Wood decaying fungi gain competitive strength through competition

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Master’s thesis

Two years

Major Subject: Biology

Title: Wood decaying fungi gain competitive strength through competition

Fahmidazaman Irin

2 MID SWEDEN UNIVERSITY

Department of Natural Sciences Supervisor: Fredrik Carlsson Examiner: Bengt Gunnar Jonsson Author: Fahmidazaman Irin

Degree program: Master by Research Main field of study: Biology

Semester: Spring 2019

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Table of contents

1. Abstract

5 2. Introduction

5 2.1. What are Saprotrophic basidiomycetes?

5 2.2. Different types of decay

6 2.3. Interactions between fungi and other soil microorganisms

6 2.4. Mycelial interactions and nutrient transfer between

interacting fungi 7

2.5. Different types of mechanisms fungi use during competition

with others 7

2.6. Factors responsible for outcome of interactions

9 2.7. Objectives as our interests

9 3. Method and Materials

10 3.1. Fungal Strains

10 3.2. Media preparation

11 3.3. Experimental design

11 3.4. Measurement of Growth

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3.5. Statistical Analysis 14

4. Results

14 5. Discussion

17 Acknowledgement

19 References

20 Appendix

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List of Table:

Table 1: Wood-decaying fungi used in fungus-fungus interaction experiment

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Table 2: Hagem Agar Media composition with amount of ingredients 11 Table 3: Two-way analysis of variance for the effect of competitive state and

opponent on the growth at day six of the experiment

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List of Figure:

Fig 1: a) Control cultures without competition b) First competitive batch c) Second competitive batch- one inoculum from control without competition, known as ‘non-competitive isolate’ and another inoculum from control with competition (1st competitive batch), known as

‘competitive isolate’ were inoculated in one petri dish.

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Fig 2: ImageJ was used to measure the size of each isolate on the agar plates by delineating the areas occupied by the isolate”.

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Fig. 3. Six combinations of competitive interactions are shown. 14 Fig.4. The overall general picture for all species. Here, X-axis represents

incubation period such as 4

, 6

and 8

day. The Y-axis refers the mean with SE values of % area covered by different isolates.

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Fig:5: Growth after six days of the four species growing individually without and with competition experience (first two bars for each species) and (second two bars) during competition with the three other species being either previously exposed to competition (pre-int) or not (non-int) .

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Appendix

Table 4: Set up of pair wise combinations in the First competitive batch 23 Table 5: Setup of pairwise combinations for second time as the Second

competitive batch

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5

Wood decaying fungi gain competitive strength through competition

1. Abstract:

Competition is the most common type of interaction occurring between wood-decaying higher fungi. Fungal species compete for space and nutrients growing on organic matter, resulting in morphological changes and strong biochemical reactions in interacting mycelia.

The main goal of this study was to focus on how much their competitiveness increased when isolates were exposed to competition on a new substrate. For this purpose, in Hagem agar cultures, four different species of wood decaying fungi were cultivated to compete with each other. Three species, Gloeophyllum sepiarium, Fomitopsis rosea, and Fomitopsis pinicola are brown-rot fungi while the fourth species, Phlebiopsis gigantea is a white-rot fungus. We measured the growth rate during competition and compared it between competitive isolates that had been allowed to compete and non-competitive isolates which had not been exposed to competition. In general, the results showed that the growth rate of competitive isolates was higher than controls without competition experience. In addition, the different species showed different degree of increased competiveness depending on which species these were exposed to. For example, the competitive Phlebiopsis gigantea showed increased competitiveness compared to the other three competitive species. The mechanisms are not well understood, but possibly linked to up-regulation of gene expression associated with antagonism during interaction.

2. Introduction

Fungi have a wide range of important roles in nature such as pathogens of crops, plants and humans, as decomposer of dead wood matter [1]. They are also used as experimental ‘model organisms’ for investigating genetics and cell biology. They can produce many important metabolites and they are used as commercial biological control agents for combating insect pests, nematodes and plant-pathogenic fungi. All fungi are eukaryotic. Some fungi typically grow as filaments termed hyphae that extend at the tips. Some exhibit apical growth by repeated cell divisions. Fungal hyphae branch form a network known as mycelium. However, some fungi grow as single-celled yeasts (e.g. Saccharomyces cerevisiae) which reproduce by budding. Fungi are heterotrophs (chemo-organotrophs). They require organic compounds as energy sources and for cellular mechanisms [3,4,5]. They produce enzymes to degrade complex organic compounds and absorb the dissolved nutrients for energy [2].

2.1. What are Saprotrophic basidiomycetes?

The fungal kingdom is subdivided into four groups: basidiomycetes, ascomycetes, zygomycetes and chytrids. Ligninolytic basidiomycetes inhabit wooden substrates [11, 6].

According to nutrient acquisition, fungi are categorized as (a) by growing as a parasite or a

pathogen of another living organism, (b) by growing as a symbiont in association with other

organism or, (c) by growing as a saprotroph on nonliving materials [8]. Saprotrophic fungi live

on dead organic matter and produce wide range of enzymes to decompose those matters into

soluble nutrients for their survival [7]. In the present study, we will focus on only saprotrophic

basidiomycetes.

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In terrestrial ecosystems, most Basidiomycota form their predominant mycelium, as an interconnected series of apically extending tubes-hyphae. Hyphae makes a large surface volume that favors the secretion of enzymes for extracellular digestion and uptake of other small molecules [8]. Hyphal tips take up a large amount of mineral nutrients, carbon and energy sources. When nutrients are needed, the hyphal tips embedded within organic resources start to forage externally for new resources and translocate from these sources to sites of demand [9]. Mycelia show remarkable physiological and morphological plasticity after local environmental effects. In terrestrial ecosystem, the organic resources are discrete. These organic resources are distributed heterogeneously in both space and time. For survival, saprotrophic fungi are able to use these discontinuously dispersed resources [10].

2.2. Different types of decay

Wood consists of an orderly arrangement of cells with cell walls composed of different amounts of cellulose, hemicellulose and lignin [12]. Different morphology and chemical composition of wood depend on the diversity of woody plants. Different types of physical, chemicals and morphological changes occur in wood depending on types of decay process [13, 14].

According to decay patterns happened by different fungi, three categories can be defined to separate the types of wood decay process. White and brown rot fungi are two major groups of decay process and they are taxonomically classified under the subdivision Basidiomycota.

White rot fungi can degrade all components of cell wall, including lignin. They often change normal wood coloration [13]. The thousands of species responsible for white rots are a heterogeneous group that may deteriorate greater or lesser amounts of specific cell wall components. Some preferentially degrade lignin from wood leaving white pockets of degraded cells consisting of cellulose entirely, while others degrade lignin and cellulose simultaneously. These white rots are common parasites of heartwood in living trees and are aggressive decomposers of woody debris in forest ecosystem. Brown rot fungi degrade cellulose rapidly in early stages of wood colonization [15]. They weaken the wood strength very early in the decay process. During decay, cell wall carbohydrates are depolymerized by brown rots leaving modified, lignin-rich substrates. The residual wood is brown in color and when it gets dry, it has cracks into cubical pieces [16].

Fungi responsible for soft rot are taxonomically classified in the subdivisions, Ascomycota and Deuteromycota. Soft rots can occur in dry environments and macroscopically may be similar to brown rots. After attacking by the soft rot fungi, the wood loses its strength largely [17].

Cavities formed in the wood as well as extensive cellulose deformation can result after decaying by soft rot fungi. As decay progresses, loss of extensive carbohydrate and increased concentration of lignin are found in the residual wood [15].

2.3. Interactions between fungi and other soil microorganisms

When fungi are growing on organic resources and soil, basidiomycete’s mycelia frequently can sense and be exposed to the mycelia of others (either of the same or of different species).

After recognizing non-self (both inter and intraspecifically), basidiomycetes result in exhibit

antagonistic responses. They change their morphology and produce extracellular enzymes,

volatile and diffusible secondary metabolites that is visually expressed as bright pigmentation

at interaction zone in mycelia on culture substratum [9]. Fungi also interact with a wide variety

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of organisms such as bacteria, invertebrates and plants [22]. Some produce small molecules known as secondary metabolites, which take part in cell signaling, pigmentation and in defense against predation. For example, some bacteria are mycophages having a negative effect on fungal growth and activity. In contrast, basidiomycetes may benefit from some bacteria to supply nitrogen and detoxify mycotoxins [18].

During wood decomposition, basidiomycetes produce reactive oxygen species and secondary metabolites, which acidify the environment [19, 20]. Bacteria have some special properties to survive in this selective environment. Basidiomycetes also interact with invertebrates and these interactions are highly dynamic. Both are affected by each other directly and indirectly.

For instance, invertebrates graze directly on mycelium or fruit bodies of fungi. Some basidiomycetes kill invertebrates and then utilize their body contents. Fungi and invertebrates interact indirectly with different effects on each other. Sometimes fungi arrest the activity of invertebrates and improve the nutritional environment or decrease carbon: nutrient ratio. In other side, invertebrates change the microbial community and also physiology as well as metabolism [21].

2.4. Mycelial interactions and nutrient transfer between interacting fungi

Lynne Boddy, 2000, did review on the interactions between Basidiomycetous fungi and the potential use of these fungi as biocontrol agents against forest pathogens. When mycelia of two different wood decomposing fungi are inoculated on an agar plate, an antagonistic reaction is triggered [23]. These interactions shift mycelial morphology, increase branching and create a dense mycelium in the interaction zone. Sometimes, pigmentation are visible in interaction zone [24]. In some cases, interacting mycelia avoids the site of contact. There can be a deadlock situation if the mycelia are in close proximity [25]. On the other hand, one fungus can is successful to overcome the antagonistic reactions against of its opponent. In addition, such cases, it overgrows with its mycelial fans or rhizomorphs to the other mycelium of fungus through lysis process. Only during transient periods, mycelia of two different individuals coexists within the same volume of a limited substrate [26].

Most studies show that fungi compete mainly for energy resources. Saprotrophic fungi compete with each other for resources rich in cellulose [27]. Fungi, however, compete not only for carbon resources but also for other nutrients. Ectomycorrhizal fungi have radical mycelium to forage and compete for nutrients in the soil. Saprotrophic fungi degrade wood or plant litter to obtain nutrients as well as carbohydrates. In a nutrient rich environment, fungal mycelia can deplete the substrate as they take up nutrients and translocate them or incorporate them into the mycelium [29, 30]. In a nutrient poor environment, fungi can enrich the substrate through nutrient translocation or recalcitrant mobilization (e.g. nutrients are sequestered into recalcitrant polyphenolic complexes), and the mycelium itself constitutes a high quality nutrient source available to other organisms [28].

2.5. Different types of mechanisms fungi use during competition with others

Competition is the most common type of interactions between wood-decaying higher fungi.

This competition can be defined as the negative effect which one organism has upon another by consuming or controlling access to a resource that is limited in availability [33]. Competition is commonly termed as either interference competition or exploitation competition [31].

Exploitation competition is a situation where one organism inhibits another by the production

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of allelopaths or by shading out of slower growing plants, this happens when one organism uses a resource and consequently reduces the availability to another [32].

Fungal competition in organic resources can be further categorized into primary resource capture and combat. The primary resource capture is termed as gaining initial access to and influence over an available un-colonized resource and combat is to capture territory from fungi already colonizing a resource or define territory from potential invaders [34]. Success in primary resource capture is occured by various factors including good dispersal, rapid spore germination, rapid mycelial extension and ability to utilize organic compounds available in previously uncolonized resources. Success in combat depends on various antagonistic mechanisms. Antagonistic interactions can be activated at a distance or hyphal interaction [35]. Antagonism at a distance induces to produce diffusible or volatile compounds via long distance. Mutual inhibition refers as the hyphae of one mycelium often become degenerate and are replaced by the other mycelium. This interspecific hyphal interaction may happen in different ways. Mostly, reactions vary depending on species combinations. Sometimes, fungi behave in this way by which one hypha by another is preceded by contact and this can recognize the host by a lectin or agglutinin-carbohydrate interaction [36]. This is followed by penetration or appression to and continued growth over the hyphal surface of the fungi.

Nutrients can then be absorbed from the host biotrophically or nectrophically. When the fungi are parasitic, they have possession of this domain to uptake their nutrition by decomposing the wood and they compete aggressively with other fungi by gross mycelial contact [37].

A particularly important type of interaction between wood-decaying higher fungi is gross mycelial contact. Gross mycelial contact covers all terms of interactions that means mycelial morphology changes dramatically during competitions. Such changes include producing aerial tufts, barrages, mycelial cords, pigments [40]. In agar culture, this takes the different forms of stationary “barrages” that is resistant to invasive mycelial fronts, mycelial fans, linear organ- cords and rhizomorphic structures. All of these forms can sometimes be expressed in a single interaction [38]. “Barrages” consists of stationary, relatively homogeneous assemblages of dense aerial and submerged hyphae. “Replacement fronts” are unilaterally spreading regular arrays of diffuse to loosely associated hyphae with a clearly defined margin that invade opposing mycelia from broad sections of interaction interfaces. “Mycelial cords” consist of quasi-linear aggregations of hyphae, arising from localized foci in interaction interfaces, which invades opposing mycelia more rapidly, but more irregularly, than replacement fronts [39]. In self-pairing, the colonies usually merge to form a uniform mycelial mat. Cord-forming fungi are known as an extremely successful group of soil microorganisms showing some activities such as “securing territory: combat, foraging strategies and reallocation of mycelial biomass and acquisition and reallocation of nutrients” [38].

Saprotrophic cord-forming fungi play a key role in the functioning of ecosystems. Their manipulation might help nutrient relocation, release, and control tree root pathogens. Inter and intracellular pigmentation is visible during interactions in agar plates by changing the phenol-oxidizing activity [41]. When cord systems make contact, there is often a defined yellow or brown discoloration and lytic response in one or both of the cord segments involved.

These mycelial responses are defined as “mycelial interference”, indicating their affinity with

“hyphal interference” between individual hyphae [30, 33]. Gross outcomes of combative

interactions can be either replacement, where one fungus gains the territory of the other

fungus, or deadlock where neither fungus gains headway. Sometimes partial replacement will

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result where initially one fungus gains headway but subsequently deadlock ensues, or where both species invades into the territory previously held by other antagonist [38].

2.6. Factors responsible for outcome of interactions

Outcome of interactions depends on many factors such as water potential of solutes, gaseous regime and to a lesser extent temperature as well as different species combinations. Size and quality of the resources that the combatants have access to is another major factor- influencing outcome of interactions [46]. For example, fungi occupying large wood resources have a higher chance of success in combat than those occupying smaller resources when challenged with the same species [45].

Microclimate, resource size and quality all can shift the balance between opponents. Thus, in the natural environment, the outcome of interactions between species can differ, leading to differences in community structure and dynamics [38]. Within the spectrum of outcomes of combative behavior there are transitive and intransitive hierarchies [42]. For example, a transitive hierarchy refers to the situation where fungus 1 is more combative than fungus 2 and both are more combative than fungus 3. The intransitive is that situation where fungus 1 is more combative than fungus 2, fungus 2 is more combative than fungus 3, but species 3 is more combative than fungus 1. Interaction modification is another factor that represents the situation where the outcome of interaction between two species is altered by the presence of a third species. According to the hierarchy of combative ability, some fungi are generally poor and others are very good at both in attack (one fungus trying to grow over the other and capture the other’s territorry) and defense (they save their territory from others by using different mechanisms). On the other hand, some are good in attack but poor in defense, while others even though poor at attack are good at defending their territory that they capture [43].

These hierarchies are broadly interconnected with fungal succession. Interspecific interactions can change the pattern of mycelial functions, including mycelial search patterns, distribution and reallocation of nutrients within mycelia, and respiration [44]. Carbon and mineral nutrients are transported to different parts of mycelial cord systems depending on supply and demand. Changes in metabolic activity of mycelia might be allowed during combative interactions in order to defend against or overcome an opponent. Interspecific interaction not only play a major role in fungal community development and decomposition processes but they have prospects as biological control agents of tree pathogens [38].

2.7. Objectives as our interests

In the ecosystem, saprotrophic fungi interact with other soil microorganisms for nutrients that

can influence fungal-mediated nutrients distribution within soil [48]. These fungi regulate

their physiological responses against grazers through changing hydrolytic enzyme production

and respiration rates. These affect directly mineralization of nutrients and CO

flux between

terrestrial and atmospheric pools [47]. In this present study, fungal competition was observed

as a small scale in the laboratory. Because, many factors may influence competitive pressure

among microorganisms in the ecosystem. In the laboratory experiment, we will focus on only

on fungal interactions on nutrients agar for a certain period. It will help us to understand their

competitive behavior during interaction in ecosystem. In this experiment, we are going to use

using four wood-rot fungi (Gloeophyllum sepiarium, Fomitopsis rosea, Fomitopsis pinicola,

Phlebiopsis gigantean). Therefore, the goal of this project is to highlight the combative

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behavior between fungi from competitive batch and from non-competitive batch during prolonged interaction under room temperature. Finally, the main research questions we have tried to find out according to this experiment are mentioned given below:

▪Will fungi obtain increased competitiveness when exposed to a competition?

▪If so, will this increased competiveness persist when exposed to a new competitive situation?

▪Is there any difference between the growth rate of fungi exposed to competition and isolates not exposed to competition?

3. Method and Materials 3.1. Fungal Strains

Four fungal species, presented by Table 1, were chosen for this experiment (Gloeophylum sepiarium (GS), Fomitopsis rosea (FR), Fomitopsis pinicola (FP) and Phlebiopsis gigantea (PG))

These were obtained from the culture collection of Mid Sweden University in Sweden. Ten isolates of each species were used in this experiment. One isolate of Fomitopsis pinicola was contaminated in the case of individual culture. Hence, only nine isolates of Fomitopsis pinicola were used in this experiment and one isolate (FP5) was repeated twice in pairwise combination. All isolates were collected from ‘Central Sweden’ and all are early succession saprotrophs.

Table 1: Wood-decaying fungi used in fungus-fungus interaction experiment

Species Name Rot-type Succession Main host tree

Gloeophyllum sepiarium (GS)

Brown Early Spruce

Fomitopsis pinicola (FP) Brown Early Spruce

Fomitopsis rosea (FR) Brown Early Spruce

Phlebiopsis gigatea (PG) White Early Pine and Spruce

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3.2. Media preparation

In this experiment, HAGEM agar was used for all fungal cultures (Table 2).

Table 2: Hagem Agar Media composition with amount of ingredients

Ingredients g/1000ml

Agar 20

Glucose 5

Malt Extract 5

NH

NO

0.5

MgSO

.7H

O 0.5

KH

PO

0.5

All components were added to 1 L of distilled deionized water. After mixing, the media were autoclaved at 15 psi pressure and 121°C. The media was then poured into sterile 9-cm diameter petri dishes and when the agar plates were polymerized, the plates were wrapped with sterile bags and preserved at 4°C until inoculation. After inoculation, the plates were constantly checked for bacterial contamination.

3.3. Experimental design

At the beginning, every isolate of the four species were grown individually on Hagem agar

known as the “control batch”. In the controls, the inoculum was placed in the center of the

dish (Fig 1). The pair wise interactions were performed into two different ways. One is termed

the “first competitive batch” and the other termed as the “second competitive batch”. For

non-self-interactive pairing, the inoculum from two different species, a 0.8 cm diameter

mycelium plug, were cut from actively growing mycelium edge and placed 3 cm apart on

another agar plate. For the first competitive batch, the inoculum from two different isolates

of different species interacted with each other in a single plate (based on pairwise

combinations in Appendix, Table 4).

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Fig 1: a) Control cultures without competition b) First competitive batch c) Second competitive batch- one inoculum from control without competition, known as ‘non-competitive isolate’ and another inoculum from control with competition (1st competitive batch), known as ‘competitive isolate’ were inoculated in one petri dish.

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After 8 days incubation, second competitive batch was initiated and the species were again allowed to compete with each other. One inoculum was taken from the control batch and another inoculum taken from first competitive batch (Appendix, Table 5). During 8 days, their interactions were observed. In all cases, the Petri dishes were sealed with Para film to avoid drying of the agar and the cultures were incubated at 21°C. All competitive outcomes were monitored for final results.

3.4. Measurement of Growth

Digital images were captured three times during their growth period using a Canon EOS 350D DIGITAL camera, mounted on a stand at a height of 40 cm. The photos were taken to estimate growth rate from the control batch, 1

competitive and 2

competitive batch. The competitiveness was analyzed based on the photographs taken from 1

and 2

competitive batch on the 4

, 6

, 8

day. During incubation period on 8th day, most of them reached the edges of the petri dishes and some inhibited the growth of others. Due to this, 6

day was chosen to analyze the difference of the growth among competitive and non-competitive isolates.

All photos were analyzed using the Java based IMAGEJ (Fernanda Oliveira da Silva. 2017).

ImageJ can calculate area and pixel value statistics of user-defined selections and intensity- thresholded objects, measuring distances and angles. We used ImageJ to analyze the area covered by individual mycelia (for examples see Fig: 2).

Fig 2: ”ImageJ was used to measure the size of each isolate on the agar plates by delineating the areas occupied by the isolate”.

In Fig 2, at first, the photo was selected from the file. Then “Image” was selected to draw the area covered by individuals in one plate. This was done for each colony on all plates. The measure from the analyze option was selected to analyze the total area covered by the isolate.

Growth rates were determined for each interaction until mycelia from any replicate reached the opposing one. By using Image J, their growth was calculated and compared according to the control without competition and control with competition batch.

The results were expressed as mean area of the 10 replicates and their associated standard

error, to assess the different growth of fungal isolates from competitive and non-competitive

batch during interactions.

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3.5. Statistical analysis

Statistical analyses were performed in R version 3.4.2. Different responses of fungi on culture media during interaction experiments under different incubation periods were subjected to analyses of variance (ANOVA). This data from the growth rate (% of petri dish at day 6) of single species cultured at day 6 for control samples (not exposed to competition) and single species cultures after being exposed to competition were analysed by two way ANOVA and t- test was also done. This data was also complied to test the effect on growth (% of petri dish at day 6) between pairs of species with different competitive background (competitive or non- competitive). ANOVA model with subsequent TukeyHSD test was also used in R.

4. Result

The mycelia expansion (growth) rate of different wood decaying fungi was varied among control batch, first competitive batch and second competitive batch. Fig 3 illustrates the competitive outcomes during interaction.

Fig. 3. Six combinations of competitive interactions are shown. (a) non-competitive Fomitopsis pinicola (FP) and competitive Gloeophyllum sepiarium (GS) competed with each other and vice versa. Similarly the interactions are shown for (b) Fomitopsis pinicola with Fomitopsis rosea (FR); (c) Fomitopsis rosea with Gloeophylum sepiurium; (d) Phlebiopsis gigantean (PG) with Fomitopsis pinicola; (e) Phlebiopsis gigantea with Fomitopsis rosea; (f) Phlebiopsis gigantea with Gloeophyllum sepiarium.

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These figures represent the second competition experiment between pairs of different species. Non-competitive isolates covered less area than competitive isolates (Fig.3.). During competition, they produced some barrages in the interaction zone between non-competitive and competitive isolate. Six pairwise combinations are shown in this figure 3. The interactions have different patterns depending on different pairwise combinations among fungal species.

Fig.4. The overall general picture for all species. Here, X-axis represents incubation period such as 4^th, 6^th and 8^th day. The Y-axis refers the mean with SE values of % area covered by different isolates.

Figure 4 illustrates the growth rate across all species is for the different combinations of

cultures. The growth rate was lowest for the control isolates ( ) and highest for competitive

isolates ( ) competing against other species. The similar trend was followed by non-

competitive isolates ( ) when competing with competitive isolates. Furthermore, the

highest growth rate was observed for competitive isolates when competing with non-

competitive isolates. Finally, the growth rate of isolates from the first competitive batch (

) was only somewhat lower than among competitive isolates from the second batch. It reveals

that i.e. competitive isolates were stronger than isolates not exposed to competition ( ).

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Fig:5: Growth after six days of the four species growing individually without and with competition experience (first two bars for each species) and (second two bars) during competition with the three other species being either previously exposed to competition (pre-int) or not (non-int) .

In figure 5, the first two bars for each species represent the two states of the four species growing individually when one group was a control not exposed to competition and the other group exposed to competition. The individuals (second bar) exposed to competition showed significantly higher growth than the control not exposed to competition (T-test, p <0.001 for all four species). The increase in growth of individuals exposed to competition varied from 260% for F. rosea to 380% for F. pinicola.

0 10 20 30 40 50 60 70 80 90 100

F. pinicola F. rosea G. sepiarium P. gigantea

Percent area covered (%)

Species

Control Pre-single Pre-int. Non-int.

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Table:3: Two-way analysis of variance for the effect of competitive state and opponent on the growth at day six of the experiment

Fomitopsis pinicola Sums of squares Df F-value p-value

State 829.38 1 24.8550 <0.01

Opponent 412.37 2 6.1790 <0.01

Interaction 57.40 2 0.8601 0.428840

Residuals 1801.92 54

Fomitopsis rosea Sums of squares Df F-value p-value

State 11.19 1 1.1078 0.297

Opponent 596.97 2 29.5468 <0.01

Interaction 98.01 2 4.8508 0.012

Residuals 545.51 54

Gloeophyllum sepiarium Sums of squares Df F-value p-value

State 341.59 1 43.2151 <0.01

Opponent 129.41 2 8.186 <0.01

Interaction 47.46 2 3.002 0.058

Residuals 426.84 54

Phlebiopsis gigantea Sums of squares Df F-value p-value

State 1.4 1 0.0290 0.866

Opponent 3652.8 2 38.2122 <0.01

Interaction 40.7 2 0.4262 0.655

Residuals 2581.0 54

The other two bars for each species in Figure 5 show the growth during competitive interaction. In Table 3, the ANOVA on the effect on growth of competitive state and opponent species is demonstrated. In F. pinicola and G. sepiarium, competitive state significantly influenced growth of the competitive individuals showing high growth than non-competitive individuals. For the other two species, F. rosea and P. gigantean, competitive state did not have any effect on growth. Opponent species significantly influenced growth for all four species. In F. rosea as an example, the interaction between competitive state and opponent species was very significant. The strongest competitor was P. gigantea followed by F. pinicola, while F. rosea and G. sepiarium was equally weak competitors (Fig 5).

5. Discussion

Saprotrophic basidiomycetes show combative behavior during interaction. The outcome of

the combative ability varies and depends on different factors such as different species

combination, nutrient resources, environmental factors and state of decay of the resources,

where the interaction is occurring, e.g. artificial media, wood or soil, and activity of the

organisms present in the substrate (El Ariebi et al. 2016). Cooke & Rayner (1984) suggested

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that two individuals of the same or different species show combative behavior in defending area by primary resource capture. In primary resource capture, the larger volume of mycelium has a greater chance to reach nutrient pockets (Holmer et. al. 1993). In this case, area occupied is also an important factor for secondary resource capture by affecting the outcome of competition. In general, a larger volume of mycelia have a relatively higher chance of success in competition (Holmer & Stenlid 1993, 1997). During interspecific interaction, saprotrophic basidiomycetes having well-developed combative ability (Stahl et. al. 1991) are also able to restrict other opponents to capture resources and space by producing secondary metabolites (mycotoxin). Replacement of its opponent is an obvious goal related to this combative strategy (Rayner and Boddy, 1988). During interaction, the competitors react to info chemical signals by triggering the antifungal and antagonistic mechanism at the onset of interaction (El Ariebi et al. 2016). Moreover, the outcome of competition is dependent on substrate (wood or artificial media) as well as different abiotic and biotic factors.

In our experiment, some species increased their growth rate after competition, but the pattern was not clear for all isolates of four species. It seems that some species increases growth rate in response to antagonism (Evans et al., 2008). As an example, increased growth rate was observed in Phlebiopsis gigantea, Gloeophyllum sepiarium and Fomitopsis pinicola after competition, except Fomitopsis rosea. This result shows that some species gain competitiveness after interaction. Moreover, all species had a higher growth rate when grown individually after 1

time interaction.

It is interesting that there was a significant difference between both the species and the

treatments. Competitive interactions may result from stress of resources or requirement for

the same resource. During interactions, the fungi may use different strategies to survive and

invade additional territory. In our experiment, the competitive isolates, taken from first

competitive batch, showed strong combative behavior, by increasing their growth rate. This

might be related to a boost in antagonistic metabolism, which can be regulated in response

to the present needs (Simon HAROLD et. al. 2005). During competition, fungi activate

extracellular enzymes, change their growth rate, alter their gross morphology and produce

antagonistic metabolites such as volatile and diffusible organic compounds (Simon HAROLD

et. al. 2005). These compounds may be produced constitutively during interaction and may

either inhibit growth of others or stimulate their own growth. That might be occurred in our

experiment by showing significantly different variances during second competition

experiment. It appears that isolates of four fungi gained competitive strength, after

interaction. The mechanism behind this short time adaptation is not clear from our

experiments. The increased growth rate will theoretically make them more likely to succeed

against opponents. However, there was a difference in response among species when exposed

to competition before interaction; such as, Fomitopsis pinicola and Gloeophylum sepiarium

showed significantly higher growth values when they were in competitive state. While,

Fomitopsis rosea and Phlebiopsis gigantea did not show any differences between competitive

and non-competitive states. In the present study, we used Hagem agar medium to carry on

our experiment that was quite faster. Because, the fungi can break down nutrients easily from

agar than from wood. It took only one week for complete growth on the Petri dish. Therefore,

we chose to analyze the plates after 6 days. The results from our experiment on artificial media

may be different compared to those from other’s experiments on wood, which is more similar

to natural conditions (Holmer et al., 1993).

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To conclude, we used four different species in our experiment (three white-rots and one brown-rot fungi) to observe the outcomes of competition. Our result shows that isolates of different species exposed to competition are more competitive than others not exposed to competition. In addition, increased growth rate will allow them for more efficient resource capture. Further studies are needed to understand their defense mechanisms (e.g. VOC compounds as a chemical signal) influenced by combative ability achieved during time exposed to competition. These studies will contribute toward a greater understanding of antagonistic metabolism in community complexes of more than two species in the nature.

Acknowledgement

Praise to Allah is the most compassionate the most merciful for giving me persistence and potency to accomplish this Master By Research study.

The path toward this dissertation has been circuitous. I would like to give thanks for its completion to those special people who challenged, supported and stuck with me along the way. I am tremendously fortunate to have such kind Professor Bengt Gunnar Jonsson who allowed me to be a part of this biology department. Without his support, it was impossible for me to accomplish this course. I was new in abroad and in that time without his guidance and support, I could not continue my study.

I would like to express my gratitude to my supervisor Dr. Fredrick Carlsson for his unwavering support, guidance and insight throughout this research project.

Special thanks to others who supported me very much. Also thanks to Dr. Edman Mattias, other Master By Research students, current PhD student Matilda Lindmark who has a very good heart, specially Dr. Jennie Sandström (former PhD student), Lab personnel Torborg Jonsson who provided practical advice and participated in discussion with me in several times.

Truthful thanks to my parents who have made me strong in personality, my husband who always helps me to take challenges and friends for being a great source of support and encouragement to complete this work.

Last but not least, I should be really grateful to Dr. Svante Holm, my current PhD supervisor,

who is my mentor. Without his support, guidance, advice, it was impossible for me to finish

this study successfully. I am really lucky to have such kind, friendly person as a PhD supervisor

and I am really thankful to him to give me time to finish my Master’s thesis.

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Appendix

Table 4: Set up of pair wise combinations in the First competitive batch. FP4 was contaminated and hence FP5 (Fomitopsis pinicola 5) was used instead of FP4 (Fomitopsis pinicola 4) isolate.

Table 5: Setup of pairwise combinations for second time as the Second competitive batch. FP4 (Fomitopsis pinicola 4) was absent here because of contamination and replaced with FP5 (Fomitopsis pinicola 5).

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