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A study on sclerotia and mesofauna relationships in forest soils described by ectomycorrhizal fungal

community profiles

by

Anzilni Fathia AMASYA

Supervisor: Professor Makiko WATANABE

Department of Geography

Graduate School of Urban Environmental Sciences Tokyo Metropolitan University

March 2015

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Abstract iii

Chapter 1 Introduction 1

1.1 Microbes as soil biota 1

1.2 Mesofauna roles and functions in soil 2

1.3 Mesofauna and sclerotia-forming ectomycorrhiza 3

1.4 Molecular methods to describe ectomycorrhizal fungal communities 5

1.5 Objective, structure of this study 6

Chapter 2 Ectomycorrhiza communities in sclerotia 8

2.1 Introduction 8

2.2 Materials and Methods 9

2.2.1 Study area and soil sampling techniques 9

2.2.2 Sclerotia extraction 17

2.2.3 Isolating fungal DNA in sclerotia 17

2.2.4 Terminal Restriction Fragment Length Polymorphism 18

2.3 Results and discussions 19

2.3.1 Sclerotia characteristics 19

2.3.2 Fungal community profiles of sclerotia 22

2.3.3 T-RFLP method applied to determine sclerotia identity 23

2.4 Conclusion 25

Chapter 3 Ectomycorrhiza communities in soil mesofauna 26

3.1 Introduction 26

3.2 Materials and Methods 27

3.2.1 Study area and soil sampling techniques 27

3.2.2 Soil mesofauna extraction 27

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3.2.3 Isolating fungal DNA in soil mesofauna 30

3.2.4 Statistical analyses 30

3.3 Results and discussions 31

3.3.1 Soil mesofauna characteristics 31

3.3.2 Fungal community profiles in soil mesofauna 34

3.3.3 Acari and Collembola as predominant mesofauna and their roles in

the soil ecosystem 39

3.4 Conclusion 46

Chapter 4 Sclerotia and mesofauna relationships in forest soils described by ectomycorrhiza communities

4.1 Introduction 47

4.2 Materials and methods 48

4.2.1 Fungal community profiles in soil 48

4.2.2 Soil pH and exchangeable aluminum content 48

4.3 Results and discussions 49

4.3.1 Ectomycorrhizal fungal community profiles in soil, sclerotia, and mesofauna 49 4.3.2 Environmental factors regulating sclerotia formation 57 4.3.3 Model of sclerotia and mesofauna relationships through ectomycorrhizal fungal

communities 61

4.4 Conclusion 67

Chapter 5 Conclusion 68

Acknowledgements iv

References v

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A BSTRACT

Many important processes in the rhizosphere affecting nutrient cycling and plant performances occur between soil microbes and mesofauna. Among the soil microbes, the fungi associates with plant roots forming mycorrhizas, which also have the ability to survive periods of time through hardening their hyphae into a spherical structure called sclerotia.

There have been many studies on interaction between sclerotia-forming ectomycorrhizas and plants, whereas sclerotia are produced by well-known plant pathogens. Relationships between plants and mesofauna have also been studied mainly on how mesofauna contribute to decomposition processes of plant debris and nutrient cycling for plant root uptake in the soil system. However there still lack information on the interactions between sclerotia-forming ectomycorrhizas and soil mesofauna. This study aimed to understand relationships between sclerotia-forming ectomycorrhizas and soil mesofauna through the analysis of ectomycorrhizal fungal community compositions in sclerotia and soil mesofauna.

In this study, first, ectomycorrhizal fungal community profiles in soil mesofauna were analyzed. Second, ectomycorrhizal fungal community profiles in sclerotia were obtained through sclerotia extraction. Finally the relationship between soil mesofauna and sclerotia-forming ectomycorrhizas was described through a proposed model.

Ectomycorrhizal fungal community profiles were obtained through the Terminal Restriction Fragment Length Polymorphism (T-RFLP) method, and samples were taken from Japanese Beech (Fagus crenata) forest soils at a plateau near Lake Tazawa of Akita Prefecture, Mt. Chokai located between Akita and Yamagata Prefecture, Mt. Iwaki of Aomori Prefecture, and Shinshu University Research Forest near Mt. Kisokomagatake in Nagano Prefecture.

This research suggested a relationship model between sclerotia and soil animal through ectomycorrhiza community profiling within a pH gradient. In soils with higher pH value or less acidic soils, it was assumed that the environment had relatively low stress and there was no necessity to form sclerotia. As the pH turned lower the slight decrease of relative abundance of ectomycorrhiza in soil animals were observed, while at the same time sclerotia were available. In areas with low pH, sclerotia were found abundant, but the same ectomycorrhiza species in soil animals were found in low abundance or none in sclerotia.

This study showed that fungal community profiling of sclerotia and mesofauna was an

effective approach not only to describe fungal resource partitioning among soil mesofauna

and identify sclerotia, but also contributed to broaden the understanding of the relationships

between sclerotia and mesofauna in the forest soil.

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C HAPTER 1

I NTRODUCTION

1.1 Microbes as soil biota

The soil biota can be understood as the biological engine of the earth, driving and modulating many of the key processes that occur within soils (Ritz et al. 2004). Soil biota consists of microbes and also a wide range of larger multicellular organisms, and the entirety interacts via series of complex food webs (Van der Putten et al. 2001). Microbes function as primary decomposers and biochemical transformers, while larger organisms provide higher-order ecosystem services such as comminution of organic matter, decomposition, and also ecosystem engineering (Sakrabani 2012).

The most studied microbes are bacteria and fungi. Bacteria are single celled prokaryotes that require soil water films to live and move within the soil matrix, whereas filamentous fungi are less constrained in this manner and can cross air-filled pored spaces (Ritz 2008). Bacteria and fungi play a major role in soil as the primary degraders of organic matter, which determines both the rate at which nutrients become available to plants and the amount of carbon stored in soils (Wurst et al. 2012). Both microbes are particularly abundant in the rhizosphere, or the layer of soil surrounding plant roots, and provide many essential ecosystem functions, including decomposition, carbon and nutrient cycling, disease suppression, and regulation of plant growth and primary productivity (Wurst et al. 2012).

Fungi, the second major microbial primary decomposer group, have the ability to establish a

relationship with plants that seems to be mutually beneficial in most instances. These symbiotic

relationship is known as mycorrhiza (“myco” for the fungi; and “rhiza” for the roots). There are two

kinds of mycorrhiza, endomycorrhiza and ectomycorrhiza (Allen 1991). The former penetrate into and

continue to live in the plant root cells, whereas the latter form a mat around the roots with minimum

cell penetration. Over 80% of plant species form root associations with mycorrhizas, which provide

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the plant with essential nutrients for growth in exchange for plant photosynthesis products (Smith and Read 2008).

Many ectomycorrhizal fungi also have the ability to survive periods of time through hardening their hyphae into a spherical structure called sclerotia. Sclerotia are thought to serve as resting structures that can survive and remain quiescent in adverse environmental conditions until circumstances become favorable for fungal growth (Coley-Smith and Cooke 1971, Willetts 1971).

Recent studies on sclerotia have been focusing on their distribution along coastal pine forest soils (Matsuda et al. 2009), the use of sclerotia as biocontrol agents (Zachow 2011), and identifying active parasites of Cenococcum geophilum sclerotia (Obase et al. 2014). However, studies on interactions between sclerotia-forming ectomycorrhiza and other soil organisms such as soil fauna as an attempt to depict a part of the complexity of the soil ecosystem have not been fully explored.

1.2 Mesofauna roles and functions in soil

Although the ultimate decomposers in soil are microorganisms, the role of soil animals is important and should not be ignored. For example, decomposition and humification will be delayed remarkably if the crushing and mixing of plant remains by soil animals do not occur beforehand (Kumada 1987). Often it is not realized that this living fraction of soil organic matter is of such great importance. The tendency in the past has been to emphasize the chemical nature of organic matter to a far greater extent than its biological aspects, even though it is the microorganisms and soil fauna that are largely responsible for the chemical nature of the rest of the material (Allison 1973).

Soil fauna are categorized based on their body sizes as microfauna, mesofauna, macrofauna,

and megafauna; with body width of less than 100 µm, between 100 µm to 2 mm, 2-20 mm, and larger

than 20 mm, respectively (Swift et al. 1979). Among these groups, soil mesofauna such as Acari,

Collembola, Tardigrada, Symphyla, Diptera, etc., affect the structure and activity of microbial

communities and enhance nutrient turnover (Coleman 1986, Verhoef and Brussaard

1990, and Lussenhop 1992). These animals also directly interact with soil microbes, affecting fungal

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communities mainly through fungal grazing, which changes fungal growth pattern marked by the release of enzymes of fungi (Bengtsson et al. 1995). The selective grazing by mesofauna may also alter the competitive relationships between fungi (Newell 1984) and thus contribute significantly to decomposition processes and nutrient turnover (Visser 1981).

1.3 Mesofauna and sclerotia-forming ectomycorrhiza

Many studies have been conducted on the interactions between mesofauna and ectomycorrhiza. Setälä (1995) reported that by consuming ectomycorrhizal fungi, soil mesofauna may exert control over the development of the plant-fungus symbiosis. Mesofauna can affect the performance of the plant–fungus symbiosis between ectomycorrhizal fungi and vascular plants (Moore et al. 2003). Feeding by mesofauna upon ectomycorrhizal fungi has been shown to increase plant primary production (Harris and Boerner 1990, Setälä 1995). As the rate of ectomycorrhizal infection increases, photosynthetic rates in leaves and often plant size increase as well (Allen 1991, Staddon et al. 1999). Furthermore, ectomycorrhizae could mediate the interactions between fungal-feeding fauna and aboveground herbivores (White 1984, Price 1991). Although studies have showed the ecological significance of fungal-feeding mesofauna can reach far beyond the rhizosphere (Moore et al. 2003), there still lack information on mesofauna relationships specifically with sclerotia-forming ectomycorrhiza. With the ability of sclerotia to survive long periods of time, information on these ectomycorrhiza may also contribute to sclerotia life history. Furthermore, understanding sclerotia and their associations with mesofauna may lead to a better understanding of the food web complexity and biological processes in soil.

Fig 1.1 describes the general relationship of ectomycorrhiza with plant roots, soil mesofauna,

and sclerotia in the forest soil.

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Fig. 1.1 Ectomycorrhiza associations with two soil organisms. During unfavorable environmental

conditions, the mycelia of ectomycorrhiza harden and form sclerotia for survival purposes. On the

other hand, ectomycorrhiza is also known to be grazed by mesofauna such as Acari, Collembola and

Nematodes.

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1.4 Molecular methods to describe ectomycorrhizal fungal communities

Mycorrhizal fungi occur in highly diverse communities (Bruns 1995) with fine-scale spatial partitioning (Dickie et al. 2002; Dickie and Reich 2005; Genney et al. 2006). Due to these difficulties, there has been an increasing reliance on molecular methods for identifying species from belowground structures (Horton and Bruns 2001), initially with restriction fragment length polymorphism analysis (RFLP) which is also known as amplified ribosomal DNA, rDNA, restriction analysis (ARDRA).

Another technique called denaturing gradient gel electrophoresis (DGGE) is also commonly used (Kowalchuk et al. 2002; Opik et al. 2003; Bougoure and Cairney 2005; Landeweert et al. 2005; Ma et al. 2005; Pennanen et al. 2005), as well as clone libraries (Landeweert et al. 2003; Renker et al. 2006), and terminal restriction fragment length polymorphism (T-RFLP). It has been suggested that T-RFLP is more sensitive than DGGE for fungi (Brodie et al. 2003; Singh et al. 2006), although obtaining sequences directly from samples may be more easily performed with DGGE (Ma et al. 2005).

T-RFLP also has significant advantages in cost over clone libraries, although clone libraries are likely the most accurate method of identifying species (Dickie and FitzJohn 2007). Using clone libraries together with T-RFLP may permit both techniques to be used to their full potential: using T-RFLP to process large numbers of samples and clone libraries on selected samples to obtain identities of key species (Lindahl et al. 2006; Widmer et al. 2006).

Generally, T-RFLP refers to the use of fluorescently labeled primers combined with

restriction digests to visualize sequence variation in either single or mixed species DNA samples

(Dickie and FitzJohn 2007). The T-RFLP technique was first developed in Liu et al. (1997) as a tool

for assessing bacterial diversity and comparing the community structure of bacteria in environmental

samples (Marsh 1999; Lukow et al. 2000; Kitts 2001). The data obtained are the sizes of the

fragments of polymerase chain reaction (PCR) amplicons that contain the labeled primer (the terminal

fragment lengths), observed as electropherogram “peaks”. Variation in the presence and location of

cutting sites results in different species having terminal fragments of different lengths. In T-RFLP, as

used by Liu and colleagues, a single fluorescent label and a single restriction digest are used. The data

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are then analyzed based on the number of peaks and the similarity of peak profiles across samples (Dollhopf et al. 2001; Edel-Hermann et al. 2004; Mummey et al. 2005). In this study the T-RFLP method is utilized to describe ectomycorrhizal fungal communities in sclerotia and mesofauna.

1.5 Objective, structure of this study

One of the ways to understand the complexity of food web and functions of biota in soil may be through apprehending the interactions between sclerotia-forming ectomycorrhiza and their associations with mesofauna. This study aimed to describe the relationships between sclerotia and mesofauna through the T-RFLP analysis of ectomycorrhizal fungal communities existing both in sclerotia and mesofauna. In soils where both sclerotia and mesofauna are present, this research will determine:

1. Which ectomycorrhiza associates with sclerotia, 2. Which ectomycorrhiza associates with mesofauna,

3. The relationship between sclerotia and mesofauna described through ectomycorrhiza communities.

Chapter 1 is the general introductive section for sclerotia, mesofauna, ectomycorrhiza

communities and the profiling technique of T-RFLP to lead to the objective of this study. Chapter 2

focuses mainly on ectomycorrhiza communities found in sclerotia and how T-RFLP method enables

to determine sclerotia identity and life history. Chapter 3 discusses about ectomycorrhiza communities

in mesofauna, and how T-RFLP approach may be applied on understanding niche differentiation

between two most predominant mesofauna groups in soil. In Chapter 4, a model of the relationship

between sclerotia and mesofauna through ectomycorrhiza communities is proposed using two

parameters, the soil pH and exchangeable aluminum content. Finally in Chapter 5, conclusions of the

study and recommendations for future steps will be presented. The structure of this study is shown in

Fig 1.2.

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Fig. 1.2 Structure of this study.

Objective:

To understand the relationships between sclerotia and mesofauna through the analysis of ectomycorrhiza communities both in sclerotia and mesofauna

Chapter 1 Introduction

Chapter 2

Ectomycorrhiza communities in sclerotia

Chapter 3

Ectomycorrhiza communities in mesofauna

Chapter 4

Sclerotia and mesofauna relationships in forest soils described by ectomycorrhiza communities

Chapter 5

Conclusion

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C HAPTER 2

E CTOMYCORRHIZA COMMUNITIES IN SCLEROTIA

2.1 Introduction

Fungal species may respond to environmental stress such as desiccation through hardening their mycelium and form a compact mass appearing as resting structures. These resting structures are called sclerotia. Sclerotia are loosely described as morphologically variable, nutrient-rich, multi hyphal structures which can remain dormant or quiescent when their environment is adverse and then when conditions improve, germinate to reproduce the fungus (Willets and Bullock 1992).

The remarkable ability of sclerotia to survive long periods in soil has been associated with the occurrence of an outer cell layer, the rind, composed of empty melanized cells (Chet 1967). There have been limitations in identifying sclerotia mainly because they grow slowly in culture (Chen et al.

2007) and they are difficult to isolate in axenic culture (Dickie et al. 2002). On the other hand, during their growth, sclerotia accumulate relatively high concentrations of carbohydrates, fats, and proteins which may provide good nutritional niches for other microorganisms associated with sclerotia to develop (Willets 1971). These sclerotia-associated microorganisms presents valuable information for studies concerning microbial diversity in the rhizosphere, biocontrol for plant pathogens (Zachow et al. 2011), and also functional heterogeneity in the adaptations towards drought (Jany et al. 2002).

As sclerotia are formed from fungal species, analyzing sclerotia-associated fungal

communities in sclerotia may confirm the identification of sclerotia, based on the fungal community

profile. Many ectomycorrhiza also form sclerotia and recent studies have been conducted on

identifying fungal species extracted from sclerotia (e.g. Obase et al. 2014, Zachow et al. 2011, Ohta et

al. 2003). In this chapter, ectomycorrhiza in sclerotia of forest soils in Japan were studied. The study

aim of this chapter was to understand which ectomycorrhiza species are associated with sclerotia in

four forest soils of Japan through T-RFLP method. Furthermore, this chapter will discuss how T-RFLP

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method can be applied to determine the identity of sclerotia.

2.2 Materials and methods

2.2.1 Study area and soil sampling techniques

Soil samples were taken from forests in the cool temperate zone in four Prefectures of Japan (Fig 2.1). These forests are predominated by Japanese Beech (Fagus crenata) trees. Therefore in this study the word “forest” in the studied areas refers to forests dominated by F. crenata in the cool temperate zone of Japan. The four studied areas were: a plateau near Lake Tazawa of Akita Prefecture, or further on will be stated as “Akita” (Fig. 2.1.1), Mt. Chokai located between Akita and Yamagata Prefecture, which will be stated in following sections as “Chokai” (Fig. 2.1.2), Mt. Iwaki of Aomori Prefecture, or will be further described as “Iwaki” (Fig. 2.1.3), and Shinshu University Research Forest near Mt. Kisokomagatake in Nagano Prefecture which will be further referred as “Nagano”

(Fig. 2.1.4).

Vegetation of the site in Akita (Fulvic Andosols, WRB/FAO-Unesco) was predominated by Fagus crenata and Quercus crispula with the forest floors mainly consisting of Sasa kurilensis.

Studied area in Mt. Chokai (Cambic Podzols, WRB/FAO-Unesco) were predominated by Fagus crenata, and less dominant plants were Viburnum furcatum and Lindera umbellata, while the forest floors were dominated by Sasa kurilensis. Meanwhile the site in Mt. Iwaki (Dystric Cambisols, WRB/FAO-Unesco) consisted mostly of Quercus serrata, Fagus crenata, and Betula ermanii trees and Sasa kurilensis dominated the forest floors. In Nagano (Dystric Cambisols, WRB/FAO-Unesco) the studied area consisted of Fagus crenata, Quercus serrata, and Pinus pumila while the forest floors were also dominated by Sasa kurilensis.

Within a 10x10 m

2

quadrat, nine points were selected evenly. Each point was a 20x20 cm

2

square where litter, F, and H layers were removed, and A horizons were collected using a bulb planter

(Spear and Jackson, Sheffield, UK) taken twice with the volume of approximately 800 cm

3

each (Fig

2.2). The bulb planter will further be mentioned as a “cylinder” in the following texts.

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Fig. 2.1 Study area across Japan

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Fig. 2.1.1 Study area at Lake Tazawa Plateau, Akita Prefecture, indicated by the red star. Bottom left

is the sampling location with predominant Fagus crenata trees and the forest floors mainly covered by

Sasa kurilensis, bottom right is the sub plot where A horizons were collected.

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Fig. 2.1.2 Study area at Mt Chokai, Yamagata Prefecture. The red star represents the sampling site.

Bottom left is the sampling location with predominant Fagus crenata trees, bottom right shows the

forest floors mainly covered by Sasa kurilensis.

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Fig. 2.1.3 Study area at Mt Iwaki, Aomori Prefecture. The red star indicates the sampling location.

Bottom left is the sampling location covered with mixed vegetation of Fagus crenata, Quercus

serrata, Betula ermanii trees, bottom right shows the forest floors mainly covered by Sasa kurilensis.

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Fig. 2.1.4 Study area near Mt Kisokomagatake, Nagano Prefecture. Sampling location indicated by

the red star. Bottom left is the sampling location covered with mixed vegetation mainly consisting of

Fagus crenata trees, bottom right shows the forest floors predominantly covered by Sasa kurilensis.

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Table 2.1 Location and characteristics of the study area

*Data obtained from The Japan Meteorological Agency.

** Data are mean ± standard errors from 9 repeats in each area.

Akita Chokai Iwaki Nagano

Study Area Lake Tazawa

Plateau

Mt. Chokai Mt. Iwaki Mt.

Kisokomagatake Coordinates

39° 46' 21.81"N, 140°

47' 2.51"E

39° 6' 42.53"N, 139° 58' 0.61"E

40° 39' 0.37"N, 140° 16' 33.60"E

35° 45' 25.56"N, 137° 51' 20.73"E

Vegetation

Fagus crenata, Quercus crispula

Fagus crenata, Viburnum furcatum, Lindera umbellate

Fagus crenata, Quercus serrata,

Betula ermanii

Fagus crenata, Quercus serrata,

Pinus pumila Floor vegetation Sasa kurilensis Sasa kurilensis Sasa kurilensis Sasa kurilensis

Soil type (FAO, 1988)

Light colored Kurobou soils

(Fulvic Andosols)

Brown forest soils (Cambic

Podzols)

Brown forest soils (Dystric Cambisols)

Brown forest soils (Dystric Cambisols)

Av. soil pH (H

2

O)** 4.10

± 0.05 4.60 ± 0.09 3.96 ± 0.04

4.20

± 0.06

Elevation (m) 760 730 790 780

Annual Rainfall (mm)* 2260 2362 1570 1070

Mean annual

temperature(°C)* 7.3 6.8 5.7 7.2

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Fig. 2.2 Soil sampling methodology. Soil samples were taken within a 10x10 m

2

plot with

approximately same distance between each sub plot (left) using a cylinder (bulb planter, Spear and

Jackson, Sheffield, UK, right). Two cylinders were taken from each sub plot for sclerotia collection.

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2.2.2 Sclerotia extraction

Sclerotia were handpicked from the A horizons then the surface of sclerotia was sterilized using the technique performed by Ohta et al. (2003). Every grain was washed with 1 ml of sterile water in a microtube for 1 minute in a shaker (IKA, Osaka, Japan) then the washing solution was removed from the tube with a sterile pipette, and this procedure was repeated ten times.

Surface-sterilized sclerotia were weighed until approximately 100 mg for each studied area, and placed in a sterile 2 mL centrifuge tube.

Sclerotia characteristics were also observed. The diameter was measured by a video microscope (Keyence, Osaka, Japan) and count density was obtained through calculating the number of grains collected per gram of soil. Mean grain weight data was collected by weighing 3 sclerotium grains per cylinder, and weight density was count density multiplied by mean grain weight.

2.2.3 Isolating fungal DNA in sclerotia

Sclerotia walls contain nonhydrolyzable residue consisting of highly resistant melanin-like

pigment which plays an important role in the resistance of sclerotia towards chemical and biological

degradation (Chet 1967). Therefore a more specific technique to isolate microbial DNA from sclerotia

is needed as compared to the technique performed in isolating fungal DNA from mesofauna. After

sclerotia was surface-sterilized and placed in a sterile tube, a metal crusher was inserted into the tube

and 200μL of Lysis Buffer (10 μL Tris-HCl pH 8 1M; 2μL EDTA 0,5M; 10μL Proteinase K; 100μL

SDS 10%, and 878μL H

2

O) was added and homogenized. Crushed sclerotia solution was incubated in

37˚C with shaking for 2 hours. Lysed solution was extracted with phenol/chloroform extraction, and

followed by the Binding buffer (60 gr Guanidine Thiocyanate; 10 mL Tris-HCl 1M; and 40mL

distilled water) and silica addition. Guanidine Thiocyanate was washed out using the Wash Buffer

(10mM Tris-HCl pH 7.5; 100mM NaCl:Ethanol = 1:4). PCR was conducted using the universal

primers ITS1F and ITS4 (Gardes and Bruns 1993) with iProof™ High-Fidelity PCR kit (Bio-rad

Laboratories, Hercules, CA, USA) under a hot start at 98˚C for 30 seconds, then 35 cycles consisting

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of 10 seconds at 98˚C, 30 seconds at 58˚C, and 30 seconds at 72˚C in a DNA thermal cycler (Takara Bio, Otsu, Japan). The PCR products were purified from the Seakem® GTG™ Agarose gel and continued with the silica method. The purified PCR products were then amplified through a quenching PCR using the quenching-fluorescence-labeled primers qLR21 and ITS1F. The 30 µL qPCR reaction mixture was prepared by adding 0.1 μg template sclerotia DNA, 1.0 μL of 10 pmol μL

-1

primers, Takara Ex Taq™ dNTPs, and 3 μL of optimized 10xEx buffer (Takara Bio) in a PCR cycler.

2.2.4 Terminal Restriction Fragment Length Polymorphism

The PCR for T-RFLP profiling was conducted after denaturing for 2 minutes at 98˚C, followed by 30 cycles consisted of 30 seconds at 95˚C, 45 seconds at 54˚C, and 90 seconds at 72˚C.

Aliquots of amplified fragments were then separately digested with restriction enzymes AluI, HhaI, and HaeIII (Takara Bio) according to the manufacturer’s instructions. The length of T-RFs from the amplified fragments were determined by a 3130xl DNA Sequencer (Applied Biosystems, Foster City, CA, USA) through mixing 2µL of purified T-RF DNA with 15µL of Hi-Di formamide and 0.1µL of DNA standard LIZ®600 (Applied Biosystems). The procedure was followed by denaturing at 96˚C for 2 minutes and immediately chilled on iced ahead of the electrophoresis using ABI automated sequence analyzer.

Lengths of the fluorescently labeled T-RFs were determined after electrophoresis by comparing with internal standards using GeneMapper® software (version 3.7, Applied Biosystems). T-RFLP profiling of the fungal communities in sclerotia resulted in peaks ranging from base sizes 50-650 base pairs.

The dominant T-RF peaks were determined by analysis of clone libraries. T-RF solutions were ligated using pGEM®-T Easy Vector (Promega, Madison, WI, USA), and Escherichia coli DH5α High Efficiency Competent Cells was used as hosts for recombinant plasmids and grown at 37˚C in LB agar, previously added 100µg mL

-1

of ampicillin, IPTG and Xgal to a final concentration of 40µg mL

-1

. White colonies were selected and sequences were determined using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and read on an Applied Biosystems 3130xl Genetic Analyzer.

The primer M13 Primer RV was used in sequencing reactions to obtain partial DNA sequences. The

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DNA sequences were aligned using MEGA version 5 (Tamura et al. 2011), and all sequences determined were compared to NCBI databases by using the BLAST program.

2.3 Results and discussions 2.3.1 Sclerotia characteristics

Sclerotia from Akita were rounded, brownish black and relatively small with a diameter ranging between 0.4 - 2.8 mm (average 0.98±0.21 mm; n=54). While those collected from Mt.

Chokai were also rounded, gray-brownish-black, having a diameter range between 0.4 - 2.6 mm (average 1.33±0.62 mm; n=54). On the other hand Mt. Iwaki sclerotia were not all perfectly rounded, their colors were shiny-black and relatively large in diameter, with a range between 0.8 - 4.4 mm (average 2.73±1.13 mm; n=54). And sclerotia found in Nagano were also round and grayish-black, found with a diameter range of 0.4 - 2.8 mm (average 1.07±0.29 mm; n=54).

Based on their morphological characteristics, sclerotia found in all studied areas correspond to most of the descriptions reported by Trappe (1962) which were 0.05 – 4 mm or more in diameter, jet-black, hard, smooth, and mostly spherical. Based on the observed collected sclerotium grains, the average sizes of sclerotia collected from Mt. Iwaki were significantly larger than those collected from the rest of the studied areas (p < 0.01 by Student’s t-test). According to Matsumoto and Tajimi (1988) the size of sclerotia responds to the fungal strategy to adapt environmental changes, whereas certain species in area where it is difficult to forecast environmental changes tend to form smaller sclerotia with less reserve substances in active term. Therefore the larger size of sclerotia found in Mt. Iwaki may indicate that this area, compared to Akita, Mt. Chokai, and Nagano, may have less environmental changes. Larger sclerotia diameter may also indicate high exchangeable aluminum (Al

Ex

) content in the soil, or had experienced some event which enriched Al

Ex

in the past (Sakagami 2008) which is shown by the lower pH in soils of Mt. Iwaki compared to that of other areas.

From each cylinder, collected grains of sclerotia in Akita ranged 0 - 19 grains (average 8.05

±1.31) per 800 cm

3

. In Mt. Chokai the number of sclerotia ranged 15 - 27 grains (average 19.83±

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0.81) per 800 cm

3

while in Mt. Iwaki sclerotia were found more abundant, ranging from 12 - 47 grains (average 33.56±2.36) per 800 cm

3

. From Nagano, sclerotia grains ranged between 0 - 52 grains (average 9.39±2.85) per 800 cm

3

.

Sclerotia in Mt. Iwaki also showed highest count density, mean grain weight and weight

density among other study sites (Table 2.2). With Mt. Iwaki also having the lowest soil pH among the

study sites (Table 2.1), this result is in accordance with Watanabe et al. (2001) where the development

of sclerotia is influenced by high active aluminum in soils occurring in low pH.

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Table 2.2 Sclerotia properties

Samples Akita Chokai Iwaki Nagano

Count density (grains/g soil) 0.09 ± 0.01 0.14 ± 0.06 0.16 ± 0.03 0.13 ± 0.07

Mean grain weight (mg) 0.76 ± 0.11 0.83 ± 0.04 1.80 ± 0.05 0.79 ± 0.16

Weight density (mg/g soil) 0.07 ± 0.13 0.11 ± 0.02 0.29 ± 0.04 0.10 ± 0.12 Diameter (mm) (n=54) 0.98 ± 0.21 1.33 ± 0.62 2.73 ± 1.13 1.07 ± 0.29

Number of sclerotia (grains) 8.05 ± 1.31 19.83 ± 0.81 33.56 ± 2.36 9.39 ± 2.85

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2.3.2 Fungal community profiles of sclerotia

Fig. 2.3 depicts that in Akita, fungal community profiles of sclerotia showed to be predominated by Laccaria sp. (Basidiomycota: Agaricales) an ectomycorrhiza commonly studied for mycorrhizal symbiosis (Plett et al. 2011, Martin et al. 2008).

Fungal community profile of sclerotia from Mt. Chokai showed one dominant peak which was identified as Arthrinium arundinis (Ascomycota: Sordariales), a species described as a dematiaceous or having dark-walled septate hyphae hyphomycetes (Pan et al. 2009). Species of Sordariales was also found abundant and frequent in sclerotia collected from forests dominated by Quercus and Pinus in Florida and Georgia, USA (Obase et al. 2014), and has been recognized as one of the soil fungal species responsible for bamboo degradation (Kim et al. 2010). Nakashizuka (1988) reported that the survival rate of Fagus crenata seedlings on the floor where dwarf bamboo had withered was much higher than that on the floor where dwarf bamboo survived. Moreover, removal of understory dwarf bamboo increased net carbon gain and transpiration rates of overstory trees (Kobayashi et al. 2006) hence removal of dwarf bamboo in relatively young stands may greatly enhance productivity of overstory trees in the long-term (Ishii et al. 2008). Based on these reports the presence of A. arundinis in sclerotia of beech forest floor in Mt. Chokai is suggested to play an important role in the survival of F. crenata.

Results from Mt. Iwaki showed one major peak which was identified as Inonotus sp.

(Basidiomycota: Hymenochaetales), a fungus known to be one of the common diseases in birch trees, including Betula ermanii. According to Yamaguchi (1989), a white-rot fungus, I. obliquus, the casual microorganism of canker disease of Japanese birch, is found in Honshu and northern Japan, especially in Hokkaido. Hattori (1990) reported to have isolated I. obliquus in sclerotium tissue of B. platyphylla Sukachev var. japonica (Miq) Hara in Tochigi, North Japan. Based on this information, isolated Inonotus sp. from sclerotia of Mt. Iwaki may serve as a pathogen for the birch trees observed in the studied site, even though the pathogenicity of a target species could not be confirmed.

In Nagano, a major peak was identified as Tuber sp. (Ascomycota: Pezizales). Ecologically,

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Tuber species are obligate ectomycorrhizal fungi and form ectomycorrhizas with pine (Pinus spp.), fir (Abies spp.), birch (Betula spp.), aspen (Populus spp.), oak (Quercus spp.), hazel (Corylus avellana L.), and rockrose (Cistus spp.) and also form mycorrhizas with orchids (Bidartondo et al. 2004, De Roman et al. 2005). The study site in Nagano was a mixed forest where Pinus and pumila and Quercus serrata were also observed, suggesting that Tuber sp. in sclerotia may possibly be the ectomycorrhizal fungi of P. pumila or Q. serrata in the study site.

2.3.3 T-RFLP method applied to determine sclerotia identity

This sub chapter was partially reported in Amasya et al. (2015) on the analysis of sclerotia-associated fungal communities found in Mt. Chokai and Mt. Iwaki. One of the most common soil fungal species forming sclerotia is Cenococcum geophilum, which is reported to be distributed worldwide (Dickie 2007, Jany et al. 2002) with host plants estimated at more than 200 species (LoBuglio et al. 1996). Sclerotia of C. geophilum were also found abundant and studied in many parts of the world. In old-growth Norway spruce forests of south Sweden, sclerotia of C. geophilum biomass were estimated to be 440 kg ha

-1

(Dahlberg et al. 1997), while in a second-growth Douglas-fir stand in the Oregon Coast Range it was reported to be 2785 kg ha

-1

(Fogel and Hunt 1979). The distribution of C. geophilum sclerotia was also studied in forest soils of Harz Mts., Germany (Sakagami 2009, Watanabe et al. 2004). In Japan, sclerotia of C. geophilum were distributed in volcanic ash soils in central Japan (Ohta et al. 2003, Watanabe et al. 2001, Watanabe et al. 2004), Andosol profiles in central Japan (Watanabe et al. 2002) and were found to be abundant in Pinus thurnbergii of coastal pine forests of Japan (Matsuda et al. 2009). Interestingly, these studies identified sclerotia as the resting bodies of C. geophilum based on the morphological characteristics description provided by Trappe (1962) and Kumada and Hurst (1967) without further molecular identification studies, while in fact a broad range of fungal species have the ability to form sclerotia.

Therefore in this sub chapter T-RFLP method combined with clone library analysis was applied to

identify sclerotia collected from studied areas.

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Fig. 2.3 T-RFLP profile of sclerotia in four study sites, using restriction enzyme HhaI.

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As shown in Fig. 2.3, T-RFLP results suggested that sclerotia of Akita was predominated by Laccaria sp., Mt. Chokai by A. arundinis, Mt. Iwaki was by Inonotus sp., and those of Nagano by Tuber sp. This suggests either: (i) observed sclerotia were formed by Laccaria sp. in Akita, A.

arundinis in Mt. Chokai, Inonotus sp. in Mt. Iwaki, and by Tuber sp. in Nagano; or (ii) sclerotia were originally formed by C. geophilum but later on occupied by other species after C. geophilum germinated, or failed to survive due to competition with those other fungal species. The latter suggestion was supported by Obase et al. (2014), stating that sclerotia-associated fungi may be specialized mycoparasites or saprobes that preferentially decay fungal tissues or act as endophytes which colonize Cenococcum sclerotia without any aggressive interactions. However, given the low success rate for Cenococcum isolations (Obase et al. 2014), and considering the lack of it in this study, the possibility that observed sclerotia were formed by Laccaria sp. in Akita, A. arundinis in Mt.

Chokai, Inonotus sp. in Mt. Iwaki, and by Tuber sp. in Nagano cannot be ruled out. Further studies need to be conducted in order to confirm either suggestion.

2.4 Conclusion

Fungal community profiles of ectomycorrhiza in sclerotia were identified as Laccaria sp. in

Akita, Arthrinium arundinis in Mt. Chokai, Inonotus sp. in Iwaki, and Tuber sp. in Nagano. This study

showed that fungal community profiling of sclerotia was an effective approach not only to identify

sclerotia but also contributed to broaden the understanding of the roles and functions of sclerotia in

the forest soil.

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C HAPTER 3

E CTOMYCORRHIZA COMMUNITIES IN SOIL MESOFAUNA

3.1 Introduction

Soil mesofauna is the most diverse component of the soil ecosystem. Animals living among the litter and inside the microscopical crevices of the soil have a fundamental role as processors and translocators of the organic matter that ends up forming the humus. Across soil ecosystems, the soil mesofauna contribute significantly to decomposition processes and nutrient turnover (Visser 1981).

Many taxa are represented, including several orders of insects and their larvae, as well as Myriapoda, Crustacea, Thysanura, Tardigrada, and others. But three of them (Acari, Collembola, and Nematoda) dominate in terms of numerical abundance and diversity. Soil mesofauna such as Acari or mites feed on microorganisms and regulates the dispersal of microbial propagules (Behan and Hill 1978). The total species number of Acari is estimated to be up to 100,000 (Schatz 2002). In total about 50-120 species are found in most forest ecosystems (Wunderle 1992). Acari are important decomposers in almost all habitats; their distribution ranges from arid coniferous forests over floodplain forests to salt marshes (Weigmann 1971, Usher 1975, Mitchell 1979). The least diverse of the three mentioned groups, Collembola (‘springtails’), includes more than 7,600 known species: more than all mammal species, or three quarters of the known number of bird species. Collembola contribute to the regulation of fungal populations (Warnock et al. 1982) and also in establishing relationships with mycorrhizae (Gange 2000) in the soil.

Microbial community profiling methods such as T-RFLP enables fungal species in soil

mesofauna to be detected. The aim of this chapter was to understand which ectomycorrhiza species

were associated with soil mesofauna through T-RFLP method and to further describe how these

animals share the fungal resources in the soil.

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3.2 Materials and Methods

3.2.1 Study area and soil sampling techniques

The study area was the same as described in Chapter 2.2.1. The location description and characteristics of the study areas is listed on Table 2.1. Soil samples were collected within a 10x10 m

2

from forest soils in the studied areas. From each site, nine cylinders, each with the capacity of approximately 800 cm

3

, were used to obtain soil from the A horizon for mesofauna extraction (Fig.

3.1).

3.2.2 Soil mesofauna extraction

Soil samples for mesofauna extraction were transported in a cotton bag instead of plastic

containers to ensure mesofauna viability before extraction from the soil. Soil mesofauna were then

extracted using a modified Berlese-Tullgren funnel (Macfadyen 1953). The basic principle of a

Berlese-Tullgren funnel is to create a temperature gradient over a soil sample in an attempt to force

mobile organisms to move away from the higher temperature and fall into a collecting vessel. In this

study the heat was produced by a 5 Watt light bulb and the heat gradient was increased by an

aluminum funnel around the soil sample (Fig 3.2). The length of the funnel used in this study was

approximately 14 cm. The collecting vessel was filled with 70% ethanol for surface sterilization and

to preserve the mesofauna for further examination. Collections of animals were identified under a

digital high density video microscope (VH-7000, Keyence, Osaka).

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Fig. 3.1 Soil sampling methodology. One cylinder of soil was taken from each sub plot for mesofauna

extraction.

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Fig. 3.2 Modified Berlese-Tullgren funnel apparatus used to isolate soil mesofauna from soil.

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3.2.3 Isolating fungal DNA in soil mesofauna

Three to five individuals of each animal from each sub-plot were initially crushed using a metal crusher and fungal DNA was isolated using Prepman™ (Applied Biosystems, Foster City, CA).

This process was repeated 3 times for each location. The ribosomal DNA Internal Transcribed Spacer (ITS) regions were then amplified by the PCR using primer pair ITS-1F/LR21 and DNA Taq Polymerase enzymes (Applied Biosystems) with conditions as follows: a hot start at 96°C for 30 seconds, then 35 cycles consisting of 10 seconds at 96°C, 30 seconds at 55°C, and 30 seconds at 72°C.

This procedure was continued with T-RFLP as described in Chapter 2.2.4. Aliquots of the amplified DNA were digested with restriction endonucleases to obtain ITS-RFLPs; each sample was digested with Hae III and HhaI (Promega).

3.2.4 Statistical analyses Mesofauna diversity

Diversity of mesofauna was estimated using two indices, the Shannon-Wiener (H’) Diversity Index (Hill 1973) and the Inverse Simpson Index (D) (Simpson 1949). The number of individuals of each mesofauna Order represents species abundance, and the number of identified Orders represents species richness.

The Shannon-Wiener index was calculated as follows, where p

i

is the proportion of characters belonging to the i

th

species:

On the other hand, the Inverse Simpson Index (D) was calculated as follows:

The higher the Inverse Simpson Index (D), the higher the diversity.

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Diversity of fungal communities

Shannon-Wiener (H’) Diversity Index (Hill 1973) was calculated to quantify the diversity of fungal communities in soil and mesofauna, with the T-RF fragment sizes representing fungal species richness and the T-RF peak heights represent the fungal species abundance.

3.3 Results and discussions

3.3.1 Soil mesofauna characteristics

Soil mesofauna collected from Berlese-Tullgren funnels in all study areas belonged to the Orders Acari, Collembola, Nematoda, Symphyla, Diptera, Coleoptera, Hymenoptera, Chilopoda and Diplopoda, except in Akita where there were no Hymenoptera collected. Fig. 3.3 shows the average abundance of soil mesofauna across study areas. In each site, Acari and Collembola were found to be predominant. Shannon (H’) Diversity Index (Hill 1973) and Inverse Simpson Index (D) was calculated to quantify soil mesofauna diversity as shown in Fig 3.4. Among study sites, Nagano (H’=

1.51; D = 3.65) showed the highest mesofauna diversity. According to MacArthur (1955) more

diverse communities will enhance ecosystem stability. Therefore, the high mesofauna diversity in the

forest soils of Nagano indicates a soil ecosystem with more stability among other studied areas.

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Fig. 3.3 Average abundance of soil mesofauna

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Fig 3.4 Soil mesofauna diversity across study sites estimated by Shannon Diversity Index (H’) and

Inverse Simpson Index (D).

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3.3.2 Fungal community profiles in soil mesofauna

T-RFLP profile of fungal species extracted from soil mesofauna of forest soils is shown in Fig 3.5.1 to Fig 3.5.4. Fungal community profile in mesofauna from Akita is shown in Fig 3.5.1, communities of fungal species collected from Chokai mesofauna is shown in Fig 3.5.2., those from Iwaki is shown in Fig 3.5.3 and from Nagano is shown in Fig 3.5.4.

The peak heights in the y-axis show the intensity, or in other words describing the

abundance of fungal communities in mesofauna. The x-axis on the other hand shows fragment sizes of

restricted fungal DNA, with each base pair length representing a specific fungal species. From the

peak heights alone, Acari of Akita in Fig 3.5.1 showed a unique peak of Laccaria sp. From Fig 3.5.4

Acari of Nagano also showed a uniqe peak of Laccaria sp. and Collembola showed unique peaks of

Paxillus sp.

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Fig. 3.5.1 T-RFLP profile of fungal species extracted from soil mesofauna in Akita. Hymenoptera

were not found in Akita samples.

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Fig. 3.5.2 T-RFLP profile of fungal species extracted from soil mesofauna in Chokai.

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Fig. 3.5.3 T-RFLP profile of fungal species extracted from soil mesofauna in Iwaki.

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Fig. 3.5.4 T-RFLP profile of fungal species extracted from soil mesofauna in Nagano.

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3.3.3 Acari and Collembola predominance and their roles in the soil ecosystem

The Order Oribatida of Acari, identified by having fungivorous mouth parts, were selected, while Acari with other types of mouth parts (e.g. having chelate or sub chelate pedipalps) were not included in this study. Most of the Collembola found belonged to the Family Entomobrydae (69%) and the rest belonged to the Order Poduromorpha (31%). Acari collected had the length of 0.6 – 0.9 mm, while Collembola were about 1.3 – 1.5 mm in length, as shown in Fig. 3.6.

Sampling site of Nagano showed the highest average weight of litter layer, and had the highest mesofauna abundance (Fig. 3.6). The number of individuals responded to the average weight of litter layer, whereas the more litter piled up the more abundant both animals tend to be extracted.

The mean abundance of Acari and Collembola in this study which was conducted in temperate deciduous forests, translates into a density of 11,043 individuals/m

2

and 10,774 individuals/m

2,

respectively. This is lower than those reported in most temperate areas of central Japan (Hijii 1994), and some tropical sites (e.g., Seastedt 1984, Gonzalez et al. 2001). In Chokai and Iwaki, Acari were found more abundant. Similar findings of Acari having higher abundance compared to Collembola in forests of central Japan was reported, and was said to be related with the characteristics of the forest having heavy rainfall, acidic soils, a large amount of litter accumulation, and slow decomposition rates (Takeda and Abe 2001; Lin et al. 2002). Acari was found less abundant compared to Collembola in Akita and Nagano, which is in accordance with the findings by Hijii (1994) who reported ratio of springtail to mites (ca. 1.12) in coniferous forests of Japan.

With the predominance of Acari and Collembola in all sites (Fig 3.3), it is estimated that the

proportions of fungal uptake by Acari and Collembola among other mesofauna. The peak heights of

each fungal species consumed by every mesofauna in each area were compared. The result is shown

in Fig 3.7. From this result it can be perceived that Acari and Collembola not only predominates the

soil in their abundance, but their uptake of ectomycorrhiza in soil also showed to be highest among

other mesofauna. This figure shows that Acari and Collembola may play a major role in the regulation

of ectomycorrhiza in forest soils.

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Fig. 3.6 Mean number of Acari and Collembola captured in each sampling area per gram soil, as

compared to the average litter layer weight. Left-above: Acari; left-below: Collembola (Amasya

2014).

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Fig. 3.7 Proportions of fungal species uptake by mesofauna of forest soils. Data shows Acari and

Collembola have the highest proportion among other mesofauna in the fungal species uptake.

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Fungal communities extracted from Acari and Collembola were estimated whether they differed from those extracted from other soil mesofauna. Fig. 3.8 showed R-analysis resulting in stress value 0.0078 suggesting that Acari and Collembola are associated with specific ectomycorrhizal fungi, while other animals with a broader range of fungal species. This finding also suggests Acari and Collembola may serve as indicators for specific ectomycorrhiza species.

With the tendency of Acari and Collembola to associate with specific ectomycorrhiza and their significance in the uptake of ectomycorrhiza in soil, this research focused on identifying the ectomycorrhiza of Acari and Collembola from the four studied areas. The results are shown in Fig. 3.9.

In Akita, Acari showed a high peak of Laccaria sp. Meanwhile Collembola of Akita showed a high peak of Paxillus sp., and Tuber sp. was detected. In Chokai, Acari were predominated by Phyllactinia sp., Tuber sp., and Laccaria sp., while the Collembola of the same area showed specific peaks of Inocybe sp. and Paxillus sp. Meanwhile in Iwaki, predominant ectomycorrhiza of Acari were Trichoderma sp. and Tuber sp., while in Collembola, Inocybe sp. was detected. In Nagano, Acari showed to be predominated by Laccaria sp. and Collembola was predominated by Paxillus sp.

As mentioned in Chapter 3, Inonotus sp. (Basidiomycota: Hymenochaetales) is a fungus known to be one of the common diseases in birch trees, including Betula ermanii, while Laccaria sp.

(Basidiomycota: Agaricales) is an ectomycorrhiza commonly studied for mycorrhizal symbiosis (Plett

et al. 2011, Martin et al. 2008) which is reported to be intermediately preferred to be grazed on by

Acari (Schneider 2005). Trichoderma sp. is a fast-growing soil fungal species reported to have

effectiveness in biocontrol of plant-pathogenic fungi and soil-borne disease (Wells et al. 1972), while

Phyllactinia sp. is a fungus from Ascomycota known to cause a powdery mildew on leaves and stems

on a broad range of host plants, including Quercus serrata (Homma 1937). Tuber species are obligate

ectomycorrhizal fungi and form ectomycorrhizas with pine (Pinus spp.), fir (Abies spp.), birch (Betula

spp.), aspen (Populus spp.), oak (Quercus spp.), hazel (Corylus avellana L.), and rockrose (Cistus

spp.) and also form mycorrhizas with orchids (Bidartondo et al. 2004, De Roman et al. 2005). In

Japan, Inocybe sp. (Basidiomycota: Agaricales) are reported to be common in roadsides near forests,

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collected from Abies mariesii, Pinus koraiensis, and Betula ermanii forests (Kobayashi 2002).

Meanwhile, Paxillus sp. (Basidiomycota: Boletales) is widely distributed mainly under deciduous trees (Populus, Betula, Salix, and Quercus) and some species of Paxillus form sclerotia (Jargeat et al.

2014). Collembola grazing on Paxillus sp. has also been reported by Ek et al. (1994) where it was

shown that low densities of collembolans induced a shift towards a larger proportion of Paxillus

involutus growth.

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Fig. 3.8 R-analysis was used for analyzing the fungal community structure of soil mesofauna showing the stress value 0.0078. Nematoda, Chilopoda, Coleoptera, Diplopoda, Diptera, Hymenoptera, Symphyla shared the same fungal communitiy structure which were different from Acari and Collembola.

-60 -50 -40 -30 -20 -10 0 10

-10 -5 0 5 10 15

D imensi on B

Dimension A Acari

Collembola Nematoda, Chilopoda, Coleoptera, Diplopoda, Diptera,

Hymenoptera, Symphyla

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Fig 3.9 Ectomycorrhiza communities in Acari and Collembola from studied area.

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3.4 Conclusion

Mesofauna in forest soils were mainly predominated by Acari and Collembola. These animals not only predominates the soil in their abundance, but their uptake of ectomycorrhiza in soil also showed to be highest among other mesofauna. This shows that Acari and Collembola may play a major role in the regulation of ectomycorrhiza in forest soils.

Ectomycorrhiza associated with soil mesofauna in this study were Laccaria sp. in Acari of

Akita, Chokai, and Nagano, Inonotus sp. in Acari of Akita, Paxillus sp. in Collembola of Chokai and

Nagano, Phyllactinia sp. in Acari of Chokai, Tuber sp. in Acari of Chokai and Iwaki, Inocybe sp. in

Collembola of Chokai and Iwaki, and Trichoderma sp. in Acari of Iwaki and Collembola of Akita and

Iwaki.

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C HAPTER 4

S CLEROTIA AND MESOFAUNA RELATIONSHIPS IN FOREST SOILS DESCRIBED BY ECTOMYCORRHIZA COMMUNITIES

4.1 Introduction

Sclerotia and mesofauna are only two out of a wide variety of organisms in the soil ecosystem. Each organism has interrelated roles and functions contributing to important soil processes involving nutrient cycles in the rhizosphere. Both sclerotia and mesofauna are related to ectomycorrhiza, through sclerotia formation and fungal grazing by mesofauna. However, there have not been many studies on the relationships between sclerotia and mesofauna through analyzing the ectomycorrhizal fungal communities existing in both organisms. The knowledge of this relationship may contribute further to the understanding of sclerotia formation influenced by mesofauna.

The previous chapters discussed the importance of sclerotia and mesofauna, and the ectomycorrhiza communities from both organisms in each study area were described. As both organisms occupy the soil as their direct environment, the fungal community profiles of ectomycorrhiza in soil were also investigated as a comparison to those extracted from sclerotia and mesofauna.

The environmental conditions that have been reported to play a role in sclerotia formation has been soil acidity and exchangeable aluminum (Al

Ex

) content (Watanabe et al. 2001, Watanabe et al. 2002, Sakagami 2008, Sakagami 2009). It has been known that soluble Al is present in the soil when the pH begins to drop below pH 5.5, and the amount of soluble Al increases dramatically in nearly all soils as the soil pH drops below pH 5.0 (Kinraide 1991).

Based on Table 2.1 the studied areas show to be acidic with relatively low pH (H

2

O) values.

In order to check the presence of soil aluminum, in this chapter the pH (KCl) and also the Al

Ex

content

were measured.

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Therefore, in this chapter, a model to describe the relationships between sclerotia and mesofauna in forest soils through the ectomycorrhizal fungal community profiles obtained by T-RFLP method was proposed as an attempt to introduce the role of mesofauna as one of the biological factors contributing to sclerotia formation, under observed soil acidity conditions.

4.2 Materials and methods

4.2.1 Fungal community profiles in soil

Soils were collected in the study area described in Chapter 2.2.1. All four studied areas were conducted in cool forest zones with soils having relatively low pH values. In order to compare with areas of a higher soil pH outside of the cool forest zones, soil samples from Tokyo Metropolitan University campus in Minamiosawa at Matsuki Hinata ryokuchi were selected, where the floor vegetation was predominated by Sasa sp. In this area, sclerotia abundance was also investigated, and mesofauna were also collected. The procedure was also continued with performing T-RFLP analysis of ectomycorrhiza obtained in mesofauna. The methodology used was the same as described in Chapter 3.2.

Nine cylinders were collected from each study area with a sampling method described in Fig.

3.1. To obtain fungal communities in the soil, fungal DNA was extracted using ISOIL for Beads Beating Soil DNA Extraction Kit (Nippon Gene Co., Ltd, Toyama, Japan). This extracting solution also uses both chemical lysis by a surface-active agent and physical disruption of cells by beads beating as the DNA extraction method. Extracted soil DNA was followed by T-RFLP analysis to obtain ectomycorrhizal fungal communities using the methodology as described in Chapter 2.2.4.

4.2.2 Soil pH and exchangeable aluminum content

In this study pH values were determined by the KCl method to obtain an index of soil acidity.

This method is more popular in those regions that have extremely acid soils and in which KCl is used

as an extractant of Al

Ex

. The KCl pH indicates the pH at which Al is extracted. Therefore both pH and

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Al

Ex

content were measured.

Sclerotia contains a relatively high concentration of Al, and the formation of sclerotia was reported to be regulated by the content of exchangeable aluminum and the status of active Al in the soil, regardless of soil type (Watanabe et al. 2002). Thus in order to analyze Al

Ex

content of soil as the substrate occupied by sclerotia, initially sclerotia were removed from the soil samples of four studied areas defined in Chapter 2.2.1. Then, soil samples were extracted with 1M KCl according to the method of Bertsch and Bloom (1996).

Al

Ex

content of soil with sclerotia was also analyzed as an attempt to confirm the contribution of Al

Ex

of sclerotia in soil. For soil with sclerotia, collected sclerotium grains were crushed with mortar and pestle, then added and mixed together with soil samples ahead of the KCl extraction following Bertsch and Bloom (1996) method. Al

Ex

content was determined using Inductively Coupled Plasma Atomic Emission Spectrometer (ICPE-9000, Shimadzu Corp, Kyoto).

4.3 Results and discussions

4.3.1 Ectomycorrhiza community profiles in sclerotia, soil mesofauna, and soil

Sclerotia were not detected in soil samples from Minamiosawa. One of the reasons was presumably due to the soil acidity level (pH = 6.11 ± 0.21 in Table 4.1), because it has been suggested that low soil pH is important for sclerotial formation (Aycock 1966). Because no sclerotia were collected, the T-RFLP analysis of ectomycorrhiza in sclerotia was not conducted. As for the rest of the studied areas, the fungal community profiles of ectomycorrhiza in sclerotia were identified as Laccaria sp. in Akita, Arthrinium arundinis in Mt. Chokai, Inonotus sp. in Iwaki, and Tuber sp. in Nagano.

Meanwhile ectomycorrhiza associated with soil mesofauna were Laccaria sp. in Acari of

Akita, Chokai, Nagano, and Minamiosawa; Inonotus sp. in Acari of Akita; Paxillus sp. in Collembola

of Chokai, Nagano, and Minamiosawa; Phyllactinia sp. in Acari of Chokai; Tuber sp. in Acari of

Chokai, Iwaki, and Minamiosawa; Inocybe sp. in Collembola of Chokai and Iwaki; and Trichoderma

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viridae in Acari of Iwaki and Collembola of Akita and Iwaki. Ectomycorrhiza communities in soil were also obtained. A summary of ectomycorrhiza in soil, sclerotia and mesofauna of four studied areas is shown in Fig. 4.1.1 to Fig. 4.1.5. The vertical red boxes in Fig. 4.1.1 to Fig 4.1.4 focus on the ectomycorrhiza species found in sclerotia as compared to the profile of the same peak in Acari, Collembola and soil. Interestingly, for Chokai, Iwaki and Nagano, when ectomycorrhiza was found relatively abundant in sclerotia, the same species were shown to be relatively less abundant or undetected in Acari and/or Collembola. The opposite is shown for Akita, where ectomycorrhiza species Laccaria sp. was found to be more abundant in Acari compared to that in sclerotia. A simplified graph of this result is shown in Fig. 4.2

Based on Fig. 4.2, ectomycorrhiza tend to be relatively abundant in mesofauna when they were found relatively low in sclerotia, and vice versa. Thus results demonstrated that ectomycorrhiza abundance in sclerotia and mesofauna showed the tendency to be inversely proportional. In Akita, when Laccaria sp. was found high in Acari, it was found in low abundance in sclerotia. Schneider (2005) reported Laccaria laccata as one of the intermediately preferred ectomycorrhiza by Acari. It was suggested that Laccaria sp. was grazed by Acari before its mycelia had the chance to form sclerotia. As for Arthrinium sp. and Inonotus sp., there had been no reports that this species are preferred for grazing by Acari or Collembola. However for Tuber sp. found in Nagano, Queralt et al.

(2014) who worked on the relationships between Tuber melanosporum and Oribatid mites, reported

that in some cases, mites have been seen carrying spores attached to their bodies. In this study, the

mesofauna were surface-sterilized with 70% ethanol before performing DNA isolation and T-RFLP

methods. Therefore the reason why Tuber sp. was not detected in the bodies of mesofauna of Nagano

was presumably due to the surface-sterilization stage in the methodology conducted in this research.

(55)

Fig. 4.1.1 Ectomycorrhiza in soil, sclerotia, and mesofauna in Akita observed through T-RFLP

peaks

(56)

Fig. 4.1.2 Ectomycorrhiza in soil, sclerotia, and mesofauna in Chokai observed through T-RFLP

peaks

(57)

Fig. 4.1.3 Ectomycorrhiza in soil, sclerotia, and mesofauna in Iwaki observed through T-RFLP

peaks

(58)

Fig. 4.1.4 Ectomycorrhiza in soil, sclerotia, and mesofauna in Nagano observed through

T-RFLP peaks

(59)

Fig. 4.1.5 Ectomycorrhiza in soil, sclerotia (not found), and mesofauna in Minamiosawa observed through T-RFLP peaks

Sclerotia

Soil

(60)

Fig. 4.2 Ectomycorrhiza abundance in Acari, Collembola, soil and sclerotia collected from the

study sites. Abundance of ectomycorrhiza in sclerotia and mesofauna showed the tendency to be

inversely proportional.

Fig. 2.1 Study area across Japan
Fig. 2.1.1 Study area at Lake Tazawa Plateau, Akita Prefecture, indicated by the red star
Fig.  2.1.2  Study  area  at  Mt  Chokai,  Yamagata  Prefecture. The  red  star  represents  the  sampling  site
Fig.  2.1.3  Study  area  at  Mt  Iwaki,  Aomori  Prefecture.  The  red  star  indicates  the  sampling  location
+7

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