial ryegrass has shown, that endophyte
infection interacts with levels of lipids, carbon and nitrogen, amino acids, magnesium, organic acid,
chlorogenic acid, nitrogen availability to alter nitrate and water soluble carbohydrates (Rasmussen
et al. 2008).
Endophyte-symbiotic meadow fescues may survive better in salinity-stress environments compared
to endophyte-free meadow fescues (Sabzalian & Mirlohi 2010). Fungal endophyte helps also to
prevent virus infections in its host grass, probably by deterring of herbivorous insects, which carry
plant viruses (Lehtonen et al. 2006). It has been shown that endophyte infection increases root hair
length and decreases root diameter in tall fescue grasses. That may increase root surface area for
water and mineral acquisition (Malinowski & Belesky 1999).
Endophyte-symbiotic meadow fescue has more panicles, greater reproductive shoot mass and
higher total seed mass than endophyte-free meadow fescue, but this is context dependent. Plant
genotype (e.g.cultivar) and environmental conditions (e.g. nutrients) are affecting to outcome of the
symbiosis (Wäli et al. 2008). The total biomass of endophyte-symbiotic meadow fescue
monocultures was 89% higher than endophyte-free meadow fescue monocultures in high nutrient
conditions (Dirihan et al. 2014). The effect of the endophyte on the annual herbage yield of the
meadow fescue monocultures varies between the cultivars (Takai et al. 2010). On the other hand,
in pastures growing multiple grass species, when meadow fescues have been young, their fungal
endophytes have not increased their ability to compete with other grass species and against
6herbivores, but in older pastures and ungrazed fields fungal endophytes have increased grasses
ability to compete with other grass species and against herbivores (Saari et al. 2010; see also Fuchs
et al. 2016; Fuchs et al. 2017; Hewitt et al. 2020.)
1.5 Glyphosate based herbicides
Today glyphosate based herbicides (GBHs) are globally the most used herbicides
(Luonnonvarakeskus 2019; Uusi-Kämppä 2019). Glyphosate, N-(phosphonomethyl) glycine, has
been in use extensively the past 40 years. It was introduced for weed control in agricultural
production fields in 1974 (van Bruggen et al. 2018). Glyphosate is a non-selective, systemic, post-
emergence herbicide, and it acts as an inhibitor of the enzyme, 5-enolpyruvylshikimate-3-phosphate
synthase (EPSPS), in the shikimate pathway (Duke & Powles 2008). Shikimate pathway is a
metabolic pathway plants use for the biosynthesis of aromatic amino acids (Gill et al. 2017). Besides
plants shikimate pathway is found in many microbes (Leino et al. 2020). Scarcity of the enzyme
leads to the deficiency of aromatic amino acids, which in turn affects various metabolic functions of
the plant and causes the plant to wither (Tu et al. 2001). Glyphosate depletes pools of compounds
needed for carbon fixation, which causes a general disruption of the organism’s metabolism (Duke
& Powles 2008). In addition usage of glyphosate can affect negatively to chlorophyll levels of plants,
even when the plants are glyphosate-resistant (Zobiole et al. 2012). In 2018 1261 000 kilograms of
glyphosate based herbicides were applied on Finnish fields. GBHs are frequently used for weed
control before establishing new perennial grass fields in Finland. GBHs can be sprayed after the
harvest in the autumn or before sowing in the spring (Luonnonvarakeskus 2019; Uusi-Kämppä
2019).
Intensive glyphosate use has led to the selection of glyphosate-resistant weeds and microorganisms
(Powles & Preston 2006). There are also reported health effects in animals associated with chronic,
ultra-low doses related to accumulation of glyphosate and its breakdown product aminomethyl
phosphonic acid (AMPA) in the environment (Shushkova et al. 2009; van Bruggen et al. 2018).
Glyphosate’s half-life in soil ranges from 2 to 215 days, and an aquatic half-life ranges from 2 to 91
days (Giesy et al. 2000; Grunewald et al. 2001; Vera et al. 2010). Glyphosate degrades primarily by
microbial processes to AMPA. AMPA, like glyphosate, is very water soluble, and it degrades slower
than glyphosate (Grunewald et al. 2001). AMPA’s half-life  in soil ranges from 60 to 240 days, and
an aquatic half-life is similar than glyphosate’s aquatic half-life (Giesy et al. 2000; Bergstrom et al.
2011). AMPA degrades to inorganic phosphate, ammonium and CO2 (Borggaard & Gimsing 2008).
AMPA’s degradation process can result a substantial increase of total phosphorous in aquatic
systems (Vera et al. 2010). Glyphosate adsorbs to clay and organic matter, which slows its
7degradation by soil microorganisms, and leads to accumulation in soils over time (Banks et al. 2014;
Sviridov et al. 2015; Okada et al. 2016).
Glyphosate and AMPA inhibit antioxidant enzyme activities and induce the accumulation of reactive
oxygen species (ROS), which result to physiological dysfunction and cell damage (Gomes et al.
2016). Glyphosate and AMPA decrease photosynthesis. Glyphosate increases chlorophyll
degradation, and AMPA disturbs biosynthesis of chlorophyll (Gomes et al. 2016). Even sublethal
glyphosate concentrations in plants, for example from residues in soil or water, decrease plant
resistance to pathogens. Infection by Fusarium species is often more severe in fields treated by
glyphosate before planting compared with untreated control fields (Kremer et al. 2005; St. Laurent
et al. 2008; Kremer & Means 2009; van Bruggen et al. 2015). Besides reduced plant resistance,
indirect effects of glyphosate and AMPA to the health of plants are possible through changes in the
endophytic and rhizosphere microbiome (Kremer et al. 2005; Kuklinsky-Sobral et al. 2005; Berg et
al. 2014; van Bruggen et al. 2016), because many microbes are sensitive to glyphosate (Leino et al.
2020). Soil microbes play diverse and critical role in soil systems and plants performance (Aislabie
& Deslippe 2013; Wall et al. 2015; Wagg et al. 2019).
Surfactants and other adjuvants are added to commercial glyphosate formulations to enhance their
efficacy. Commercial GBH formulations can be more toxic than pure glyphosate due to the toxicity
and action of the surfactants and other adjuvants used (Giesy et al. 2000; Edginton et al. 2004;
Bringolf et al. 2007; Mesnage et al. 2012; Moore et al. 2012; Sihtmäe et al. 2013). For example the
Roundup® formulation is more toxic than glyphosate or AMPA for all taxa tested (Giesy et al. 2000).
1.6 The aims and hypothesis of this study
Grasses have evolved to tolerate high pressure from vertebrate grazers. In agricultural fields
perennial grasses are used in pastures for animals grazing or cut for animals as a form of hay or
silage. I chose meadow fescue for my study species, because it is commonly used in Finnish
pastures and silage fields, and has not been studied as often as for example tall fescue or perennial
ryegrass. GBH are commonly used herbicides in Finland for example in autumn when finishing the
pasture or silage field or crop field before sowing it again. However, the effects of glyphosate
residues in the soil on the growth and biomass production of meadow fescue have not been studied
before. To better understand whether the effect of GBH in soil on meadow fescue plants is connected
to its antimicrobial effect on the soil biota, I added sterilized soil as a treatment, where the soil biota
is destroyed.
8Grass for silage or hay is cut usually low, ca. 5-8 cm height from the soil surface. In this study I
decided to compare uncut meadow fescue biomass production to cutting heights 5 cm and 15 cm
from the soil surface.
My study questions are:
1. Does the endophytic fungus affect biomass production of meadow fescue under cutting treatment?
2. Does cutting height affect the total green biomass production, root biomass and chlorophyll
content of meadow fescue?
3. Does soil treatment with GBH and the presence of the endophytic fungus affect to plants’ total
green biomass production, root biomass and chlorophyll content?
My first hypothesis is that endophytic fungi have a positive effect for growth and recovery after the
cutting treatment based on their mutualistic nature. In addition my hypothesis is that endophyte-
symbiotic grasses will produce more chlorophyll than grasses that are endophyte-free.
My second hypothesis is that cutting height affects the growth, green biomass production and root
biomass production. I predict that cutting at 5 cm height will decrease grass after growth, the total
aboveground and root biomass compared to grasses cut to a height of 15 cm. In addition I predict
that cutting height and the presence of endophytic fungus affect the chlorophyll content of the plants.
My third hypothesis is that GBH and sterilized soil treatments decrease the total aboveground
biomass and root biomass. Furthermore, I predict that growing in GBH treated soil and sterilized soil
decreases the chlorophyll content of the plants compared to the control plants.
92 Material and methods
2.1 Study design and treatments
Meadow fescue seeds used for the study were collected in the previous year (2020) from meadow
fescue plants with known endophyte status, that grew on an experimental field at the Ruissalo
Botanical Garden in Turku. Seeds were checked for the endophyte status before they were used. 3-
5 seeds from each plant were placed over night into an Eppendorf tube containing 1 ml of liquid
(made by 0.9 ml of water, 0.1 ml of ethanol and 0.025 g of Sodium hydroxide). Next day, seeds were
rinsed with tap water, then slightly crushed and microscopically analyzed. In endophyte-symbiotic
plants the endophytic fungal hyphae was detected between the embryonic cells (Figure 1).
Figure 1. Epichloë uncinata hyphae between embryonic cells of the meadow fescue seed
Seeds from seven endophyte-symbiotic plants (E+) and seven endophyte-free (E-) plants were used
in this study. Seeds from the seven E+ grasses were pooled, and similarly seeds from the seven E-
plants were pooled before randomly selected sowing. Seeds were sown on 21.04.2021 to two
planting trays that were filled with Kekkilä Garden Viherkasvimulta planting soil and watered. Each
potting tray consisted of 260 units, and each unit was sown with 3-5 seeds. Units were thinned to
one seedling on 04.05.2021. Both trays had 130 units with E+ seeds and 130 units with E- seeds
that were randomly chosen from seed mixtures (Figure 2). The trays were covered with transparent
plastic to keep the moisture in until the seeds germinated.
10
Figure 2. Meadow fescue seedlings growing on the trays
The soil used for planting the seedlings was collected from a long-term field experiment, which had
been established in 2013 at the Botanical Garden of Ruissalo (Helander et al. 2019). The soil type
of the field was medium clay. Before collecting the soil it was treated with lime (Tarhurin
Puutarhakalkki, 10 kg/100 m2, which had been spread in every spring on the field) and was tilled on
21.04.2021. The soil had been treated with a permitted dose of Roundup Gold® (450 g/l
isopropylamine glyphosate salt, application rate: 6.4 l/ha) that had been applied twice per year (in
the spring and in the autumn) since the year 2014. The control soil received the same amount
sprayed tap water without Roundup® application. The last treatments before collecting the soil for
this study were done on 29.04.2021.
The nutrient values of the soil were (based on an analysis made in 2016): pH 6.2 mg/l, phosphorus
4.2 mg/l, kalium 250 mg/l, calcium 1900 mg/l, magnesium 570 mg/l, sulfur 10.6 mg/l, zinc 2.74 mg/l,
copper 7.5 mg/l, manganese 15 mg/l (Viljavuustutkimus 2016).
Soil for the pots from the experimental field was collected on 11.05.2021. Soil was collected from
the area treated with GBH (glyphosate based herbicide, in this case Roundup®) and from the control
(C) area. Part of the soil from the control area was heat-sterilized (S) in an autoclave (120 °C for 20
minutes). Soil was put in 1.5 liter pots which were put on three separate tables (one table for control
pots, one for glyphosate pots and one for sterilized pots) to prevent contamination. Meadow fescue
seedlings were transferred to the pots on the same day (11.05.2021). There were 60 pots of GBH
soil, 60 pots of control (C) soil and 60 pots of sterilized (S) soil, each group contained 30 E+ seedlings
11
and 30 E- seedlings. After the first clipping treatment the grasses were placed on the same table in
a random order to guarantee similar environment (Figure 3).
Figure 3. Meadow fescues from the different soil treatments, endophyte statuses and cutting treatments on
the table in a random order
Plants were grown in a greenhouse with ambient light and temperature (20-26 °C). On 25.05.2021
the pots were weeded. High amount of weeds were growing in the GBH treated pots. Control pots
had fewer weeds, and sterilized pots none. I also replaced dead plants (6 GBH and 1 control). Early
stages of the study 5 grasses growing in GBH treated soil (8.3% of all grasses growing in GBH
treated soil) and 1 grass growing in control soil died (1.7% of all grasses growing in control soil).
Four of the dead plants died on the GBH group were from 5 cm cutting treatment (6.7% of all grasses
growing in GBH treated soil), 1 was from the group that was cut from the height of 15 cm. The only
dead plant in the control group was from 5 cm cutting treatment. Pots were weeded again on
06.06.2021, and GBH pots contained a high amount of weeds. Meadow fescues on GBH pots were
smaller and thinner than meadow fescues on control and sterilized pots (personal observation).
Unlike control and sterilized pots, GBH pots contained moss growth. Pots were regularly watered to
avoid drought stress. Plants were grown in a greenhouse chamber until harvest of the aboveground
biomass on 08.09.2021 (Figure 4).
12
Grasses were cut with normal scissors to the height of 5 cm and 15 cm (Figure 4). The cutting height
was measured with a soft tape measurer. Grasses were cut on 18.06.2021, 09.07.2021 and
05.08.2021.
Figure 4. Ending the experiment by harvesting the green biomass
2.2 Biomass and chlorophyll content sampling
To study the effects of glyphosate residues in soil as well as soil sterilization treatment on plant
performance the following plant parameters were recorded. The length of the longest leaf from
meadow fescues was measured, the amount of tillers was counted and the SPAD value was
measured on three occasions 17.06.2021, 08.07.2021 and 04.08.2021. The SPAD meter (Soil Plant
Analysis Development) measures the difference between the transmittance of a red (650 nm) and
an infrared (940 nm) light through the leaf, which enables the estimation of the amount of chlorophyll
present on the leaf. The first assessments were carried out on sunny days; 17.06.2021 the air
temperature outside was between 19°C and 22°C (in the greenhouse warmer) (Foreca 2021),
08.07.2021 the air temperature outside was between 21 °C and 25 °C (in the greenhouse warmer)
(Foreca 2021), the third assessment on 04.08.2021 was carried out when it was a cloudy and partly
rainy, when outside temperature was between 15 °C and 18 °C (Foreca 2021). The last day when
SPAD values were taken (06.09.2021) the weather was mostly cloudy and temperature between
+14 °C and +17 °C (Foreca 2021).
13
The SPAD (Minolta SPAD-502Plus meter) evaluations were taken on three randomly chosen leaves
per plant, whereby the meter was placed randomly on leaf mesophyll tissue. Three SPAD readings
were taken per plant and averaged to provide a single SPAD value. Diurnal chloroplast movements
in response to light, affecting the degree of heterogeneity in the chlorophyll distribution, and therefore
the SPAD values, should be minor, because all SPAD-measurements were conducted during 10:00-
16:00 o’clock.
The fresh cut aboveground biomass parts from the cutting treatment plants (5 cm and 15 cm) were
weighed on 18.06.2021, 09.07.2021 and 05.08.2021. After weighing the cut biomass was dried in
oven at 65 °C for at least 48 hours and weighed again to get the dry weight. The scale used for
weighing was same every time, Mettler Toledo AX204.
The experiment was concluded for 17 weeks after planting (06.-10.9.2021). The green biomass was
cut and the fresh biomass was weighed, then leaves were dried and weighed again to gain the dry
weight. Roots were carefully separated from the soil by rinsing them gently in water until they were
clean. After washing, roots were dried with paper towels and weighed, then dried in the oven in the
same manner as the leaves (at 65 °C for 48 hours in the drying oven) and weighed again.
I tested the effects of clipping and endophyte status, as well as their interaction term on total
aboveground biomass, root biomass and chlorophyll content (SPAD value) via Analysis of Variance
(ANOVA), before applying Tukey’s Posthoc test comparing single treatments. Same response
variables were analyzed with soil treatments and endophyte status as predictor variables. Total
aboveground biomass was correlated with root biomass and a curve was fitted to the graph.
Statistical analyses (ANOVA and correlation) were done in R.
14
3 Results
3.1 Effect of cutting height and endophyte status on plant parameters
Cutting height significantly affected the total aboveground plant biomass, root biomass and
chlorophyll content of the meadow fescue plants (Table 1). Symbiotic endophytic fungi did not affect
the biomass or chlorophyll content in any of the treatments.
Table 1. ANOVA summary table of plant responses to cutting treatment, endophyte treatment and their
interaction term.
Response
variable
Effector variable Df F p
Total
aboveground
biomass
Cutting 2.168 75.74 <0.0001
Endophyte 1.168 0.160 0.690
Cutting*Endophyte 2.168 0.795 0.453
Root biomass Cutting 2.168 110.1 <0.0001
Endophyte 1.168 2.056 0.154
Cutting*Endophyte 2.168 0.437 0.647
Chlorophyll
(SPAD)
Cutting 2.168 38.35 <0.0001
Endophyte 1.168 3.374 0.068
Cutting*Endophyte 2.168 1.395 0.251
Uncut meadow fescues produced the largest total aboveground dry biomass, which was significantly
higher compared to total aboveground dry biomass from grasses cut to 15 cm or 5 cm. The grasses
cut to the height of 5 cm had the smallest total aboveground dry biomass, which was further
significantly smaller compared to grasses cut to 15 cm (Figure 5). Similarly uncut meadow fescues
produced a largest root dry biomass, which was significantly higher compared to root dry biomass
from grasses cut to 15 cm or 5 cm (Table 2). The grasses cut from the height of 5 cm had the smallest
root dry biomass, which was further significantly smaller compared to grasses cut to 15 cm (Figure
6). In addition uncut meadow fescues had the biggest chlorophyll content, which was significantly
higher compared to the chlorophyll content from grasses cut to 5 cm. Uncut meadow fescues did not
have significantly bigger chlorophyll content compared to the chlorophyll content from grasses cut to
15 cm (Table 2). The grasses cut from the height of 5 cm had the smallest chlorophyll content, which
was further significantly smaller compared to grasses cut to 15 cm (Figure 7).
15
Figure 5. Total aboveground biomass was significantly different between the cutting heights (0= uncut, 5 cm,
15 cm), but was not affected by endophyte symbiosis. Letters indicate significant differences following
Posthoc Tukey’s tests (Table 2).
Figure 6. Root biomass was significantly different between the cutting heights (0= uncut, 5 cm, 15 cm), but
was not affected by endophyte symbiosis. Letters indicate significant differences following Posthoc Tukey’s
tests (Table 2).
16
Figure 7. Chlorophyll content measured as SPAD was significantly different between the cutting heights (0=
uncut, 5 cm, 15 cm), but was not affected by endophyte symbiosis. Letters indicate significant differences
following Posthoc Tukey’s tests (Table 2).
Table 2. Results from a Posthoc Tukey’s test testing pairwise comparisons of plant parameters between the
clipping treatments.
Total aboveground dry biomass T p
Uncut Cut 5 cm 12.197 <0.0001
Uncut Cut 15 cm 4.232 0.0001
Cut 5 cm Cut 15 cm 8.008 <0.0001
Dry root biomass
Uncut Cut 5 cm 14.232 <0.0001
Uncut Cut 15 cm 10.609 <0.0001
Cut 5 cm Cut 15 cm 3.797 0.0006
Chlorophyll (SPAD)
Uncut Cut 5 cm 7.911 <0.0001
Uncut Cut 15 cm 0.867 0.661
Cut 5 cm Cut 15 cm 7.030 <0.0001
17
3.2 Effect of soil treatment and endophyte on plant parameters
Soil treatment significantly affected the total aboveground biomass (Table 3). Plants produced higher
amount of total aboveground biomass when growing in a sterilized soil (S) compared to control (C)
or glyphosate based herbicide treated (G) soil (Figure 8). Grasses growing in sterilized soil also had
bigger leaf area compared to control or GBH group. Root biomass was not significantly affected even
though there was a trend to higher biomass in the sterilized soil treatment (Table 3). Chlorophyll
content measured as SPAD values were highest in plants growing in sterilized soil (Figure 9), but
significantly different only in comparison to control plants (Table 4).
Table 3. ANOVA summary table of plant responses to soil treatment, endophyte treatment and their
interaction term
Response
variable
Effector variable Df F p
Total
aboveground
biomass
Soil treatment 2.168 8.461 0.0003
Endophyte 1.168 0.032 0.859
Soil*Endophyte 2.168 0.182 0.834
Root biomass Soil treatment 2.168 2.433 0.090
Endophyte 1.168 0.654 0.420
Soil*Endophyte 2.168 0.568 0.568
Chlorophyll
(SPAD)
Soil treatment 2.168 4.392 0.013
Endophyte 1.168 2.552 0.859
Soil*Endophyte 2.168 0.226 0.834
18
Figure 8. Total aboveground biomass was significantly different between soil treatments (C= control, G=
glyphosate based herbicide, S= sterilized), but was not affected by endophyte symbiosis. Different letters
indicate significant differences following Posthoc Tukey’s tests (Table 4).
Figure 9. SPAD value was significantly different between soil treatments (C= control, G= glyphosate based
herbicide, S= sterilized), but was not affected by endophyte symbiosis. Different letters indicate significant
differences following Posthoc Tukey’s tests (Table 4).
19
Table 4. Results from a Posthoc Tukey’s test testing pairwise comparisons of plant parameters between the
soil treatments.
Total aboveground dry biomass T p
Sterilized soil Control 2.744 0.018
GBH Control 1.350 0.370
GBH Sterilized 4.050 0.0002
Chlorophyll (SPAD)
Sterilized Control 2.774 0.017
GBH Control 0.461 0.899
GBH Sterilized 2.262 0.064
20
Figure 10. Correlation between root biomass and total aboveground biomass showing that higher root
biomass is connected to higher aboveground plant biomass
Correlation between root biomass and total aboveground biomass shows that higher root biomass
is connected to higher aboveground plant biomass (Figure 10). However, small root system can
result in relatively large plants, which is displayed in the steep slope at low root biomass values.
21
4 Discussion
4.1 Effect of cutting height, soil treatment and endophyte status on plant
parameters
My results support the hypothesis that grass management practices impact the carbon sequestration
and storage potential of pastures. Cutting height significantly affected to the total above ground plant
biomass, root biomass and chlorophyll content of the meadow fescues. The grasses cut to height of
5 cm produced 95.24% less total aboveground biomass, 322.23% less root biomass and had 32.98%
lower chlorophyll content compared to the meadow fescues cut to 15 cm height. The uncut meadow
fescues produced clearly the highest total biomass and root biomass; the uncut meadow fescues
had 3.51% more total aboveground dry biomass than meadow fescues cut to 15 cm height, 50.58%
more total aboveground dry biomass than meadow fescues cut to 5 cm height, and 67.48% more
root biomass than meadow fescues cut to 15 cm height, and 92.30% more root biomass than
meadow fescues cut to 5 cm height. Uncut meadow fescues had 37.0% higher chlorophyll content
compared to the meadow fescues cut to 5 cm height, and 3.03% higher chlorophyll content
compared to the meadow fescues cut to 15 cm height. My results are in concordance with my
hypothesis and supported by the resource allocation hypothesis (Oesterheld 1992; Holland et al.
1996; Thornton & Millard 1996). Grasses have a capability to recover well after defoliation, but cutting
too low and too often results in poor regrowth rates. After being eaten or cut, grasses need to take
strength for growing from the roots, which means that part of the roots will die. If this resource
allocation is experienced often, plant does not have viable roots for regrowth and it dies (Oesterheld
1992; Holland et al. 1996; Thornton & Millard 1996). Similarly to in high total aboveground biomass
production in uncut plants, their chlorophyll content was high. Plants have reduced ability to capture
energy and carbon needed for photosynthesis when a herbivore eats leaf tissue or if part of the plant
is cut away (Gurevitch et al. 2006), which in turn leads to a decreased chlorophyll content and growth,
as noticed especially with grasses cut to 5 cm height.
Perennial grasses have high organic matter content and they can mitigate greenhouse gas
emissions through soil carbon sequestration and improve other ecosystem responses, water quality,
nutrient cycling, and support biological diversity (Franzluebbers 2012). Cutting grasses for silage or
animals grazing on pasture diminish momentarily the amount of green biomass. My results
demonstrate that cutting too low leads to poor regrowth rates and small biomass. If cut or let animals
to graze grass too low, it is harder for the grass to recover and it does not sequestrate as much
carbon as it would when leaves are longer. Grazing intensity of pastures should be regulated to
maintain adequate leaf area for maximum plant growth rates throughout the grazing season
(Matches 1992).
22
Against my prediction endophyte status did not significantly affect the total aboveground plant
biomass, root biomass and chlorophyll content of the meadow fescue grasses. Usually symbiotic
endophytes offer various benefits for their hosts, for example increased growth and reproduction,
and resistance to abiotic environmental stresses (Saikkonen et al. 2004; Lehtonen et al. 2005).
Growing in a greenhouse might have affected the results at least partly. Because the grasses were
not experiencing a competition pressure from other plants, they could use all nutrients, light energy
and moisture by themselves, so endophytic fungus did not provide advantage to their host grass. In
addition the beneficial effect of endophytes is often still missing in younger plants (Fuchs et al 2017b).
The positive effect from the endophyte to the grass host is strongest in the environments with plenty
of nutrients (Saikkonen et al. 2006; Cheplick & Faeth 2009). I did not add any fertilizers during the
growing season, and during watering some nutrients might have flushed away, and that might be
one reason why grasses did not benefit the positive effects of endophytes.
Meadow fescues growing in sterilized soil grew much better than grasses growing in control or
glyphosate treated soil. From early on the grasses growing in the sterilized soil group were bigger
and more robust than in the other groups, and they produced higher amount of total aboveground
biomass and had higher chlorophyll content compared to two other groups. Grasses growing in
sterilized soil had 57.98% more total aboveground dry biomass than grasses growing in control soil
and 190.54% more total aboveground dry biomass than grasses growing in glyphosate based
herbicide treated soil. Meadow fescues growing in sterilized soil had 20.38% more root dry biomass
than grasses growing in control soil and 58.18% more root biomass than grasses growing in
glyphosate based herbicide treated soil. Grasses growing in sterilized soil had 12.59% higher
chlorophyll content than grasses growing in control soil and 10.23% higher chlorophyll content than
grasses growing in glyphosate based herbicide treated soil. Based on these figures, having residues
of glyphosate based herbicides in soil will decrease harvest of meadow fescue. In addition the death
rate of grasses growing in GBH soil was elevated compared to other groups (8.3% death rate in
GBH group, 1.7% death rate in control soil and 0% death rate in sterilized group). My results
emphasize the importance of soil health and suggest that the use of GBH decrease the resilience of
agricultural systems.
One explanation for the good growth of grasses growing in sterilized soil might be that during the
sterilization in the autoclave, the killed biota has turned into a good fertilizer and has given an
advantage to the grasses growing in the sterilized soil. There are examples of that on previous
studies as well, where grasses performed better when growing in sterilized soil (e.g. Hines et al.
2017). Grasses can cope in poor soils, and they might not need a big microbiome around them,
since they are regularly growing on yards, road verges and other nutrient poor areas, where also
microbes are scarce (Rikkinen 2014; Wagg et al. 2019). In addition harmful pathogens might have
23
lived in the soil and died during sterilization, so they did not affect to the grasses growing in sterilized
soil (van der Putten & Peters 1997).
Biomass production of plants depends on energy supplied by photosynthesis. Grasses growing in
sterilized soil had bigger leaf area and were able to acquire more sunlight and carbon, so they had
larger amount of chlorophyll compared to control group and GBH group (even though only
comparison between sterilized and control groups were significant). Glyphosate can affect the
carbon sequestration and contribute to smaller leaf area and biomass. Decreased shoot and root
biomass due to growing in GBH treated soil likely occurred because of decreased photosynthesis
rate, disrupted growth hormone biosynthesis, or lower nutrient accumulation (Bott et al. 2008;
Zobiole et al. 2010; Zobiole et al. 2012; Fuchs et al. 2021; Fuchs et al. 2022).
Correlation between root biomass and total aboveground biomass showed that higher root biomass
was connected to higher aboveground plant biomass, but that small root system can result in
relatively large plants. An explanation for that might be that on soils that contain relatively big amount
of nutrients, like agricultural soils usually do, plants do not need to grow big root systems, because
nutrients are easily available on the top soil layer.
4.2 General conclusions
Cutting grass for silage or hay and the practice how animals are grazing are underestimated when
considering pasture management. My results demonstrated that it would be possible to get more
harvest when increasing the cutting height or moving animals to other pasture before they have been
eating the grass too low.
Soil properties may determine grass productivity. My results demonstrated that soil history of
glyphosate use can determine amounts of aboveground and root biomass of grasses. Because
glyphosate is harmful for the plants (Tu et al. 2001; Duke & Powles 2008; Zobiole et al. 2012; Gill et
al. 2017), environment (Giesy et al. 2000; Grunewald et al. 2001; Simonsen et al. 2008; Shushkova
et al. 2009; Vera et al. 2010; Battaglin et al. 2014; van Bruggen et al. 2018) and animals including
humans (Daruich et al. 2001; Dallegrave et al. 2003; Richard et al. 2005; Benachour et al. 2007;
Dallegrave et al. 2007; Benachour and Seralini 2009; Paganelli et al. 2010; Koller et al. 2012;
Mesnage et al. 2012; Samsel & Seneff 2013; Guyton et al. 2015; Bai & Ogbourne 2016; Gill et al.
2018), it should be really thought through if it is absolutely necessary to use it. In addition some
plants have developed resistance against glyphosate (Powles & Preston 2006), in which case
spreading glyphosate does not have an effect to those plants.
24
As this is a thesis, my goals were also to learn how to design and conduct a working greenhouse
experiment. Working with plants has its own benefits and restrictions, of which I wanted to learn
more of. While some improvements could be done if planning a similar experiment again, I find the
methods of my one-growing-season experiment appropriate and the results very interesting.
4.3 The larger scale
Rotational grazing, which means that animals are grazing in relatively small paddocks for 1-3 days,
and then there is a break when the grass of that paddock can rest and grow for 20-60 days, enhances
productivity compared to extensive, continuous grazing. Rotational pastures can produce almost
three times as much forage as continuous pastures, and also nutritional quality has been higher on
rotational pastures comparing to continuous pastures (Paine et al. 1999; Oates et al. 2011). In
addition, it has been shown, that higher stocking densities (bigger amount of animals per hectare)
for shorter durations are associated with greater total production per hectare than lower stocking
densities. Higher stocking rate increases the nutritional value of the forage, and less pasture is
wasted due to trampling, fouling, and rejection as stocking rate is high. Using rotational grazing helps
to reduce feed costs and potentially increases profits by reducing overall costs of production (Fales
et al. 1995; Phillip et al. 2001).
Similarly, it would be more beneficial to cut grass for silage higher than conventionally. Then grasses
would have a better regrowing efficiency which increases total harvests. Also roots recover better
when cutting leaves higher. That leads to the better recovery after the defoliation, less loss of plants,
which means less overseeding and longer period before sowing a new pasture or silage crop
(Holland et al. 1996; Thornton & Millard 1996).
Future agricultural policies need to consider how to reverse the loss of soil biodiversity. Climate
change, new biotic stresses brought about by change in ecological balance, and the possible
introduction of new pests, will influence the future of forage grasses. Public attitudes will require
further reductions in synthetic pesticides and residues in primary products (Fletcher & Easton 1997.)
Grasses can be used as a powerful carbon sink, and in addition pastures can be used to recover the
biodiversity of many plant, fungus, microbe, insect, bird and mammal species. When grasses are
not overgrazed, they will recover faster and more complete, they will acquire more nutrients and
water with their bigger root systems, and thereby absorb more carbon from the atmosphere. Since
pastures and silage fields do not need to be tilled yearly but they are perennials, they also storage
more carbon in roots and soil than annual fields do. Although forests store more carbon in total
compared to grasslands, in vulnerable environments driven for example periodic fires, grasslands
might be better carbon storages (Dass et al. 2018). It is environmentally essential to grow pastures,
because they provide important ecosystem services (for example enhanced biodiversity, prevention
25
of erosion and nutrient runoffs, serving as carbon sinks, and food production on areas where only
grasses can be grown productively) (Paustian et al. 1997; Jobbágy & Jackson 2000; Guo & Gifford
2002; Soussana et al. 2014; Poeplau et al. 2018). Pastures take around 67% of global land surface,
so taking a proper care of that land would make a real difference to carbon sequestration and
biodiversity.
26
5 Acknowledgements
I want to thank my thesis instructors Dr. Benjamin Fuchs, Dr. Marjo Helander and Dr. Kari Saikkonen
for their knowledge, help and patience. They supported my work and pushed me forwards. In
addition to all the guidance, I need to acknowledge the supplies, tools and materials they provided
for my experiment. In addition I want to thank the Botanical Garden of Ruissalo for the space in one
of the greenhouses I had for the summer.
27
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