Sclerotia Grains in Soils: A New Perspective from Pedosclerotiology (Progress in Soil Science) 981334251X, 9789813342514

Citation preview

Progress in Soil Science

Makiko Watanabe  Editor

Sclerotia Grains in Soils A New Perspective from Pedosclerotiology

Progress in Soil Science Series Editors: Alfred E. Hartemink, Soil Science, University of Wisconsin, Madison, WI, USA Alex B. McBratney, Sydney Institute of Agriculture School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW, Australia

Aims and Scope Progress in Soil Science series publishes books that contain novel approaches in soil science in its broadest sense – books in the series should focus on true progress in a particular area of the soil science discipline. The scope of the series is to publish books that enhance the understanding of the functioning and diversity of soils in all parts of the globe. The series includes multidisciplinary approaches to soil studies and welcomes contributions of all soil science subdisciplines. Key themes: soil science - soil genesis, geography and classification - soil chemistry, soil physics, soil biology, soil mineralogy - soil fertility and plant nutrition - soil and water conservation - pedometrics - digital soil mapping - proximal soil sensing - soils and land use change - global soil change - natural resources and the environment. Submit a proposal : Proposals for the series will be considered by the Series Editors. An initial author/editor questionnaire and instructions for authors can be obtained from the Publisher, Dr. Robert K. Doe ([email protected]).

More information about this series at http://www.springer.com/series/8746

Makiko Watanabe Editor

Sclerotia Grains in Soils A New Perspective from Pedosclerotiology

Editor Makiko Watanabe Department of Geography, Graduate School of Urban Environmental Sciences Tokyo Metropolitan University Hachioji, Tokyo, Japan

ISSN 2352-4774 ISSN 2352-4782 (electronic) Progress in Soil Science ISBN 978-981-33-4251-4 ISBN 978-981-33-4252-1 (eBook) https://doi.org/10.1007/978-981-33-4252-1 © Springer Nature Singapore Pte Ltd. 2021 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

To Toshihide and Shoko

Preface

Every existence ύπαρξη (íparksi) in soil performs a function and is connected to the other organisms in the ecosystem. However, the functions of the sclerotia, the glittering black spheres produced as resting bodies by ectomycorrhizal (ECM) fungi and found in forest soils around the globe, remain poorly known. Why do they remain there? What can be understood from their existence in heterogeneous soil ecosystems? We began our study of fungal sclerotia grains in June 1999, while we were still studying the green pigment present in soil humic acids, known as HA Pg. As part of our joint fieldwork (Dr. M. Watanabe, Tokyo Institute of Technology, and Dr. N. Fujitake, Kobe University), we occasionally found these large, black grains, 6–7 mm in diameter, in volcanic ash soils in Fagus forest in Asadaira, Mt. Myoko, Central Japan. The area had been found to be a kind of hot spot of HA Pg by the late Dr. K. Kumada of Nagoya University, who studied fungal sclerotia as the origin of HA Pg. Our encounters with sclerotia made us realize the potential for interdisciplinary research into the role of sclerotia grains in soil. How well do we understand the role that fungal sclerotia, so abundant in soil, play in soil ecosystems? What can sclerotia grains tell us about environmental conditions in the past, over recent centuries, and up to 10,000 years ago? What can we learn from natural sclerotia grains to develop mimic techniques? To what extent are sclerotia grains that are present in soil a concern for soil researchers? We do not yet have a clear answer on the significance of the presence of sclerotia grains in soil. There are two potential pathways forward: study of their material properties and study of their presence in soil across time and space. The aim of this book was to summarize available data on the anatomy of melanized fungal sclerotia, represented by Cenococcum geophilum sclerotia found in forest soils worldwide, and the factors regulating their distribution combined with perspectives on how to further elucidate their ecological function in soil. This book comprises results and discussions provided by joint research with soil geographers, soil organic chemists, soil microbiologists, physiologists, biologists, environmental

vii

viii

Preface

chemists, isotope chemists, nuclear physician, materials engineers, civil engineers, and biotechnologists. Chapter 1 provides a definition of sclerotia grains found in soils and differentiates them from sclerotia propagules. It also provides a review of the melanized fungal sclerotia produced by Cenococcum geophilum and related species. In addition, how study of C. geophilum sclerotia has contributed to the field of earth science is introduced. Chapter 2 details molecular methods used to identify the fungi responsible for the formation of sclerotia in soils and suggests that using culture-based methods may not work to identify the fungus of origin because sclerotia persist in forest soils for a long time, and the hyphae cell they contain may be too degraded for culture or DNA extraction. Chapter 3 describes the relationships between soil mesofauna, ECM fungi, and sclerotia in forest soils by comparing fungal species extracted from soil mesofauna and sclerotia and the ECM fungal community profiles in soil. Fungivory of Acari and/or Collembola of sclerotia-forming ECM fungi is suggested as a key biological process regulating sclerotia formation in forest soils. Chapter 4 describes recent research on the diversity of microbial communities in sclerotia grains and evaluates the potential for sclerotia grains to act as bacterial carriers. Chapter 5 details the physical and chemical characteristics of sclerotium grain collected from a buried A horizon from a Japanese Andosol, using various instrumental techniques, i.e., C-NMR, FT-IR, Al-NMR, XRD, ICP-OES, ICP-MS. Chapter 6 provides an overview of the origins of soil polysaccharide, and the monosaccharide composition of various soils under different land use types. ECM fungal sclerotia are discussed as sources of polysaccharides in forest soils. Chapter 7 reports the results of accelerator mass spectrometry 14C dating of sclerotia grains, humic acid, and humin fractions from three different Andosol profiles in Japan. 14C dating allows us to determine the age of formation of individual grains. These dates are more likely to indicate the beginning of soil formation than the 14C age of the humic acids. Chapter 8 reports the micromorphological features of the interiors of sclerotia grains examined by scanning electron microscopy and transmission electron microscopy combined with energy-dispersive X-ray and microcomputed tomography analyses. Chapters 9–11 compile comparative studies of geographical distribution and properties of sclerotia grains in forest soils from Japan, Germany, and Mongolia. A clear contrast in metal enrichment was shown between sclerotia grains in low-pH forest soils and high-pH forest soils. Melanized sclerotia grains formed by ECM fungi may be widely distributed in forest soil around the globe. Chapter 12 provides concluding remarks: Investigating melanic sclerotia grains that bridge microbial and pedogenic processes may clarify the biological intent of sclerotia formation and autonomous homeostasis of soil ecosystem. Sclerotia grains, a mesoscale component in soil, may hold the key to understanding novel aspects of soil ecological systems.

Preface

ix

Space limitations prevent us from providing a complete list of the areas encompassed by our study of sclerotia grains. We wish to express our special gratitude to the late Prof. Manfred Fruehauf, Martin Luther University of HalleWittenberg; Associate Professor Urlike Hardenbicker, University of Regina, Canada; Ms. Angelika Pott, Hamburg, Germany; and Dr. Reiko Liermann, Leipzig University, for their support during fieldwork in Germany. Fieldwork in Mongolia was conducted with the support of Prof. Bayanjargal Ochirkyuyag, President of the National University of Mongolia (NUM) and a strong partnership between NUM and Tokyo Metropolitan University. We thank all graduate students at the Tokyo Institute of Technology, Ibaraki University, Kobe University, Meijo University, and Tokyo Metropolitan University, who helped with field collection of sclerotia and with lab work. M. Watanabe expresses special appreciation to Prof. Nobuhide Fujitake of Kobe University and Prof. Hiroyuki Ohta, President of Ibaraki University for their contributions to sclerotia projects. The authors thank Mr. K. Sugiura, Mr. S. Yoshimi, and Mr. M. Kato, Shimadzu Analytical & Measuring Center, Inc., Kanagawa, Japan; Dr. Shinji Sugiyama, Paleoenvironment Research, Miyazaki, Japan; Associate Prof. Yudzuru Inoue, Nagasaki Institute of Applied Science; Prof. Syuntaro Hiradate, Kyushu University; Mr. R. Ohki, Senior Technician, and Mr. J. Koki of the Technical Dept., Tokyo Institute of Technology; Mr. S. Suzuki, Mr. T. Hattori, Mr. T. Hatano, and Mr. H. Terashima of JEOL, Tokyo, Japan, for technical support in sclerotia grain observation and analyses; and Prof. Hiroyuki Matsuzaki of Micro Analysis Laboratory, Tandem accelerator, University of Tokyo, and Mr. Ryosuke Hayase of the Institute of Accelerator Analysis, Fukushima, Japan, for their support in 14C dating of sclerotia grains. M. Watanabe is grateful to Emeritus Prof. Kazuhiko Egashira of Kyushu University, Emeritus Prof. Kazue Tazaki of Kanazawa University, Prof. Katsutoshi Sakurai, President of Kochi University, Associate Prof. Akiyoshi Yamada of Shinshu University, Emeritus Prof. Masami Nanzyo of Tohoku University, Emeritus Prof. Masaki Furuya of the University of Tokyo, Dr. Nobuyasu Ito of the National Institute of Advanced Industrial Science and Technology, Japan, Dr. Tyrone L. Daulton of Washington University in St. Louis, and Prof. Tsookhuu Khinayat of the National University of Mongolia for their valuable advice on our sclerotia grain research. M. Watanabe sincerely thanks Emeritus Prof. Atsuyuki Okabe, University of Tokyo, who named our research “pedosclerotiology” and encouraged the writing of this book. M. Watanabe would like to thank Mr. Yosuke Nishida and Ms. Taeko Sato of Springer Japan, Mr. Prasad Gurunadham of Springer Nature, and Ms. Parttibane ArulVani of SPi Global for their kind support and technical assistance to realize the publishing of this contributed volume. Tokyo, Japan July 2020

Makiko Watanabe

Contents

1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Makiko Watanabe, Keisuke Obase, and Kazuhiko Narisawa

1

2

Fungal Communities of Sclerotia Grains from Forest Soils . . . . . . Kazuhiko Narisawa, Anzilni Amasya, Yaya Sasaki Nonoyama, and Keisuke Obase

17

3

Relationships Between Soil Mesofauna, Ectomycorrhizal Fungi, and Sclerotia in Forest Soils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anzilni Amasya and Kazuhiko Narisawa

4

Sclerotia Grains as Bacterial Carriers in Soil . . . . . . . . . . . . . . . . . Yaya Sasaki Nonoyama and Kazuhiko Narisawa

5

Chemical Characterization of Sclerotia Grains Collected from a Volcanic Ash Soil Profile in Japan . . . . . . . . . . . . . . . . . . . Bolormaa Oyuntsetseg, Nobuo Sakagami, Khulan Nyamsanjaa, and Makiko Watanabe

6

Origin of Soil Polysaccharides, and Ectomycorrhizal Fungal Sclerotia as Sources of Forest Soil Polysaccharides . . . . . . . . . . . . Shigetoshi Murayama and Yuki Sugiura

35 63

77

91

7

Dating of Sclerotia Grains in Andosol Profiles . . . . . . . . . . . . . . . . Makiko Watanabe, Nobuo Sakagami, and Kiminori Tonosaki

119

8

Micromorphological Features of Sclerotia Grains . . . . . . . . . . . . . Makiko Watanabe and Akira Genseki

139

9

Spatial Distribution of Sclerotia Grains in Forest Soils, Northern and Central Japan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nobuo Sakagami and Sayuri Kato

153

xi

xii

10

11

12

Contents

Spatial Distribution of Sclerotia Grains in Low-pH Forest Soils, Central Germany . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Makiko Watanabe and Nobuo Sakagami

173

Melanized Sclerotia Grains from Mongolian Steppe Forest Soils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Khulan Nyamsanjaa, Bolormaa Oyuntsetseg, and Makiko Watanabe

193

Sclerotia Grain, a Mesoscale Component of Soil . . . . . . . . . . . . . . Makiko Watanabe

209

Contributors

Anzilini Amasya, Dr Sc Mizuho Consulting Indonesia Co. Ltd., Jakarta, Indonesia Graduate School of Urban Environmental Sciences, Tokyo Metropolitan University, Tokyo, Japan Akira Genseki Materials Analysis Division, Open Facility Center, Tokyo Institute of Technology, Tokyo, Japan Sayuri Kato, MSc TMU Curator’s Course, University Education Center, Tokyo Metropolitan University, Tokyo, Japan Shigetoshi Murayama, Dr Agri Department of Environmental Bioscience, Faculty of Agriculture, Meijo University, Nagoya, Japan Present Address: Tsukuba, Ibaraki, Japan Kazuhiko Narisawa, Dr Agri Department of Biological Production Science, College of Agriculture, Ibaraki University, Amimachi, Ibaraki, Japan Yaya Sasaki Nonoyama, Dr Eng Graduate School of Urban Environmental Sciences, Tokyo Metropolitan University, Tokyo, Japan Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, Tokyo, Japan Khulan Nyamsanjaa, MSc Graduate School of Urban Environmental Sciences, Tokyo Metropolitan University, Tokyo, Japan Department of Chemistry, School of Art and Sciences, National University of Mongolia, Ulaanbaatar, Mongolia Keisuke Obase, Dr Agri Department of Mushroom Science and Forest Microbiology, Forestry and Forest Products Research Institute, Tsukuba, Japan Bolormaa Oyuntsetseg, PhD Department of Chemistry, School of Art and Sciences, National University of Mongolia, Ulannbaatar, Mongolia

xiii

xiv

Contributors

Nobuo Sakagami, Dr Sc Department of Biological Production Science, College of Agriculture, Ibaraki University, Amimachi, Ibaraki, Japan Yuki Sugiura, M Agr Department of Environmental Bioscience, Meijo University, Nagoya, Japan Present Address: Yourou-Cyo, Gifu, Japan Kiminori Tonosaki, MSc JEOL Ltd, Akishima, Tokyo, Japan Makiko Watanabe, PhD Department of Geography, Graduate School of Urban Environmental Sciences, Tokyo Metropolitan University, Hachioji, Tokyo, Japan

Chapter 1

Introduction Makiko Watanabe, Keisuke Obase, and Kazuhiko Narisawa

Abstract Sclerotia are fungal resting structures, or persistent propagules, closely related to research interests of botanist, mycologist, cytologist, histochemist, and phytopathologist. The degradability or durability of sclerotia is probably dependent on the ecological conditions, and the inactivated sclerotia propagules may become a constitution as “sclerotia grains” in soil while keeping the unadulterated structure. The black spheric resting bodies of Cenococcum spp. detected from forest soils remain as one of the soil constituents while functioning as a substrate for fungi and bacteria even though the ability to germinate has been lost. The wide habitat ranges of Cenococcum spp. on earth suggest the adaptability of the Cenococcum spp. sclerotia against severe environment. Because of their recalcitrant structure existing in soil, sclerotium is not only a material of biological resource studies but is a key material of integrated science that unravels the global history and earth system before the relationship between human and soil begins. Keywords Sclerotia · Cenococcum geophilum · Earth Sciences

M. Watanabe (*) Department of Geography, Graduate School of Urban Environmental Sciences, Tokyo Metropolitan University, Hachioji, Tokyo, Japan e-mail: [email protected] K. Obase Department of Mushroom Science and Forest Microbiology, Forestry and Forest Products Research Institute, Tsukuba, Japan e-mail: [email protected] K. Narisawa Department of Biological Production Science, College of Agriculture, Ibaraki University, Amimachi, Ibaraki, Japan e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2021 M. Watanabe (ed.), Sclerotia Grains in Soils, Progress in Soil Science, https://doi.org/10.1007/978-981-33-4252-1_1

1

2

M. Watanabe et al.

Fig. 1.1 The dead branch of Cryptomeria japonica (left) and their needle leaves infected with black oval-shaped sclerotia of Scolicosporium sp. (right)

1.1

Sclerotia: Fungal Propagule or Soil Grain?

Sclerotia are fungal resting structures, or persistent propagules which can germinate to produce mycelia, asexual spores of sporocarps such as apothecia in which sexual spores are borne (Coley-Smith and Cooke 1971). Mycelium of fungal species such as Deuteromycetes, Ascomycetes, and Basidiomycetes are known/believed to form sclerotia. Their structure enables them to survive periods of adverse conditions, which are too severe for the ordinary vegetative mycelium, such as undernutrition, low temperature, and desiccation. The growth of sclerotia proceeds to sexual reproduction at increased nutritional levels (Ferdinandsen and Winge 1925; Cochrane 1958; Scheidegger and Brunner 1995). Sclerotization is evaluated as an important method to preserve culture collections in long term with advantages of low cost and low maintenance (Nakasone et al. 2004). Sclerotia-forming fungi are ecologically diverse and many unrelated fungi with diverse trophic modes may form sclerotia (Smith et al. 2015). Sclerotia have attracted research interests of botanist, mycologist, cytologist, histochemist, and phytopathologist for a long time. Figure 1.1 shows the needle leaves Cryptomeria japonica infected with black oval-shaped sclerotia of Scolicosporium sp. Sclerotium-forming fungi are excellent targets for the discovery of antibacterial, antifungal, and anti-herbivore compounds (Smith et al. 2015). According to Townsend and Willetts (1954), many diseases of crops are caused by those fungi which normally produce sclerotia at some time in their life history. These sclerotia were recognized with great biological and phytopathological importance since they serve themselves as vegetative reproductive bodies, or reproductive structures developed from themselves. Many researches under such interests had been reported on various species: Rhizoctonia solani (Naiki and Ui 1969, 1975, 1977), Sclerotinia sclerotiorum (Maxwell et al. 1970; Saito 1977), Sclerotinia minor (Bullock et al. 1980a, b, 1983), Sclerotinia fructigena (Calonge 1968), Sclerotium rolfsii (Chet et al. 1967, 1969), Pisolithus tinctorius (Grenville et al. 1985a), Hebeloma

1 Introduction

3

sacchariolens (Fox 1986). Matsumoto and Tajimi (1988, 1990) reported life-history strategy determined by sclerotium production and continuous variation associated with habitat differences within isolates of T. incarnata and T. ishikariensis. Naiki and Ui (1977) studied the population of sclerotia of R. solani in soil, which causes “crown rot” disease of sugar beet, and reported that most of the sclerotia distributed in rhizosphere and surface soil, and the highest number of sclerotia per soil was observed in the closest to the roots of infected plants. As mentioned above, survival of sclerotia in soil has been mostly reported for plant pathogenic fungi to understand the survival structure against various conditions in soil. Naiki and Ui (1969) examined the life of sclerotia in soil using 2 types of isolates of R. solani. In terms of the observations of the germinate ratio and inner cell structure, it was concluded that sclerotia of R. solani could survive at least 1 year in soil. Experiment on the changes in the number of the propagules of Macrophomina phaseoli (Maubl.) Ashby by soil plate method demonstrated the half-life of the sclerotia in soil as 0.65–3.59 years, 1.88 years in average (Watanabe 1973). The decay of sclerotia of Sclerotium cepivorum under field conditions in New Zealand soils was observed at a significant proportion after just 2 months in soil, supposedly caused by adverse environment conditions and subsequent attack by microbes, after which numbers declined in soil slowly over a period of 2 years (Harper et al. 2002). Sclerotia of Sclerotium rolfsii are known to have involuted structure such as rind, cortex, medulla, and an intermediate layer (Chet et al. 1969). On the contrary, simple sclerotia formed by Rhizoctonia solani have uniformed structure constructed with almost ordinary cells of mycelium. Sclerotia are also known to contain “reserve substances” for germination, such as lipids in Cenococcum geophilum (Massicotte et al. 1992), Pisolithus tinctorius (Grenville et al. 1985a), Paxillus involutus (Grenville et al. 1985b; Moore et al. 1991), protein in P. involutus (Grenville et al. 1985b; Moore et al. 1991), and carbohydrates in both P. tinctorius (Grenville et al. 1985a) and P. involutus (Grenville et al. 1985b; Fox 1986; Moore et al. 1991). Fox (1986) studied the sclerotium-like bodies of Hebeloma sacchariolens and suggested that they played a major role in short-term nutrient storage. Moore et al. (1991) reported the chemical composition of reserve substance and cytoplasm of the cells of P. involutus. Protein bodies (electron opaque granules) were characterized by phosphorus (P) and accompanying cations (Mg, S, Ca, Ni), and cytoplasm was characterized by lower levels of P, Ca, and Mg. The products of sclerotia of Paxillus involutus was also analyzed in culture and the presence of phosphates associated with protein, lipid, and glycogen granules was detected inside the sclerotia (Moore et al. 1991). Chet et al. (1967) examined the possible biochemical role of the components in Sclerotium rolfsii Sacc. Fox (1986), Moore et al. (1991), and Massicotte et al. (1992) studied the morphology and ultrastructure of sclerotia in a culture of ectomycorrhizal fungi and some ectomycorrhizal fungi (e.g., Paxillus involutus, Cenococcum geophilum) are known to form sclerotia (Trappe 1964, 1969; Grenville et al. 1985b; Fox 1986; Moore et al. 1991; Massicotte et al. 1992). The development of sclerotia, which has been caught eyes of researchers since the late 19c, was confirmed to have three main types, Loose type, Terminal Type, and

4

M. Watanabe et al.

Lateral/Strand type (Townsend and Willetts 1954; Willetts 1972). Early stage in development of sclerotial initiation, ordinary growth of mycelium (branching, fusion of branches, coalescence, and septation) occurs intensively (Willetts 1972). Their morphological characteristics are identified by their specific rind and cells. Some sclerotia have characteristic spherical black bodies protected by thick and melanic pigmented walls, and are considered to be resistant against microbial attack during the period of inactivity as well as to desiccation, generally 1 month to several years (Cochrane 1958; Gray and Williams 1971; Fox 1986). The degradability or durability of sclerotia propagule is probably dependent to the ecological conditions, which is not well known yet. The inactivated sclerotia propagules, so called “dead sclerotia”, may become a constitution as “sclerotia grains” in soil with preserving the unadulterated structure.

1.2

Melanized Sclerotia Grains Found in Forest Soils

Cenococcum geophilum Fr. (syn. C. graniforme Ferd. and Wing.) is one of the most frequently encountered ectomycorrhizal (or ectoendomycorrhizal) fungi in nature (Trappe 1962, 1964), which colonize plant roots of specific taxonomical groups (such as Pinaceae, Fagaceae, and Betulaceae) and form symbiotic associations with their hosts (LoBuglio 1999). As C. geophilum was originally described from its black sclerotia by J. Sowerby in 1800 (under the name Lycoperdon graniforme), the fungus exists not only as sterile dematiaceous mycelia but also melanized sclerotia in forest soils (Trappe 1964; LoBuglio 1999). Even though sexual or asexual spores, which are important taxonomic criteria in fungal classification, have never been convincingly recorded for C. geophilum (but see Fernandez-Toiran and Agueda 2007), recent phylogenetic studies revealed that C. geophilum is monophyletic sister group of Glonium in Dothideomycetes of Ascomycota (Spatafora et al. 2012) and includes several genetically closely-related cryptic species (e.g., Obase et al. 2016, 2017). Cenococcum geophilum forms ectotrophic and ectendotrophic associations with unusually large numbers of tree, shrub, and herbaceous genera and has a worldwide distribution including sub-tropical, temperate to arctic-alpine climatic zones (Trappe 1964; Obase et al. 2017). C. geophilum has even been observed above the Arctic Circle in Alaska and Canadian High Arctic (75
330 N, 84
400 W) and as an important symbiont of trees at timber line in the Washington and Oregon Cascade mountain range (Trappe 1964, 1988; Bledsoe et al. 1989). C. geophilum is also often dominantly found in habitats exposed to high drought stress, including coastal pine forests (e.g., Matsuda et al. 2009; Obase et al. 2011), seasonally dry woodlands (Smith et al. 2007), and volcanic deserts (Wu et al. 2005). Moreover, C. geophilum is found in serpentine soils that have high levels of phytotoxic stress (Panaccione et al. 2001). Sclerotia of C. geophilum, together with sclerotia of a species of Morchella, were more numerous in burned area than a nearby unburned forest for 2 years following fire (Miller et al. 1994). C. geophilum was able to grow in acidic soils and survive

1 Introduction

5

under exposure of simulated rain at pH 2.5 (Meier et al. 1989). These wide habitat ranges of C. geophilum suggest the adaptability of the fungus against severe environment. Shaw and Sidle (1982) reported that the ability of live sclerotia to survive for several years can provide sufficient inoculum to colonize host species effectively. Indeed, C. geophilum sclerotia can be one of the most common resistant propagules of ectomycorrhizal fungi in forest soils; they can survive drought treatments in forest soils and germinate and form ectomycorrhizal roots on bioassay seedlings (Glassman et al. 2015). Furthermore, Obase et al. (2018) demonstrated that genotypic diversity of C. geophilum is significantly higher in sclerotia than ectomycorrhizal roots in the same soil samples. The results of these experiments indicate excel abilities of C. geophilum sclerotia for maintaining genetic diversity of the fungus and functions in forest ecosystems. The features of sclerotia of C. geophilum has been precisely noted by Trappe (1969) and Massicotte et al. (1992), which tend to be particularly abundant near Cenococcum mycorrhiza, as 0.05 ~ 4 mm or more in diameter, and viable, mature ones are jet-black, hard, smooth, mostly spherical, and often with emergent hyphae. Trappe (1969) also noted “live sclerotia” are denser than water and have a high content of ethanol-soluble oil (supposedly reserve substances), while “dead sclerotia”—which can persist indefinitely in soil—look much like live ones but float in water and lack the ethanol-soluble oil. Figure 1.2 shows mycelia hyphae, mycorrhiza, sclerotia of Cg associated with thin root of various arborescent species. Sclerotium grains can be easily found in natural forest soil, due to their shining black spherical structure (Fig. 1.3). Attentions to sclerotium grains are able to be paid

Fig. 1.2 Top: Mycelial hyphae of C. geophilum isolate (Cg AT 353) from Pinus mycorrhiza. Bottom Left: Mycorrhiza, hyphae, and sclerotia of C. geophilum associated with the seedling roots of Quercus crispula Blume. Bottom Right: Thin roots of Salix reinii (Mineyanagi) attached by jet-black hyphae and sclerotium of Cg. Sclerotium grain is denoted by an arrow

6

M. Watanabe et al.

Fig. 1.3 Cg sclerotia grains found from forest soils. Arrows point to sclerotia grains

Fig. 1.4 Features of the section of Cg sclerotia grain observed by a digital microscope (Keyence VHS 6000) and the 3D imagery of the transverse wall structure (right)

on the displayed monolith specimens of forest soils in museums, characterized by the black spherical grain; and on specimens of thin sections, characterized by their hollowed round structure. Figure 1.4 shows the digital microscopic imagery of the surface of Cg sclerotia grain. The morphological features of C. geophilum sclerotia sliced into half pieces are shown in Figs. 1.5 and 1.6. They have honeycomb structure in their transverse wall constructed by the empty cells contacting each other. Histopathological coiled structure cannot be recognized, but a hollow

1 Introduction

7

Fig. 1.5 Features of Cg sclerotia grain with a hollow internal structure and honeycomb transverse wall observed by an optical microscope and scanning electron microscope (right two)

Fig. 1.6 SEM observation of some Cg sclerotia grains sliced into half

structure can be frequently observed inside sclerotium. Figure 1.6 illustrates variety of internal structure of sclerotia grain. The biochemical properties of C. geophilum sclerotia that allow them to remain in the soil and retain their structure are unclear. The possible contributions of various components, including a melanin-like pigment, have been discussed to explain the resistance of Sclerotium rolfsii Sacc. sclerotia against biological and chemical degradation (Chet et al. 1967); and melanin deposited in cell walls has also been implicated in the degradation resistance of C. geophilum (Malik and Haider 1982). In addition, a particular fungal melanin, dihydroxynaphthalene (DHN) melanin, known as polyketide melanin, is believed to be involved in the high resistance of

8

M. Watanabe et al.

various fungi, including C. geophilum, to environmental stresses such as water stress (Butler and Day 1998; Fernandez and Koide 2013). Melanin deposited in fungal cell walls forms complexes with other cell wall components such as chitin, β-glucans, and proteins (Butler and Day 1998). Most of the organic constituents of fungal cell walls and sclerotia are believed to be labile compounds (Dallies et al. 1998). For example, neutral saccharides account for 6.0–16% of the C content of C. geophilum sclerotia collected from soil on Mount Ontake, central Japan (Sugiura et al. 2017). Melanic tissues that directly colonize C. geophilum root tips, extramatrical mycelium, and sclerotia are likely to be large and stable pools of carbon in forest soils (Dahlberg et al. 1997). Sclerotia of Cenococcum geophilum can significantly contribute to the fungal biomass of forest situations, and thus represent an important source of assimilated carbon from host species (LoBuglio 1999). Vogt et al. (1981, 1982) estimated the biomass of sclerotia in a 23- and 180-year-old Abies amabilis stand to be 2300 kg ha1 year1 and 3000 kg ha1 year1, respectively. It is also known that mycorrhizal root tips and sclerotia have their maximum production in autumn (e.g., Vogt et al. 1981, 1982; Lussenhop and Fogerl 1999). The existence of large sclerotium grains (>5 mm) was noted by Kumada (1987) who collected the floating grains in snow-melt streams of Asadaira (1500 m) Mt. Myoko, central Japan. Although Cenococcum sclerotia can act as a substrate for diverse fungi (Obase et al. 2014), they persist in forest soils for a long time. The facts give us a key question “what are the Cenococcum sclerotia grains in soil?”

1.3

Sclerotia Grains in Earth Sciences Studies

Sclerotia-like grains may be able to be found in cultivated soils. According to USDA/NRCS Soil Survey Manual (Soil Science Division Staff 2017), such grains would be defined as concentrations; identifiable bodies within the soil that form and accumulate due to pedogenesis. Pedogenic processes responsible for concentration development in the soil include chemical dissolution and precipitation, oxidation/ reduction, and accrual due to physical or biological processes. Biological concentrations are discreet bodies accumulated by biological process, for example fecal pellets and worm casts (Soil Science Division Staff 2017). Sclerotia are rather studied in earth sciences; paleopedology, geochemistry, sedimentology, and geology, with a different viewpoint from soil microbiology and soil ecology. Sclerotia of Cenococcum geophilum, mainly, can be a resistant marker of environmental archives, as both facies fossils and index fossils. Retallack (1990) introduced fungal sclerotium, and/or sclerotinite, together with spherosiderite, pyrite framboid, botryococcoid algal colony, which could be grouped as some common constituents of coal and waterlogged paleosols. He also described that some resting stages of fungi, sclerotia, are distinctive having small woody balls with vermiform hollows common in peats and coals. The physical persistence of vesicular-arbuscular mycorrhizal spores (grains of fungal products like sclerotia)

1 Introduction

9

was demonstrated in research on paleosols. Holmqvist and Schlyter (2000) examined the chronosequence of closely superimposed paleosols from an arctic/alpine meadow soil in northern Sweden with radiocarbon (14C) dating of asexual spores of vesicular-arbuscular mycorrhizae, and determined the long-term (i.e., over a millennium time span) loss rate of organic carbon. Hormes et al. (2004) used 14C dating to estimate that C. geophilum “spores” found from buried soils in glacial sediments in northern Sweden were 5000–6000 years. BP, and this information was used to clarify the geochronology of glacier fluctuations in the region. (Note, however, that the “spores” were actually sclerotia; C. geophilum does not produce spores [LoBuglio 1999]). Kobayashi et al. (2015) studied the Late Holocene peaty sediment in Rishiri Island, Hokkaido, Japan and found the distribution of Cg sclerotia grains from the bottom to top of the sediment having the highest density in the sediment of ca. 3 ka. They suggested that the fluctuation of density of Cg grains indicates the past environmental change of dry-wet condition, i.e., the favorable condition for the possible host plant, Pinus pumila. A case of C.geophilum sclerotia contribution in recent earth science appears in the discussion of the trigger of Younger Dryas event. The Younger Dryas is named for the climate change that abruptly occurred in the Northern Hemisphere about 14,500 years ago until 11,500 years ago, bringing back near-glacial conditions in the period when Earth’s climate began to shift from a cold glacial world to a warmer interglacial state. Other proxy records, including varved lake sediments in Europe, also display these abrupt shifts. The Younger Dryas is clearly observable in paleoclimate records from many parts of the world (https://www.ncdc.noaa.gov/ abrupt-climate-change/The%20Younger%20Dryas). Regarding the cause of the event, one theory suggests: During the transition from the last glacial period into the present interglacial, the North American ice sheet (Laurentide Ice Sheet) was rapidly melting and adding freshwater to the ocean. Geochemical evidence from ocean sediment cores supports this idea. A more northerly routing of meltwater has a greater impact on the salinity and density of the surface ocean in the North Atlantic, which can cause a slowing of the ocean’s thermohaline circulation and climate changes around the world. As the meltwater flux abated, became less intensive, the thermohaline circulation strengthened again and climate recovered (https://www. ncdc.noaa.gov/abrupt-climate-change/The%20Younger%20Dryas). While, another theory suggests: A cometary or meteoritic body or bodies hit and/or exploded over North America 12,900 years ago, causing the Younger Dryas climate episode. Cosmic impact objects such as magnetic spherule, carbon spherule, nanodiamond particle, soot material, and some more were found from the YD stratigraphy sediments and ice cores (Firestone et al. 2007; Kinzie et al. 2014; Wolbach et al. 2018a, b). Counter evidences against extraterrestrial theory were provided to prove that the YD event was triggered by terrestrial impact and not by cosmic impact, in terms of precise examination of the C. geophilum sclerotia grains collected from the YD stratigraphy (Scott et al. 2010; Pinter et al. 2011). Daulton et al. (2016) determined the so-called “nanodiamond” as graphene in Cg sclerotia grains. The grains of C.geophilum sclerotia, archiving natural history have become to play a key role in the discussions.

10

M. Watanabe et al.

It can be said that sclerotia grains are hardly recognized as soil constituents by soil scientists, except Kumada who was the pioneer scholar introducing Cg sclerotia as an origin of Pg, the green soil pigment. Comparative soil humus chemistry highlighted C. geophilum sclerotia. Kumada and Hurst (1967) examined British, Japanese and Swedish Podzols and showed that the presence of sclerotia (mean diameter 0.50.4 mm in British Podzols; 1.10.4 mm in Japanese and Swedish Podzols) in horizons A and B. Their interest to sclerotia came up with their suggestion that the source of the green fraction of P type soil humic acid, so-called “Pg,” derives from fungal metabolites and the skeletal formula of Pg was assumed as DHPQ, dihydroxyperylenequinone 4,9-Dihydroxyperylene-3,10-dione. Various quinone compounds are found in soil and sediment and known to be metabolites of higher plants, soil fungi and lichens. Kumada and Hurst (1967) reported that P type humic acid commonly present in podzolic soils and alpine grassland soils had characteristic absorption band resulting from the presence of green pigment. Furthermore, it was suggested that the source of Pg was small spherical black fungal sclerotia and this fungal species was tentatively identified as Cenococcum graniforme (currently Cenococcum geophilum from their morphological characteristics). Approximately 50 years after the Kumada’s assumption, C. geophilum sclerotia have become geochemist’s interest, to discuss the origin of Perylene. Perylene (Perilene), a polycyclic aromatic hydrocarbon (PAH) displays blue fluorescence under a UV light. In 1967, almost half a century ago from now, perylene was found in sediments along the California coast. Later, many researchers found perylene in the sediments of the ocean and lake around the world, but no one was able to explain where this perylene came from. Occasionally, a small blackish grain was observed in the sediment collected from the bottom of the Lake Biwa, Central Japan (Itoh et al. 2010). In this study, fresh sclerotia grains in forest soils of the catchment area of Lake Biwa were collected to compare with the properties of the broken particles in sediment samples. By the honeycomb cell structure and the existence of septal holes in each cell, the black small grains from the Biwa lake sediment were identified as C. geophilum sclerotia. In the next step, an experiment was designed to obtain the fluorescent spectra of 3 reagents, perylene, 3,10-perylene quinone, and dihydroxyperylenequinone with the filter unit, and the observed and calculated spectra of each particle in different diagenetic stages were compared. The good correspondence between the observed and calculated spectra explained the transformation process of the particles. So, it has become clear that Cg sclerotia are the possible origin of perylene. The sclerotia grains produced in the forest soil of the catchment area flowed into Lake Biwa due to runoff, and then over a long period of time after remaining on the lake bottom, maybe not long, the chemical structure changed from DHPQ to perylene. At the present time, Lake Biwa is the only case that confirmed the relationship between sclerotia in forest soil and perylene in sediments. However, as with perylene and sclerotia exist all around the world, there is the possibility that all the perylene in the world is derived from DHPQ contained in sclerotia, the resting body of mycorrhizal fungi.

1 Introduction

11

Fig. 1.7 Sclerotia grains collected from buried A horizon of Pachic Andisols, Tochigi Prefecture, Japan (Nanjo and Kanno 2018). https://link.springer.com/chapter/10.1007/978-981-13-1214-4_4

Nanjo and Kanno (2018) confirmed sclerotia grains of a 0.1 mm diameter size and demonstrated as an aluminum dominant inorganic constituents in Andisol: Pachic melanudand profile, in Kiwadashima, Tochigi, Japan, by SEM-EDX analyses (Fig. 1.7). Their existence in the A3 horizon at depth of 30–80 cm, which is 10 cm above the 30 cm thickness layer of the Shichihonzakura Pumice derived from the eruption of 12,700 years B.P. Nantai volcano (Machida and Arai 2003), suggests the early stage of soil formation had been developed under past soil ecology with relation of Cenococcum geophilum.

1.4

Conclusion

Earth science evidence prove the occurrence of the abrupt Younger Dryas climate event in the northern hemisphere ca.12,900 years ago, when the last glacial period begins to dawn. Although there are various theories for the cause, there is no doubt that a large number of fungal sclerotia grains have been detected in sedimentary layers of the northern hemisphere corresponding to this period. It is also a clear fact that sclerotia grains transport down river systems and accumulate into lake sediments. The black spheric resting bodies of Cenococcum spp. detected from forest

12

M. Watanabe et al.

soils remain as one of the soil constituents while functioning as a substrate for fungi and bacteria even though the ability to germinate has been lost. Cenococcum spp. form visually detectable dormant particle in the life cycle as its reproductive structure. The lack of reproductive structures such as sexual and asexual spores in life cycle gives difficulty for mycologists to subcategorize Cg genus and to use molecular biology methods for identifying the fungal species responsible for the sclerotia formation. Sclerotia have become mycologists’ interests on the basis of social concern of preventing crop disease, which could have probably started since settlers opened forest to gain farmland and start cultivation. The recalcitrant structure existing in soil, formed by the worldwide ectomycorrhizal fungi Cenococcum spp., is not only a material of biological resource studies, but is a key material of integrated science that unravels the global history and earth system before the relationship between human and soil begins.

References Bledsoe C, Klein P, Bliss LC (1989) A survey of mycorrhizal plants on truelove lowland, Devon Island, N.W.T., Canada. Can J Bot 68:1848–1818 Bullock S, Ashford AE, Willetts HJ (1980a) The structure and histochemistry of sclerotia of Sclerotinia minor Jagger II. Histochemistry of extracellular substances and cytoplasmic reserves. Protoplasma 104:333–351 Bullock S, Willetts HJ, Ashford AE (1980b) The structure and histochemistry of sclerotia of Sclerotinia minor Jagger I. Light and electron microscope studies on sclerotial development. Protoplasma 104:315–331 Bullock S, Willetts HJ, Ashford AE (1983) The structure and histochemistry of sclerotia of Sclerotinia minor Jagger III. Changes in ultrastructure and loss of reserve materials during carpogenic germination. Protoplasma 117:214–225 Butler MJ, Day AW (1998) Fungal melanins: a review. Can J Microbiol 44:1115–1136 Calonge FD (1968) Origin and development of intrahyphal hyphae in Sclerotinia fructigena. Mycologia 60:932–942 Chet I, Henis Y, Mitchell R (1967) Chemical composition of hyphal and sclerotial walls of Sclerotium rolfsii Sacc. Can J Microbiol 13:137–141 Chet I, Henis Y, Kislev N (1969) Ultrastructure of sclerotia and hyphae of Sclerotium rolfsii Sacc. J Gen Microbiol 57:143–147 Cochrane VW (1958) Physiology of fungi. Wiley, New York and London, 524 pp Coley-Smith JR, Cooke RC (1971) Survival and germination of fungal sclerotia. Annu Rev Phytopathol 9:65–92 Dahlberg A, Jonsson L, Nylund JE (1997) Species diversity and distribution of biomass above and below ground among ectomycorrhizal fungi in an old-growth Norway spruce forest in South Sweden. Can J Bot 75:1323–1335 Dallies N, Francois J, Paquet V (1998) A new method for quantitative determination of polysaccharides in yeast cell wall. Application to the cell wall defective mutants of Saccharomyces cerevisiae. Yeast 14:1297–1306 Daulton TL, Amari S, Scott AC, Hardiman M, Pinter N, Anderson RS (2016) Comprehensive analysis of nanodiamond evidence relating to the younger Dryas impact hypothesis. J Quater Sci 32:7–34

1 Introduction

13

Ferdinandsen C, Winge O (1925) Cenococcum Fr. A monographic study. K VetLandbohojsk Arskr 1925:332–382 Fernandez CW, Koide RT (2013) The function of melanin in the ectomycorrhizal fungus Cenococcum geophilum under water stress. Fungal Ecol 6:479–486 Fernandez-Toiran L, Agueda B (2007) Fruitbodies of Cenococcum geophilum. Mycotaxon 100:109–114 Firestone EB et al (2007) Evidence for an extraterrestrial impact 12,900 years ago that contributed to the megafaunal extinctions and the younger Dryas cooling. Proc Natl Acad Sci 104:16016–16021 Fox FM (1986) Ultrastructure and infectivity of sclerotium-like bodies of the ectomycorrhizal fungus Hebeloma sacchariolens, on birch (Betula SPP.). Trans Br Mycol Soc 87:359–369 Glassman SI, Peay KG, Talbot JM, Smith DP, Chung JA, Taylor JW, Vilgalys R, Bruns TD (2015) A continental view of pine-associated ectomycorrhizal fungal spore banks: a quiescent functional guild with a strong biogeographic pattern. New Phytol 205:1619–1631 Gray TRG, Williams ST (1971) Soil micro-organisms. Oliver and Boyd, Edinburgh, 240 p Grenville DJ, Peterson RL, Piché Y (1985a) The development, structure, and histochemistry of sclerotia of ectomycorrhizal fungi. I Pisolithus tinctorius. Can J Bot 63:1402–1411 Grenville DJ, Peterson RL, Piché Y (1985b) The development, structure, and histochemistry of sclerotia of ectomycorrhizal fungi. II Paxillus involutus. Can J Bot 63:1412–1417 Harper GE, Frampton CM, Stewart A (2002) Factors influencing survival of sclerotia of Sclerotium cepivorum in New Zealand soils. N Z J Crop Hortic Sci 30:29–35 Holmqvist J, Schlyter P (2000) Vesicular-arbuscular mycorrizae for dating multiple paleosols and carbon loss rate determination. Geoderma 97:125–133 Hormes A, Karlen W, Possnert G (2004) Radiocarbon dating of palaeosol components in moraines in Lapland, northern Sweden. Quater Sci Rev 23:2031–2043 Itoh N, Sakagami N, Torimura M, Watanabe M (2010) Perylene in Lake Biwa sediment originating from Cenococcum geophilum in its catchment area. Geochim Cosmochim Acta 85:241–251 Kinzie CR et al (2014) Nanodiamond-rich layer across three continents consistent with major cosmic impact at 12,800 cal BP. J Geol 122:475–506 Kobayashi H, Momohara A, Ohmori A, Kondo R, Sato M (2015) Fossil sclerotium (Cenococcum geophilum) from the late Holocene peaty sediment and its relationship with vegetation in and around Giboshi pond, Rishiri Island, Hokkaido. Risiri Kenkyu 34:85–90. (In Japanese with English abstract) Kumada K (1987) Recollection—the forty years for humus research. Fertil Sci 10:1–57. (in Japanese) Kumada K, Hurst HM (1967) Green humic acid and its possible origin as fungal metabolite. Nature 214:631–633 LoBuglio KF (1999) Cenococcum. In: Cairney JWG, Chambers SM (eds) Ectomycorrhizal fungi: key genera in profile. Springer-Verlag, Berlin Lussenhop J, Fogerl R (1999) Seasonal change in phosphorus content of Pinus strobus— Cenococcum geophilum ectomycorrhizae. Mycologia 91:742–746 Malik K, Haider K (1982) Decomposition of 14C-labeled melanoid fungal residues in a marginally sodic soil. Soil Biol Biochem 14:457–460 Massicotte HB, Trappe JM, Peterson RL, Melville LH (1992) Studies on Cenococcum geophilum. II. Sclerotium morphology, germination, and formation in pure culture and growth pouches. Can J Bot 70:125–132 Machida H, Arai F (2003) Atlas of Tephra in and around Japan, revised edn. University of Tokyo Press, Tokyo. ISBN 978-4-413-060745-2 Matsuda Y, Noguchi Y, Ito S (2009) Ectomycorrhizal fungal community of naturally regenerated Pinus thunbergii seedlings in a coastal pine forest. J For Res 14:335–341 Matsumoto N, Tajimi A (1988) Life-history strategy in Typhula incarnata and T. ishikariensis biotypes a, B and C as determined by sclerotium production. Can J Bot 66:2485–2490

14

M. Watanabe et al.

Matsumoto N, Tajimi A (1990) Continuous variation within isolates of Typhula ishikariensis biotypes B and C associated with habitat differences. Can J Bot 68:1768–1773 Maxwell DP, Williams PH, Maxwell MD (1970) Microbodies and lipid bodies in the hyphal tips of Sclerotinia sclerotiorum. Can J Bot 48:1689–1691 Meier S, Robarge WP, Bruck RI, Grand LF (1989) Effects of simulated rain acidity on ectomycorrhizae of red spruce seedlings potted in natural soil. Environ Pollut 59:315–324 Miller SL, Torres P, McClean TM (1994) Persistence of basidiospores and sclerotia of ectomycorrhizal fungi and Morchella in soil. Mycologia 81:89–95 Moore AEP, Ashford AE, Peterson RL (1991) Reserve substances in Paxillus involutus sclerotia determination by histochemistry and X-ray microanalysis. Protoplasma 163:67–81 Naiki T, Ui T (1969) On the survival of the sclerotia of Rhizoctonia Solan i Kühn in soil. Bull Fac Agric Hokkaido Univ 6:430–436. (In Japanese with English abstract) Naiki T, Ui T (1975) Ultrastructure of sclerotia of Rhizoctonia Solan i Kühn invaded and decayed by soil microorganisms. Soil Biol Biochem 7:301–304 Naiki T, Ui T (1977) Population and distribution of sclerotia of Rhizoctonia solani Kühn in sugar beet field soil. Soil Biol Biochem 9:377–381 Nakasone KK, Pereson WS, Jong C-S (2004) Preservation and distribution of fungal cultures. In: Mueller GM, Bills FG, Foster SM (eds) Biodiversity of fungi: inventory and monitoring methods. Elsevier, Amsterdam, pp 37–47 Nanjo M, Kanno H (2018) Inorganic constituents in soil. In: Basics and visuals. Springer Open Access, Berlin. https://doi.org/10.1007/978-981-13-1214-4 Obase K, Lee JK, Lee SY, Chun KW (2011) Diversity and community structure of ectomycorrhizal fungi in Pinus thunbergii coastal forests in the eastern region of Korea. Mycoscience 52:383–391 Obase K, Douhan GW, Matsuda Y, Smith ME (2014) Culturable fungal assemblages growing within Cenococcum sclerotia in forest soils. FEMS Microbiol Ecol 90:708–717 Obase K, Douhan GW, Matsuda Y, Smith ME (2016) Revisiting phylogenetic diversity and cryptic species of Cenococcum geophilum sensu lato. Mycorrhiza 26:529–540 Obase K, Douhan GW, Matsuda Y, Smith ME (2017) Progress and challenges in understanding the biology, diversity, and biogeography of Cenococcum geophilum. In: Biogeography of mycorrhizal symbiosis. Springer, Cham, pp 299–317 Obase K, Douhan GW, Matsuda Y, Smith ME (2018) Isolation source matters: sclerotia and ectomycorrhizal roots provide different views of genetic diversity in Cenococcum geophilum. Mycologia 110:473–481 Panaccione DG, Sheets NL, Miller SP, Cumming JR (2001) Diversity of Cenococcum geophilum isolates from serpentine and non-serpentine soils. Mycologia 93:645–652 Pinter N, Scott AC, Daulton TL, Podoll A, Koeberl C, Anderson RS, Ishman SE (2011) The younger Dryas impact hypothesis: a requiem. Earth Sci Rev 106:247–264 Retallack GJ (1990) Soils of the past—an introduction to paleopedology. Cambridge University Press, Cambridge, p 520 Saito I (1977) Studies on the maturation and germination of sclerotia of Sclerotinia scletotiorum (lib.) de Bary, a causal fungus of bean stem rot. In: Report of Hokkaido Prefectural Agricultural Experiment Station, vol 26, pp 1–106. (in Japanese and English summary) Scheidegger C, Brunner I (1995) Electron microscopy of ectomycorrhiza: methods, applications, and findings. In: Varma A, Hock B (eds) Mycorrhiza. Springer-Verlag, Berlin, pp 205–228 Scott AC, Pinter N, Collinson ME, Hardiman M, Anderson RS, Brain AP, Smith SY, Marone F, Stampanoni M (2010) Fungus, not comet or catastrophe, accounts for carbonaceous spherules in the younger Dryas “impact layer”. Geophys Res Lett 37:L14302 Shaw CG, Sidle RC (1982) Evaluation of planting sites common to a Southeast Alaska clear-cut. II Available inoculum of the ectomycorrhizal fungus Cenococcum geophilum. Can J Bot 73: S760–S767

1 Introduction

15

Smith ME, Douhan GW, Rizzo DM (2007) Ectomycorrhizal community structure in a xeric Quercus woodland based on rDNA sequence analysis of sporocarps and pooled roots. New Phytol 174:847–863 Smith ME, Henkel TW, Rollins JA (2015) How many fungi make sclerotia? Fungal Ecol 13:211–220 Soil Science Division Staff (2017) Soil survey manual. In: Ditzler C, Scheffe K, Monger HC (eds) USDA handbook 18. Government Printing Office, Washington, DC. https://www.nrcs.usda. gov/wps/portal/nrcs/detail/soils/ref/?cid¼nrcs142p2_054253#concentrations Spatafora JW, Owensby CA, Douhan GW, Boehm EWA, Schoch CL (2012) Phylogenetic placement of the ectomycorrhizal genus Cenococcum in Gloniaceae (Dothideomycetes). Mycologia 104:758–765 Sugiura Y, Watanabe M, Nonoyama Y, Sakagami N, Guo Y, Murayama S (2017) Saccharides of ectomycorrhizal fungal sclerotia as sources of forest soil polysaccharides. Soil Sci Plant Nutr 63:426–433 Townsend BB, Willetts HJ (1954) The development of sclerotia of certain fungi. Trans Br Mycol Soc 37:213–221 Trappe JM (1962) Cenococcum graniforme—its distribution, ecology, mycorrhiza formation, and inherent variation. PhD thesis. University of Washington, Seattle, Washington, USA Trappe JM (1964) Mycorrhizal host and distribution of Cenococcum graniforme. Lloydia 27:100–106 Trappe JM (1969) Studies on Cenococcum graniforme. I An efficient method for isolation from sclerotia. Can J Bot 47:1389–1390 Trappe JM (1988) Lessons from alpine fungi. Mycologia 80:1–10 Vogt KA, Edmonds RL, Grier CC (1981) Biomass and nutrient concentrations of sporocarps produced by mycorrhizal and decomposer fungi in Abies amabilis stands. Oecologia 50:170–175 Vogt KA, Grier CG, Meier CE, Edmonds RL (1982) Mycorrhizal role in net primary production and nutrient cycling in Abies amabilis ecosystem in western Washington. Ecology 63:370–380 Watanabe T (1973) Survivability of Macrophomina phaseoli (Maubl.) Ashby in naturally-infested soils and longevity of the sclerotia formed in vitro. Ann Phytopathol Soc Jpn 39:333–337 Willetts HJ (1972) The morphogenesis and possible evolutionary origins of fungal sclerotia. Biol Rev 47:515–536 Wolbach WS et al (2018a) Extraordinary biomass-burning episode and impact winter triggered by the younger Dryas cosmic impact 12,800 years ago. 1. Ice cores and glaciers. J Geol 126:165–184. https://doi.org/10.1086/695703 Wolbach WS et al (2018b) Extraordinary Biomass-Burning Episode and Impact Winter Triggered by the Younger Dryas Cosmic Impact 12,800 Years Ago. 2. Lake, Marine, and Terrestrial Sediments. J Geol 126:185–205. https://doi.org/10.1086/695704 Wu B, Nara K, Hogetsu T (2005) Genetic structure of Cenococcum geophilum populations in primary successional volcanic deserts on Mount Fuji as revealed by microsatellite markers. New Phytol 165:285–293

Chapter 2

Fungal Communities of Sclerotia Grains from Forest Soils Kazuhiko Narisawa, Anzilni Amasya, Yaya Sasaki Nonoyama, and Keisuke Obase

Abstract Sclerotia, tentatively identified as the resting bodies of Cenococcum geophilum, were obtained from cool-temperate forests on Mt. Chokai and Mt. Iwaki in northern Japan and on Mt. Ontake in central Japan, to survey sclerotia-associated fungi and attempt to identify which fungi produced the sclerotia. The sclerotia-associated fungal communities were surveyed using terminal restriction fragment length polymorphism analysis combined with construction of a clone library using the internal transcribed spacer region of ribosomal DNA. Sclerotia were also cultured, and resulting fungal colonies were identified. Fungi associated with sclerotia from Mt. Chokai and Mt. Iwaki were predominantly Arthrinium arundinis and Inonotus sp., respectively. These sclerotia-associated species either formed the sclerotia, attacked and colonized C. geophilum sclerotia, or occupied inviable sclerotia originally formed by C. geophilum. Sequencing of the clone library generated from the Mt. Ontake sclerotia suggested that C. geophilum was present among the isolated fungi, which were mostly ascomycetes. Although C. geophilum could not be cultured from the sclerotia, three dark septate endophytes, none of K. Narisawa (*) Department of Biological Production Science, College of Agriculture, Ibaraki University, Amimachi, Ibaraki, Japan e-mail: [email protected] A. Amasya Graduate School of Urban Environmental Sciences, Tokyo Metropolitan University, Tokyo, Japan Present Address: Mizuho Consulting Indonesia Co. Ltd., Jakarta, Indonesia Y. S. Nonoyama Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, Tokyo, Japan Present Address: Graduate School of Urban Environmental Sciences, Tokyo Metropolitan University, Tokyo, Japan K. Obase Department of Mushroom Science and Forest Microbiology, Forestry and Forest Products Research Institute, Tsukuba, Japan e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2021 M. Watanabe (ed.), Sclerotia Grains in Soils, Progress in Soil Science, https://doi.org/10.1007/978-981-33-4252-1_2

17

18

K. Narisawa et al.

which could be identified, formed sclerotia in culture. Thus, the sclerotia examined in this study might have been formed either by C. geophilum or by other fungal taxa. Keywords Sclerotia · Cenococcum geophilum · T-RFLP · Clone library analysis · DSE fungi

2.1

Introduction

Fungal species can respond to environmental stress such as desiccation by forming resting structures composed of compact masses of hardened mycelia known as sclerotia. One of the most common soil fungal species that forms sclerotia is Cenococcum geophilum, which is distributed worldwide (Jany et al. 2002; Dickie 2007) and has more than 200 species of host plants (LoBuglio et al. 1996). The sclerotia of C. geophilum are abundant and have been examined in many parts of the world. The biomass of C. geophilum sclerotia was estimated to be 440 kg ha
1 in old-growth Norway spruce forests in southern Sweden (Dahlberg et al. 1997) and 2785 kg ha
1 in a second-growth Douglas fir stand in the Oregon Coast Range (Fogel and Hunt 1979). The distribution of C. geophilum sclerotia has also been examined in forest soils of the Harz Mountains in Germany (Watanabe et al. 2004; Sakagami 2009a). The sclerotia of C. geophilum are distributed in Andosols in central Japan (Watanabe et al. 2002) and are also abundant in Pinus thunbergii forests in coastal area of Japan (Matsuda et al. 2009). These studies tentatively identified sclerotia as the resting bodies of C. geophilum according to a description of morphological characteristics provided by Trappe (1969) and Massicotte et al. (1992) without further molecular identification; however, a broad range of fungal species have the ability to form sclerotia (Smith et al. 2015). Identification of sclerotia can be difficult mainly because they grow slowly in culture (Chen et al. 2007) and are occasionally difficult to isolate in axenic culture (Obase et al. 2014). Furthermore, it is difficult to extract DNA from sclerotial cell because they contain a non-hydrolyzable residue consisting of a highly resistant melanin-like pigment, which plays an important role in the resistance of sclerotia to chemical and biological degradation (Chen et al. 2007). On the other hand, sclerotia accumulate relatively high concentrations of carbohydrates, fats, and proteins during their growth, which may be a good source of nutrition for the development of other microorganisms associated with sclerotia (Willetts 1971). These sclerotia-associated microorganisms provide valuable information for studies on microbial diversity in the rhizosphere, biocontrol for plant pathogens (Zachow et al. 2011), and functional heterogeneity in fungal adaptations to drought (Homma 1937). We aimed to identify the fungi that formed the sclerotia collected from Japanese forests, if possible, and to identify the members of the sclerotia-associated fungal community.

2 Fungal Communities of Sclerotia Grains from Forest Soils

2.1.1

19

Molecular Methods for Identification of Sclerotia-Associated Fungi

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), which can make them difficult to identify. Thus, researchers increasingly rely on molecular methods to identify species based on belowground structures alone (Horton and Bruns 2001), initially with restriction fragment length polymorphism (RFLP) analysis, also known as amplified ribosomal DNA (rDNA) restriction analysis. 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 are clone libraries (Landeweert et al. 2003; Renker et al. 2006) and terminal restriction fragment length polymorphism (T-RFLP) analysis. It has been suggested that T-RFLP is more sensitive than DGGE for fungal identification (Brodie et al. 2003; Singh et al. 2006), although obtaining sequences directly from samples may be easier with DGGE (Ma et al. 2005). T-RFLP also has significant cost advantages over construction of clone libraries, although clone libraries are likely the most accurate method of identifying species (Dickie and Fitz John 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 constructing clone libraries for selected samples to determine the 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 digestion to visualize sequence variation in either single- or mixedspecies DNA samples (Dickie and Fitz John 2007). The T-RFLP technique was first developed by Liu et al. (1997) as a tool for assessing bacterial diversity and comparing bacterial community structure between environmental samples (Marsh 1999; Lukow et al. 2000; Kitts 2001). T-RFLP data are visualized as an electropherogram, with the size and relative fluorescence intensity of fragments containing the labeled primer (terminal fragment lengths) observed as peaks. Variation in the presence and location of restriction 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 number of peaks and the similarity of peak profiles across samples are then analyzed (Dollhopf et al. 2001; Edel-Hermann et al. 2004; Mummey et al. 2005). Although Cg forming sclerotia are also known as ectomycorrhizal fungi, we performed T-RFLP analysis (Liu et al. 1997), coupled with ITS region clone library construction and sequencing (Schütte et al. 2008). Since this technique requires no culturing, it may offer a rapid method for identifying sclerotia and their associated fungal communities.

20

2.2 2.2.1

K. Narisawa et al.

Analysis of Sclerotia-Associated Fungal Communities by T-RFLP Profiling Materials and Methods

Sclerotia were collected from Mt. Chokai in Yamagata Prefecture (39 60 42.5300 N, 139 580 0.6100 E, site elevation: 730 m; annual rainfall: 2362 mm) and Mt. Iwaki in Aomori Prefecture (40 390 0.3700 N, 140 160 33.6000 E, site elevation: 790 m; annual rainfall: 1750 mm), both in northern Japan, where sclerotia are known to be abundant in the soil. Vegetation at the Mt. Chokai site (Andic Podzols, WRB/ FAO-Unesco) is dominated by Fagus crenata, with smaller numbers of Viburnum furcatum and Lindera umbellata; the forest floor is dominated by Sasa kurilensis. The Mt. Iwaki site (Fulvic Andosols, WRB/FAO-Unesco) mostly contains Quercus serrata, F. crenata, and Betula ermanii trees; Sasa kurilensis is the dominant plant on the forest floor. Within a 10  10 m2 quadrat, nine evenly distributed points were selected (n ¼ 9). At each point, a 20  20 cm2 square was marked from which the litter, F, and H soil layers were removed, and the A horizon was collected using a cylinder (10 cm  10 cm), total volume of approximately 800 cm3 per quadrat. Sclerotia were collected by hand from the A horizon soil and surface-sterilized using the following procedure described by Ohta et al. (2003). Briefly, every sclerotia grain was washed with 1 mL of sterile water in a microtube for 1 min with vortex mixing, the washing solution was removed with a sterile pipette, and this procedure was repeated 10 times. Sclerotia samples were collected from nine quadrat at each study site. Approximately 100 mg of surface-disinfested sclerotia from each study site were placed in a sterile 2-mL centrifuge tube. A metal crusher was inserted into the tube and 200 μL of lysis buffer (10 μL Tris-HCl, pH 8, 1 M; 2 μL EDTA, 0.5 M; 10 μL proteinase K; 100 μL SDS 10%, and 878 μL H2O) was added and homogenized. The crushed sclerotia solution was incubated at 37  C with shaking for 2 h. The lysed solution was extracted using phenol/chloroform, followed by addition of binding buffer (60 g guanidine thiocyanate; 10 mL Tris-HCl, 1 M; and 40 mL distilled water) and silica. Guanidine thiocyanate was washed out using wash buffer (10 mM Tris-HCl, pH 7.5; 100 mM NaCl: ethanol ¼ 1: 4). PCR was conducted using the universal primers ITS1F and ITS4 (Gardes and Bruns 1993) with the iProofTM High-Fidelity PCR kit (Bio-Rad Laboratories, Hercules, CA, USA) under a hot start at 98  C for 30 s, then 35 cycles consisting of 10 s at 98  C, 30 s at 58  C, and 30 s at 72  C in a thermal cycler (Takara Bio, Otsu, Japan). PCR products were purified in a Seakem GTGTM agarose gel (Lonza Group Ltd., Basel, Switzerland) and followed by further purification with the silica method. Purified PCR products were then amplified by quenching PCR using the quenching-fluorescence-labeled primers qLR21 and ITS1F. Thirty microliters of the qPCR reaction mixture were prepared by adding 0.1 μg template sclerotia DNA, 1.0 μL of 10 pmol μL
1 primers, Takara Ex TaqTM dNTPs, and 3 μL of optimized 10 Ex buffer (Takara Bio) in a thermal cycler.

2 Fungal Communities of Sclerotia Grains from Forest Soils

21

PCR for T-RFLP profiling was conducted after denaturing for 2 min at 98  C, followed by 30 cycles of 30 s at 95  C, 45 s at 54  C, and 90 s at 72  C. Aliquots of the amplified fragments were then separately digested with the restriction enzymes AluI, HhaI, and HaeIII (Takara Bio) according to the manufacturer’s instructions. Lengths of T-RFs were determined with a 3130xl DNA Sequencer (Applied Biosystems, Foster City, CA, USA) by mixing 2 μL of purified T-RF DNA with 15 μL of Hi-Di formamide and 0.1 μL of DNA standard LIZ600 (Applied Biosystems). Samples were denatured at 96  C for 2 min and immediately chilled on ice prior to electrophoresis using an ABI automated sequence analyzer. Lengths of fluorescently labeled T-RFs were determined after electrophoresis by comparison with internal standards using GeneMapper software (version 3.7, Applied Biosystems). T-RFLP peaks ranged in size from 50 to 650 base pairs. Samples with the dominant T-RF peaks were selected for identification by clone library analysis. T-RF solutions were ligated using the pGEM-T Easy Vector (Promega, Madison, WI, USA), and Escherichia coli DH5α high efficiency competent cells were used as hosts for recombinant plasmids and grown at 37  C in LB agar (Merck KGaA, Darmstadt, Germany), to which 100 μg mL
1 of ampicillin, IPTG, and Xgal had previously been added to a final concentration of 40 μg mL
1. White colonies were selected, and sequences were determined using the 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. DNA sequences were aligned using MEGA version 5 software (Tamura et al. 2011), and all sequences were compared to those in the NCBI database using the BLAST program.

2.2.2

Results and Discussion

2.2.2.1

Sclerotia Grain Density

Sclerotia from Mt. Chokai were spherical, black, with a diameter 0.4 to 2.6 mm (average 1.33  0.06 mm; n ¼ 54), whereas sclerotia from Mt. Iwaki were black, some were spherical and some were irregularly shaped, had a relatively large diameter, ranging from 0.8 to 4.4 mm (average 2.73  0.11 mm; n ¼ 54). Based on morphological characteristics, sclerotia from both areas matched the description given by Trappe (1962): diameter of 0.05–4 mm or greater, jet black color, hard, smooth, and mostly spherical. The average diameter of Mt. Iwaki sclerotia was significantly larger than that of Mt. Chokai sclerotia (p < 0.01; Student’s t-test). According to Matsumoto and Tajimi (1988), sclerotia size reflects different strategies in response to environmental changes. A larger sclerotial diameter may also indicate a high extractable aluminum content in the soil or exposure to an event that enriched extractable aluminum (Watanabe et al. 2002, 2004). The number of sclerotia ranged between 15 and 27 grains (average 19.83  0.81) per 800 cm3 at Mt. Chokai, whereas at Mt. Iwaki sclerotia were more abundant, ranging from 12 to

22

K. Narisawa et al.

Table 2.1 Soil and sclerotia properties Soil Litter weight (g) Weight of F and H horizons (g) O horizon thickness (cm) Bulk density of A horizon (g cm
3) Soil pH (H2O) Soil pH (KCl) Sclerotia Count density (grains g
1) Mean grain weight (mg)a Weight density (mg g
1)a

Mt. Chokai 77.7  13.5 287  32.9 5.08  0.36 0.45  0.04 4.60  0.09 3.88  0.04 Mt. Chokai 0.14  0.06 0.83  0.04 0.11  0.02

Mt. Iwaki 47.9  6.71 465  45.5 5.31  0.03 0.35  0.04 3.96  0.04 3.44  0.06 Mt. Iwaki 0.16  0.03 1.80  0.05 0.29  0.04

Data are mean  standard errors from 9 replicates for soils and 18 replicates for sclerotia. ap < 0.01; Student’s t-test

47 grains (average 33.56  2.37) per 800 cm3 soil. Sclerotia from Mt. Iwaki also had a higher count density, weight, and weight density than those from Mt. Chokai (Table 2.1).

2.2.2.2

Identification of Sclerotia-Associated Fungi by Molecular Methods

Extraction of sclerotial DNA with a commercially available DNA extraction kit produced poor results in terms of both quality and quantity. The phenol/chloroform method used in this study gave more reliable results but was relatively time consuming. Our analyses identified five species of fungi in the two sites: two from Mt. Chokai and three from Mt. Iwaki. The fungal community profile of sclerotia from Mt. Chokai showed one dominant peak (Fig. 2.1), which was identified as Arthrinium arundinis (Ascomycota: Sordariales), a species described as dematiaceous or having dark-walled septate hyphae (Pan et al. 2009). Species of Sordariales were also detected in sclerotia collected from forests dominated by Quercus and Pinus in Florida and Georgia, USA (Obase et al. 2014), and have 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 F. crenata seedlings on the floor where dwarf bamboo had withered was markedly higher than that on the floor where dwarf bamboo survived. Moreover, the removal of understory dwarf bamboo increased the net carbon gain and transpiration rates of overstory trees (Kobayashi et al. 2006); therefore, the removal of dwarf bamboo in relatively young stands may greatly enhance the productivity of overstory trees in the long term (Ishii et al. 2008). Based on these findings, the presence of A. arundinis in sclerotia from the beech forest floor on Mt. Chokai is suggested to play an important role in the survival of F. crenata.

2 Fungal Communities of Sclerotia Grains from Forest Soils

23

Fig. 2.1 T-RFLP profiles of sclerotia from Mt. Chokai and Mt. Iwaki using the restriction enzyme HhaI (Amasya et al. 2015)

Hypocrea lixii (Ascomycota: Hypocreales), which was found in sclerotia from both of the sites examined, is also known to be the sexual reproductive stage or teleomorph of Trichoderma harzianum (Chaverri and Samuels 2002), a fast-growing soil fungal species reported to be effective in the biocontrol of plant-pathogenic fungi and soil-borne diseases (Wells et al. 1972). The presence of H. lixii in sclerotia was not considered to be a contaminant from the sclerotial surface because, according to Elad et al. (1984), mycoparasitic species of Hypocrea/Trichoderma degrade and grow within resting structures (sclerotia) produced by a wide variety of pathogenic fungi, such as Sclerotinia spp., Typhula spp., Macrophomina phaseolina, and Verticillium dahliae. The results obtained from Mt. Iwaki showed one major peak, which was identified as Inonotus sp. (Basidiomycota: Hymenochaetales), a fungus that commonly

24

K. Narisawa et al.

causes disease in birch trees, including B. ermanii. According to Yamaguchi (1989), the white-rot fungus Inonotus obliquus, the causal microorganism of canker disease in Japanese birch, has been found in Honshu and northern Japan, especially in Hokkaido. Hattori (1990) isolated I. obliquus from the sclerotial tissue collected from Betula platyphylla var. japonica (Miq.) H. Hara in Tochigi, northern Japan. This finding suggests that Inonotus sp. isolated from Mt. Iwaki sclerotia may be a pathogen of the birch trees at the study site. The final fungus identified, Phyllactinia sp. (Ascomycota: Erysiphales), causes powdery mildew on the leaves and stems of a broad range of host plants, including Q. serrata (Homma 1937). Contrary to our expectations, C. geophilum was not detected at either site. The T-RFLP method is capable of detecting the presence of a species but cannot unequivocally indicate the absence of a species (Dickie et al. 2002). Other species, including C. geophilum, may have been present in sclerotia, but amplification may have been insufficient for detection. However, the results of our study suggest that: (i) sclerotia collected at Mt. Chokai were formed by A. arundinis and those at Mt. Iwaki were formed by Inonotus sp.; or (ii) sclerotia were originally formed by C. geophilum, but were subsequently occupied by other species. Obase et al. (2014) reported that sclerotia-associated fungi may be specialized mycoparasites or saprobes that preferentially decay fungal tissues or act as endophytes that colonize C. geophilum sclerotia without any aggressive interactions. However, given that we found no evidence of C. geophilum, the sclerotia observed may have been formed by A. arundinis on Mt. Chokai and by Inonotus sp. on Mt. Iwaki. Further investigation is required. Our current data could not clearly show the details of the interaction of C. geophilum with other fungi for the formation of sclerotia, but recent developments in molecular biology techniques will shed light on this interaction.

2.3

Fungal Community Analysis by Culture and Molecular Methods in Sclerotia from Forest Soil on Mt. Ontake, Gifu Prefecture, Japan

2.3.1

Material and Methods

2.3.1.1

Soil Sampling and Collection of Sclerotia

We sampled surface soil (Andic Podzols, WRB/FAO-Unesco) on 24 June 2006 from Yunohana Pass on Mt. Ontake in Gifu Prefecture (35 550 1400 N, 137 270 4700 E, elevation 2103 m) in a grove of northern Japanese hemlock (Tsuga diversifolia). The soil in this location has previously been reported to contain large numbers of sclerotia (Sakagami 2009b). Surface soil (0–5 cm depth) was collected using a trowel and stored at 4  C. According to wet sieving method (Brundrett 1996),

2 Fungal Communities of Sclerotia Grains from Forest Soils

25

Fig. 2.2 Microscopic images of sclerotia investigated in this study. (a) Four washed sclerotia, (b) Cross section of a sclerotium, (c) Internal structure of a sclerotium (outer shell and internal tissue). Scale bars: (a, b) 500 μm; (c): 50 μm (Nonoyama et al. 2016)

sclerotia grains were collected with diameters of 0.5 mm or greater (Fig. 2.2). The content of sclerotia was approximately 150 grain kg
1 soil.

2.3.1.2

Creation of Clone Library for Identification of Fungal Species

The majority of sclerotia obtained from the soil ranged in diameter from 1 to 2 mm and weighed between 1 and 2 mg. DNA was extracted from approximately 1 g of sclerotia using an ISOIL for Beads Beating kit (Nippon Gene Co. Ltd., Tokyo, Japan) and amplified by PCR with the ITS-1F and ITS-4 (Nishizawa et al. 2010) primers under the following conditions: 4 min at 94  C followed by 35 cycles consisting of 94  C for 35 s, 52  C for 55 s, 72  C for 2 min, and a final extension for 10 min at 72  C. PCR products were separated by gel electrophoresis, and bands were purified. A clone library was created from the purified PCR products by the method described by Nishizawa et al. (2010). PCR products were processed with a BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems), and DNA sequences were analyzed with a 3130x1 Genetic Analyzer (Applied

26

K. Narisawa et al.

Biosystems). A homology search of DNA sequences was performed with the DDBJblastn program.

2.3.1.3

Isolation of Fungi by Culturing

We conducted culture trials to directly isolate and identify fungi from sclerotia. Following the method published by Narisawa et al. (1998), after washing the surfaces of sclerotia with 0.005% Tween 20, we placed individual sclerotia on artificial growth media to isolate fungi. The three types of growth media used were cornmeal agar (CMA, 17 g L
1; Difco Laboratories, Detroit, MI, USA), CMA adjusted with sulfuric acid to pH 5.0, and modified Melin–Norkrans (MMN) medium with a glucose concentration of 0.5%. Sclerotia were incubated at room temperature (24  C) for at least 1 month under dark conditions. Hyphae growing from each sclerotium produced a single-species colony. A total of 38 isolates were obtained. Hyphae growing out of the sclerotia were transferred to 1/2 CMMY medium (8.5 g cornmeal agar, 10.0 g malt extract, 2.0 g yeast extract, and 7.5 g L
1 bacto agar; all from Difco). To identify the isolates, a portion of each colony was scraped and DNA was extracted with PrepMan Ultra Sample Preparation Reagent (Applied Biosystems CA, USA). PCR amplification was performed under the conditions described above. After purification, a sequencing reaction was performed, and the DNA sequence was analyzed.

2.3.1.4

Isolates’ Ability to Form Sclerotia and Sclerotial Morphology

To investigate isolates’ ability to form sclerotia and sclerotial morphology, isolates were cultured on three types of media (CMA, MMN, and 1/2 CMMY) at room temperature (24  C) for approximately 1 month. An isolate of C. geophilum AT353 provided by Dr. A. Yamada obtained from roots was used for morphological comparison. Slide cultures were made of isolates that formed sclerotia on the above media, and the morphological characteristics of the sclerotia were observed under a light microscope (BX51, Olympus, Tokyo, Japan).

2.3.2

Results and Discussion

2.3.2.1

Identification of Isolates by the Clone Library Method

A total of 14 fungal species were identified by the clone library method (Table 2.2). Of the 45 clones, only one was identified as C. geophilum, which has previously been reported to form sclerotia in forest soil. Three clones identified as vouchered mycorrhizal fungi exhibited strong sequence homology to C. geophilum. None of

Unidentified Total:

Basidiomycota

Phylum Ascomycota

Subphylum Pezizomycotina; Pezizomycotina; Pezizomycotina; Pezizomycotina; Pezizomycotina; Pezizomycotina; Pezizomycotina; – – – Hymenomycetes Hymenomycetes Hymenomycetes –

Class Leotiomycetes Leotiomycetes Leotiomycetes Leotiomycetes Leotiomycetes Dothideomycetes Dothideomycetes – – – Homobasidiomycetes Homobasidiomycetes Homobasidiomycetes –

Closest relatives Leohumicola minima strain DAOM 232587 Helotiales sp. Bjelland 61 Trichoglossum hirsutum isolate AFTOL-ID 64 Lophodermium sp. C1 Gyoerffyella rotula strain 130–1090 Vouchered mycorrhizae (Dothideomycetes) isolate SEQ10X Cenococcum geophilum isolate Ve-95-12 Mycorrhizal ascomycete of Rhododendron type isolate E97023 Ascomycete sp. olrim401 Ascomycete sp. BC15 Lactarius piperatus hue126 Lepista personata strain IFO 7717 Tomentella sublilacina clone 20–45 Fungal sp. TRN36

Identity (%) 94–98 91–92 90 96 98 93–94 91 92–93 87–90 93 92–96 96 89 89

No. of clone 17 3 1 2 1 3 1 2 5 1 6 1 1 1 45

Table 2.2 Classification (according to DDBJ-blastn search) and number of clones isolated from the interior of sclerotia collected from soil from Mt. Ontake, Gifu Prefecture (Nonoyama et al. 2016)

2 Fungal Communities of Sclerotia Grains from Forest Soils 27

28

K. Narisawa et al.

the species comprising the 41 remaining clones have been previously reported to form sclerotia.

2.3.2.2

Identification of Isolates by the Culture Method

The number and classification of isolates obtained from sclerotia collected from soil from Mt. Ontake cultured on CMA (pH 6.2), CMA (pH 5.0), and MMN (pH 5.8) are shown in Fig. 2.3 The majority (34) of the 38 isolates obtained were ascomycetes. CMA adjusted to pH 5.0 yielded the greatest number of isolates, likely due to the fact that the soil from which the sclerotia were collected was acidic (pH 4.86). The most commonly isolated species was Oidiodendron pilicola (Ascomycetes: Myxotrichaceae). A total of seven O. pilicola isolates were obtained using CMA adjusted to pH 5.0 and MMN adjusted to pH 5.8. Oidiodendron spp. have previously been isolated in large numbers from acidic soil (Bååth et al. 1984) but have not been reported to form sclerotia. Given that acidobacteria, which are found in low-pH

Fig. 2.3 Number and classification of isolates obtained from sclerotia collected from soil from Mt. Ontake (Gifu Prefecture). CMA cornmeal agar, MMN modified Melin–Norkrans medium with 0.5% glucose content (Nonoyama et al. 2016)

2 Fungal Communities of Sclerotia Grains from Forest Soils

29

environments, are the dominant group of bacteria found inside sclerotia (Nonoyama et al. 2009), the interior of sclerotia may constitute a low-pH environment. Our frequent isolation of O. pilicola, which prefers acidic environments, strongly suggests that it colonizes the insides of sclerotia formed by other fungi. Phialophora finlandia (Jumpponen 2001) and Meliniomyces variabilis (Ohtaka and Narisawa 2008), which are both classified as dark septate endophytic (DSE) fungi, were also isolated. Several DSE fungi have been reported to form sclerotia or microsclerotia (O’Dell et al. 1993; Stoyke and Currah 1993; Wagg et al. 2008). In addition, seven species of ectomycorrhizal fungi were isolated (Gyoerffyella rotula, Gyoerffyella rotula, Hypocrea sp., Lecythophora mutabilis, Cryptosporiopsis ericae, Cadophora sp., Helotiales sp.). No pathogenic fungi known to form sclerotia were isolated. Although C. geophilum was detected by the clone library method, it was not isolated with the culture method. To explain this disparity, we hypothesize that either the sclerotia were no longer viable or that C. geophilum, due to its slow growth on artificial media (LoBuglio 1999), was unable to compete with other species that had colonized the interior of the sclerotia. We also cannot exclude the possibility that the sclerotia were formed by a species other than C. geophilum.

2.3.2.3

Morphology of Sclerotia Formed by Isolates

When the isolates and C. geophilum AT353 were cultured on CMA, MMN, and 1/2 CMMY media, C. geophilum only formed sclerotia on MMN (Fig. 2.4a). In contrast, isolates Sc-iso-f02, Sc-iso-f06, and Sc-iso-f11, which were assigned to the mycelium radices atrovirens (MRA) complex (Fig. 2.3), formed sclerotia on all three media (Fig. 2.4b–d). Although verification through inoculation experiments is required, these isolates have been reported to be DSE (Jumpponen and Trappe 1998). These three isolates formed especially high numbers of sclerotia on CMA: hyphae spread over entire Petri dishes and began forming sclerotia at hyphal tips where further growth was hindered. Greater numbers of sclerotia were formed as desiccation of the media progressed. Obase et al. (2014) isolated 297 C. geophilum isolates and 427 isolates of species other than C. geophilum from 971 sclerotia. In contrast, Amasya et al. (2015) did not detect T-RFLP peaks associated with C. geophilum. As mentioned above, in this study, C. geophilum was detected by the clone library method but was not isolated by the culture method. This result suggests that the sclerotia may have been formed by species other than C. geophilum, including DSEs, or that many of the sclerotia were inviable and degraded, which resulted to hinder DNA extraction.

30

K. Narisawa et al.

Fig. 2.4 Sclerotia formed by Cenococcum geophilum on modified Melin–Norkrans medium with 0.5% glucose content and microsclerotia formed by unidentified fungal isolates on cornmeal agar. (a): C. geophilum AT353, (b) Sc-iso-f02, (c) Sc-iso-f06, (d) Sc-iso-f11. Scale bar: 10 mm (Nonoyama et al. 2016)

2.4

Conclusion

T-RFLP profiling of sclerotia grains collected in Fagus forest soils on Mt. Iwaki, Aomori Prefecture, and Mt. Chokai, Yamagata Prefecture, did not indicate that the sclerotia were formed by C. geophilum. Rather, it appears that the sclerotia were formed by sclerotia-associated species or that the sclerotia were originally formed by C. geophilum and subsequently occupied by sclerotia-associated species. Clone library analysis of fungal community structure indicated that multiple fungal species, including DSEs, are associated with sclerotia collected in soil from Mt. Ontake in Gifu Prefecture. These species may represent species that colonize C. geophilum sclerotia that have become inviable or species that themselves form sclerotia. Fungal community profiling by culture and clone library methods allowed us to identify many sclerotia-associated fungal species. Further investigation using this approach, combined with different types of media and culture conditions, could help to identify more sclerotia-forming species and broaden our understanding of the roles and functions of sclerotia in forest soil.

2 Fungal Communities of Sclerotia Grains from Forest Soils

31

References Amasya AF, Narisawa K, Watanabe M (2015) Analysis of sclerotia-associated fungal communities in cool-temperate forest soils in north Japan. Microbes Environ 30:113–116 Bååth E, Lundgren B, Söderström B (1984) Fungal populations in podzolic soil experimentally acidified to simulate acid rain. Microb Ecol 10:197–203 Bougoure DS, Cairney JWG (2005) Fungi associated with hair roots of Rhododendron lochiae (Ericaceae) in an Australian tropical cloud forest revealed by culturing and culture-independent molecular methods. Environ Microbiol 7:1743–1754 Brodie E, Edwards S, Clipson N (2003) Soil fungal community structure in a temperate upland grassland soil. FEMS Microbiol Ecol 45:105–114 Brundrett M (1996) Working with mycorrhizas in forestry and agriculture. ACIAR, Canberra, ACT, pp 167–168 Bruns TD (1995) Thoughts on the processes that maintain local species diversity of ectomycorrhizal fungi. Plant Soil 170:63–73 Chaverri P, Samuels GJ (2002) Hypocrea lixii, the teleomorph of Trichoderma harzianum. Mycol Prog 1:283–286 Chen L, Yan W, Xu Y (2007) Identification and preliminary analysis of the genetic diversity of Cenococcum geophilum Fr. Agric Sci China 6:956–963 Dahlberg A, Jonsson L, Nylund JE (1997) Species diversity and distribution of biomass above and below ground among ectomycorrhizal fungi in an old-growth Norway spruce forest in south Sweden. Can J Bot 75:1323–1335 Dickie IA (2007) Host preference, niches and fungal diversity. New Phytol 174:230–233 Dickie IA, Fitz John RG (2007) Using terminal restriction fragment length polymorphism (TRFLP) to identify mycorrhizal fungi: a methods review. Mycorrhiza 17:259–270 Dickie IA, Reich PB (2005) Ectomycorrhizal fungal communities at forest edges. J Ecol 93:244– 255 Dickie IA, Xu B, Koide RT (2002) Vertical niche differentiation of ectomycorrhizal hyphae in soil as shown by T-RFLP analysis. New Phytol 156:527–535 Dollhopf SL, Hashsham SA, Tiedje JM (2001) Interpreting 16S rDNA T-RFLP data: application of self-organizing maps and principal component analysis to describe community dynamics and convergence. Microb Ecol 42:495–505 Edel-Hermann W, Dreumont C, Perez-Piqueres A, Steinberg C (2004) Terminal restriction fragment length polymorphism analysis of ribosomal RNA genes to assess changes in fungal community structure in soils. FEMS Microbiol Ecol 47:397–404 Elad Y, Barak R, Chet I (1984) Parasitism of sclerotia of Sclerotium rolfsii by Trichoderma harzianum. Soil Biol Biochem 16:381–386 Fogel R, Hunt G (1979) Fungal and arboreal biomass in a western Oregon Douglas-fir ecosystem: distribution patterns and turnover. Can J For Res 9:245–256 Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes – application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118 Genney DR, Anderson IC, Alexander IJ (2006) Fine-scale distribution of pine ectomycorrhizas and their extramatrical mycelium. New Phytol 170:381–390 Hattori T (1990) National Institute of Agricultural Sciences (NIAS) Genebank. http://www.gene. affrc.go.jp/databases-micro_search_detail_en.php?maff=420279 Homma Y (1937) Erysiphaceae of Japan. J Fac Agric Hokkaido Imp Univ 38:183–461 Horton TR, Bruns TD (2001) The molecular revolution in ectomycorrhizal ecology: peeking into the black-box. Mol Ecol 10:1855–1871 Ishii HT, Kobayashi T, Uemura S, Takahashi K, Hanba YT, Sumida A, Hara T (2008) Removal of understory dwarf bamboo (Sasa kurilensis) induces changes in water-relations characteristics of overstory Betula ermanii trees. J For Res 13:101–109 Jany J, Garbaye J, Martin F (2002) Cenococcum geophilum populations show a high degree of genetic diversity in beech forests. New Phytol 154:651–659

32

K. Narisawa et al.

Jumpponen A (2001) Dark septate endophytes – are they mycorrhizal? Mycorrhiza 11:207–211 Jumpponen A, Trappe JM (1998) Dark septate endophytes: a review of facultative biotrophic rootcolonizing fungi. New Phytol 140:295–310 Kim J, Lee S, Ra J, Lee H, Huh N, Kim G (2010) Fungi associated with bamboo and their decay capabilities. Holzforschung 65:271–275 Kitts CL (2001) Terminal restriction fragment patterns: a tool for comparing microbial communities and assessing community dynamics. Curr Issues Intest Microbiol 2:17–25 Kobayashi T, Miki N, Kato K (2006) Understory removal increases carbon gain and transpiration in the overstory of birch (Betula ermanii) stands in northern Hokkaido, Japan: trends in leaf, shoot, and canopy. Proceedings of international workshop on H O and CO exchange in Siberia, pp 19– 22 Kowalchuk GA, De Souza FA, Van Veen JA (2002) Community analysis of arbuscular mycorrhizal fungi associated with Ammophila arenaria in Dutch coastal sand dunes. Mol Ecol 11:571–581 Landeweert R, Leeflang P, Kuyper TW, Hoffland E, Rosling A, Wernars K, Smit E (2003) Molecular identification of ectomycorrhizal mycelium in soil horizons. Appl Environ Microbiol 69:327–333 Landeweert R, Leeflang P, Smit E, Kuyper TW (2005) Diversity of an ectomycorrhizal fungal community studied by a root tip and total soil DNA approach. Mycorrhiza 15:1–6 Lindahl BD, Ihrmark K, Boberg J, Trumbore SE, Högberg P, Stenlid J, Finlay RD (2006) Spatial separation of litter decomposition and mycorrhizal nitrogen uptake in a boreal forest. New Phytol 173:611–620 Liu WT, Marsh TL, Cheng H, Forney LJ (1997) Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl Environ Microbiol 63:4516–4522 LoBuglio KF (1999) In: Cairney JWG, Chambers SM (eds) Ectomycorrhizal Fungi key genera in profile. Springer-Verlag, Berlin, pp 287–309 LoBuglio KF, Berbee ML, Taylor JW (1996) Phylogenetic origins of the asexual mycorrhizal symbiont Cenococcum geophilum Fr. & other mycorrhizal fungi among the Ascomycetes. Mol Phylogenet Evol 6:287–294 Lukow T, Dunfield PF, Liesack W (2000) Use of the T-RFLP technique to assess spatial and temporal changes in the bacterial community structure within an agricultural soil planted with transgenic and non-transgenic potato plants. FEMS Microbiol Ecol 32:241–247 Ma WK, Siciliano SD, Germida JJ (2005) A PCR–DGGE method for detecting arbuscular mycorrhizal fungi in cultivated soils. Soil Biol Biochem 37:1589–1597 Marsh TL (1999) Terminal restriction fragment length polymorphism (T-RFLP): an emerging method for characterizing diversity among homologous populations of amplification products. Curr Opin Microbiol 2:323–327 Massicotte HB, Trappe JM, Peterson RL, Mel Ville LH (1992) Studies in Cenococcum geophilum. II. Sclerotium morphology, germination, and formation in pure culture and growth pouches. Can J Bot 70:125–132 Matsuda Y, Hayakawa N, Ito S (2009) Local and microscale distributions of Cenococcum geophilum in soils of coastal pine forests. Fungal Ecol 2:31–35 Matsumoto N, Tajimi A (1988) Life-history strategy in Typhula incarnate and T. Ishikariensis biotypes A, B, and C as determined by sclerotium production. Can J Bot 66:2485–2490 Mummey DL, Rillig MC, Holben WE (2005) Neighboring plant influences on arbuscular mycorrhizal fungal community composition as assessed by T-RFLP analysis. Plant Soil 271:83–90 Nakashizuka T (1988) Regeneration of beech (Fagus crenata) after the simultaneous death of undergrowing dwarf bamboo (Sasa kurilensis). Ecol Res 3:21–35 Narisawa K, Tokumasu S, Hashiba T (1998) Suppression of clubroot formation in Chinese cabbage by the root endophytic fungus, Heteroconium chaetospira. Plant Pathol 47:206–210 Nishizawa T, Zhaorigetu KM, Sato Y, Kaneko N, Ohta H (2010) Molecular characterization of fungal communities in non-tilled, cover-cropped upland rice field soils. Microbes Environ 25:204–210

2 Fungal Communities of Sclerotia Grains from Forest Soils

33

Nonoyama Y, Narisawa K, Ohta H, Watanabe M (2009) Bacterial community in sclerotia of Cenococcum species and soil in sub-alpine forest, central Japan. Geophysical Research Abstracts—EGU2009, 11, EGU2009–6512-1 Nonoyama Y, Sakagami N, Narisawa K, Ohta H, Watanabe M (2016) Fungal community analysis by isolation and clone library in sclerotia from forest soil in Mt. Ontake, Gifu Prefecture, Japan. Soil Microorg 70:56–59 O’Dell TE, Massicotte HB, Trappe JM (1993) Root colonization of Lupinus latifolius Agardh. and Pinus contorta Dougl. by Phialocephala fortinii Wang & Wilcox. New Phytol 124:93–100 Obase K, Douhan GW, Matsuda Y, Smith ME (2014) Culturable fungal assemblages growing within Cenococcum sclerotia in forest soils. FEMS Microbiol Ecol:1–10 Ohta H, Yagi M, Suzuki J, Fujitake N, Watanabe M (2003) Characterization of Sphingomonas spp. found as predominant members in the culturable bacteria community of a green pigmentcontaining sclerotium grain from Mt. Myoko (Japan) volcanic ash soil. Microbes Environ 18:126–132 Ohtaka N, Narisawa K (2008) Molecular characterization and endophytic nature of the rootassociated fungus Meliniomyces Variabilis (LtVB3). J Gen Plant Pathol 74:24–31 Opik M, Moora M, Liira J, Koljalg U, Zobel M, Sen R (2003) Divergent arbuscular mycorrhizal fungal communities colonize roots of Pulsatilla spp. in boreal Scots pine forest and grassland soils. New Phytol 160:581–593 Pan H, Zhiang T, Kong J (2009) Notes on soil dematiaceous hyphomycetes from the Yellow River source area, China. Mycosystema 28:014–019 Pennanen T, Heiskanen J, Korkama U (2005) Dynamics of ectomycorrhizal fungi and growth of Norway spruce seedlings after planting on a mounded forest clear-cut. For Ecol Manag 213:243–252 Renker C, Weißhuhn K, Kellner H, Buscot F (2006) Rationalizing molecular analysis of fieldcollected roots for assessing diversity of arbuscular mycorrhizal fungi: to pool, or not to pool, that is the question. Mycorrhiza 16:525–531 Sakagami N (2009a) Analysis on formation factor of sclerotia of Cenococcum geophilum in Picea abies forest, Harz Mts., Germany. Geogr Rev Jpn Ser B 82:184–195 Sakagami N (2009b) Distributional optimum of sclerotia, resting bodies of Cenococcum geophilum in forest soils. Geogr Rep Tokyo Metropol Univ 46:63–72 Schütte UME, Abdo Z, Bent SJ, Shyu C, Williams CJ, Pierson JD, Forney LF (2008) Advances in the use of terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes to characterize microbial communities. Appl Microbiol Biotechnol 80:365–380 Singh BK, Munro S, Reid E, Ord B, Potts JM, Paterson E, Millard P (2006) Investigating microbial community structure in soils by physiological, biochemical and molecular fingerprinting methods. Eur J Soil Sci 57:72–82 Smith ME, Henkel TW, Rollins JA (2015) How many fungi make sclerotia? Fungal Ecol 13:211– 220 Stoyke G, Currah RSH (1993) Resynthesis in pure culture of a common sub-alpine fungus-root association using Phialocephala fortinii and Menziesia ferruginea (Ericaceae). Arct Alp Res 25:189–193 Tamura K, Peterson D, Peterson N, Stecher G, Ne M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 Trappe JM (1962) Cenococcum graniforme—its distribution, ecology, mycorrhiza formation, and inherent variation. Ph.D. thesis. University of Washington, Seattle, Washington, USA Trappe JM (1969) Studies on Cenococcum graniforme. I. An efficient method for isolation from sclerotia. Can J Bot 47:1389–1390 Wagg C, Pautler M, Massicotte HB, Peterson RL (2008) The co-occurrence of ectomycorrhizal, arbuscular mycorrhizal, and dark septate fungi in seedlings of four members of the Pinaceae. Mycorrhiza 18:103–110

34

K. Narisawa et al.

Watanabe M, Kado T, Ohta H, Fujitake N (2002) Distribution and development of sclerotium grains as influenced by aluminum status in volcanic ash soils. Soil Sci Plant Nutr 48:569–575 Watanabe M, Ohishi S, Pott A, Hardenbicker U, Aoki K, Sakagami N, Ohta H, Fujitake N (2004) Soil chemical properties and distribution of sclerotium grains in forest soils, Harz Mts., Germany. Soil Sci Plant Nutr 50:863–870 Wells HD, Bell DK, Jaworski CA (1972) Efficacy of Trichoderma harzianum as a biocontrol for Sclerotium rolfsii. Phytopathology 62:442–447 Widmer F, Hartmann M, Frey B, Kölliker R (2006) A novel strategy to extract specific phylogenetic sequence information from community T-RFLP. J Microbiol Methods 66:512–529 Willetts HJ (1971) The survival of fungal sclerotia under adverse environmental conditions. Biol Rev 46:387–407 Yamaguchi T (1989) Decay of Betula platyphylla var. japonica caused by Fuscoporia obliqua. Trans Meet Hokkaido Branch Jpn For Soc 37:91–93. (in Japanese) Zachow C, Grosch R, Berg G (2011) Impact of biotic and abiotic parameters on structure and function of microbial communities living on sclerotia of the soil-borne pathogenic fungus Rhizoctonia solani. Appl Soil Ecol 48:193–200

Chapter 3

Relationships Between Soil Mesofauna, Ectomycorrhizal Fungi, and Sclerotia in Forest Soils Anzilni Amasya and Kazuhiko Narisawa

Abstract In this study, we isolated sclerotia, soil mesofauna, and ectomycorrhizal fungi from soils in Fagus forests in northern and central Japan. We detected fungal species in soil mesofauna by using microbial community profiling methods, and the ectomycorrhizal fungal community profiles in soil were compared to those extracted from sclerotia and mesofauna samples. Our results indicate that Acari and Collembola play a major role in the regulation of ectomycorrhizal fungi in forest soils. We noted a relationship between soil pH and exchangeable aluminum content and sclerotia formation. Our findings also suggest that fungivory of Acari and/or Collembola toward sclerotia-forming ectomycorrhizal fungi is a key biological factor regulating sclerotia formation in forest soils. Keywords Community profiles of ECM · Acari · Collembola · Low-pH forest soils · Fungivory · Sclerotia formation

3.1

Introduction

The soil mesofauna is the most diverse component of the soil ecosystem. Animals living in the litter and the microscopic crevices in the soil play a fundamental role as processors and translocators of the organic matter that is transformed into the humus. Across soil ecosystems, the soil mesofauna contribute significantly to decomposition processes and nutrient turnover (Visser et al. 1981). Many taxa are represented,

A. Amasya Graduate School of Urban Environmental Sciences, Tokyo Metropolitan University, Tokyo, Japan Present Address: Mizuho Consulting Indonesia Co. Ltd, Jakarta, Indonesia K. Narisawa (*) Department of Biological Production Science, College of Agriculture, Ibaraki University, Amimachi, Ibaraki, Japan e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2021 M. Watanabe (ed.), Sclerotia Grains in Soils, Progress in Soil Science, https://doi.org/10.1007/978-981-33-4252-1_3

35

36

A. Amasya and K. Narisawa

Fig. 3.1 Associations between ectomycorrhizal fungi and soil organisms. When environmental conditions are unfavorable, the mycelia of ectomycorrhizae harden and form sclerotia for survival. In addition, ectomycorrhizae are grazed by mesofauna such as Acari, Collembola, and Nematoda (Amasya 2014)

including several orders of insects and their larvae, as well as Myriapoda, Crustacea, Thysanura, Tardigrada, and others. In terms of numerical abundance and diversity, the Acari, Collembola, and Nematoda dominate. Acari, or mites, feed on microorganisms and regulate the dispersal of microbial propagules (Behan and Hill 1978). The total number of species of Acari is estimated to be nearly 100,000 (Schatz 2002). Acari are important decomposers in almost all habitats; their distribution ranges from arid coniferous forests to floodplain forests to salt marshes (Usher 1975; Mitchell 1979; Weigmann 2009). The least diverse of these three groups, Collembola, or springtails, includes more than 7600 known species, more than all mammal species and three-quarters of the known number of bird species. Collembola contribute to the regulation of fungal populations (Warnock et al. 1982) and to the establishment of relationships between plants and mycorrhizal fungi (Gange 2000) in the soil. Sclerotia-forming fungi and mesofauna are only some of the wide variety of organisms in soil ecosystems. Each organism has interrelated roles and functions contributing to important soil processes involving nutrient cycles in the rhizosphere (Fig. 3.1). Some ectomycorrhizal fungi form sclerotia, and this process may occur in response to grazing by soil mesofauna. However, few studies have investigated the relationships between sclerotia and mesofauna by analyzing the ectomycorrhizal fungal community. Knowledge of this relationship may contribute to the understanding of how sclerotia formation is influenced by mesofauna.

3 Relationships Between Soil Mesofauna, Ectomycorrhizal Fungi, and Sclerotia in. . .

37

Many studies have examined the interactions between mesofauna and ectomycorrhizal fungi. Setälä (1995) reported that by consuming ectomycorrhizal fungi, soil mesofauna may influence development of plant–fungus symbioses. Mesofauna can also affect the dynamics of the symbiosis between ectomycorrhizal fungi and vascular plants (Moore et al. 2003). Mesofauna feeding on ectomycorrhizal fungi has been shown to increase primary productivity of the host plants (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 can mediate the interactions between fungus-feeding mesofauna and aboveground herbivores (White 1984; Price 1991). Although studies have shown that the ecological significance of fungus-feeding mesofauna can reach far beyond the rhizosphere (Moore et al. 2003), data are lacking on mesofaunal relationships with sclerotia-forming ectomycorrhizal fungi. Given the ability of sclerotia to survive for long periods of time, information on these ectomycorrhizal fungi may also contribute to our understanding of sclerotia life history. Furthermore, understanding sclerotia and their associations with mesofauna may lead to a better understanding of the complex food web and biological processes in the soil.

3.2

Community Profiles of Ectomycorrhizal Fungi Consumed by Soil Mesofauna

3.2.1

Materials and Methods

3.2.1.1

Study Area and Soil Sampling Techniques

The location and characteristics of the study sites are given in Table 3.1. Forest soil samples were collected from 10
10 m2 plots in the five study areas: Akita, Iwaki, Chokai, Nagano, and Minamiosawa. At each site, nine cylinders, each with a capacity of approximately 800 cm3, were used to obtain soil from the A horizon to 10 cm depth for mesofauna extraction (Fig. 3.2).

3.2.1.2

Soil Mesofauna Extraction

Soil samples for mesofauna extraction were transported in cotton bags to ensure that mesofauna remained viable until extraction. Soil mesofauna were 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 W light bulb and the heat gradient was increased by placing an aluminum funnel (14 cm in length) around the soil sample. The collecting vessel was filled with 70% ethanol for surface

a

Fagus crenata, Viburnum furcatum, Lindera umbellate Sasa kurilensis Andic Podzols 730 2362

6.8

7.3

Chokai Mt. Chokai 39º60 42.5300 N, 139º580 0.6100 E

Akita Lake Tazawa 39º460 21.8100 N, 140º470 2.5100 E Fagus crenata, Quercus crispula Sasa kurilensis Fulvic Andosols 760 2260

Data obtained from: The Japan Meteorological Agency (2011)

Floor vegetation Soil type (FAO 2015) Elevation (m) Annual rainfall (mm)a Mean annual temperature (ºC)a

Vegetation

Study area Coordinates

Table 3.1 Location and characteristics of the study area

5.7

Iwaki Mt. Iwaki 40º390 0.3700 N, 140º160 33.6000 E Fagus crenata, Quercus serrata, Betula ermanii Sasa kurilensis Fulvic Andosols 790 1570 7.2

Nagano Mt. Kisokomagatake 35º450 25.5600 N, 137º510 20.7300 E Fagus crenata, Quercus serrata, Pinus pumila Sasa kurilensis Fulvic Andosols 780 1070

14.4

Minamiosawa Tama Hill 35º370 06.9900 N, 139º220 50.3500 E Quercus acutissima Quercus serrata Sasa nipponica Melanic Andosols 140 1602

38 A. Amasya and K. Narisawa

3 Relationships Between Soil Mesofauna, Ectomycorrhizal Fungi, and Sclerotia in. . .

39

Fig. 3.2 Soil sampling methodology. One cylinder of soil was taken from each subplot for mesofauna collection (Amasya 2014)

sterilization and to preserve the mesofauna for further examination. Mesofauna were identified to genus under a digital high-density video microscope (VH-7000, Keyence, Osaka, Japan).

3.2.1.3

Isolation of Fungal DNA from Soil Mesofauna

Three to five individuals of each species from each subplot were crushed using a metal crusher (TAITEC Corporation, Saitama, Japan) and fungal DNA was isolated using Prepman™ Ultra sample reagent (Applied Biosystems, CA, USA). This process was repeated three times for each location, and results were averaged. The ribosomal DNA internal transcribed spacer (ITS) regions were then amplified by PCR using the ITS-1F/LR21 primer pair and Taq DNA polymerase (Applied Biosystems, Waltham, MA, USA) with conditions as follows: a hot start at 96  C for 30 s, then 35 cycles consisting of 10 s at 96  C, 30 s at 55  C, and 30 s at 72  C. Samples were also subjected to T-RFLP analysis. T-RFLP refers to the use of fluorescently labeled primers combined with restriction digestion to visualize sequence variation in either single- or mixed-species DNA samples (Dickie and Fitz John 2007). The T-RFLP technique was first developed by Liu et al. (1997) as a tool for assessing bacterial diversity and comparing bacterial community structure between environmental samples (Marsh 1999; Lukow et al. 2000; Kitts 2001). T-RFLP data are visualized as an electropherogram, with the size and relative

40

A. Amasya and K. Narisawa

fluorescence intensity of fragments containing the labeled primer (terminal fragment lengths) observed as peaks. Variation in the presence and location of restriction 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 number of peaks and the similarity of peak profiles across samples are then analyzed (Dollhopf et al. 2001; Edel-Hermann et al. 2004; Mummey et al. 2005). Aliquots of the amplified DNA were digested with restriction endonucleases to obtain ITS-RFLPs; each sample was digested with Hae III and HhaI (Promega Corporation, Madison, WI, USA).

3.2.1.4

Statistical Analyses

Mesofaunal Diversity Diversity of mesofauna was estimated using two indices, the Shannon–Wiener diversity index (Hill 1973) and the inverse Simpson index (Simpson 1949). The number of individuals of each mesofaunal order was used to indicate species abundance and the number of identified orders to indicate species richness. The Shannon–Wiener index (H0 ) was calculated as follows, where pi is the proportion of the population belonging to the ith species: H0 ¼ 

R X

pi : ln ðpi Þ

i¼1

The inverse Simpson index (D) was calculated as follows: 1 D ¼ PR

2 i¼1 pi

The higher the value of D, the greater the diversity.

Diversity of Fungal Communities To quantify fungal diversity in soil and that consumed by mesofauna, the Shannon– Weiner index was calculated, with T-RF fragment sizes representing fungal species richness and T-RF peak heights representing fungal species abundance.

3 Relationships Between Soil Mesofauna, Ectomycorrhizal Fungi, and Sclerotia in. . .

3.2.2

Results and Discussion

3.2.2.1

Soil Mesofauna Characteristics

41

At all study sites, soil mesofauna belonging to the Acari, Collembola, Nematoda, Symphyla, Diptera, Coleoptera, Hymenoptera, Chilopoda, and Diplopoda were identified, except at Akita where no Hymenoptera were collected. Figure 3.3 shows the average abundance (n ¼ 9) of soil mesofauna across study sites. At all sites, Acari and Collembola were predominant. The Shannon–Wiener diversity index and inverse Simpson index are shown in Table 3.2. Among study sites, Nagano showed the highest mesofaunal diversity (H0 ¼ 1.51; D ¼ 3.65). According to MacArthur (1955), more diverse communities will show enhanced ecosystem stability. Therefore, the high mesofaunal diversity found in the forest soils of Nagano indicates the soil ecosystem with the highest stability among the studied areas.

Fig. 3.3 Average abundance of soil mesofauna (Amasya 2015) Table 3.2 Soil mesofauna diversity across study sites estimated by Shannon–Wiener diversity index (H0 ) and inverse Simpson index (D) Diversity Indices Shannon (H0 ) Simpson’s D

Area Akita 3.16 1.35

Nagano 3.65 1.51

Chokai 3.32 1.40

Iwaki 3.35 1.36

42

3.2.2.2

A. Amasya and K. Narisawa

Community Profiles of Fungi Consumed by Soil Mesofauna

T-RFLP profiles of fungi extracted from forest soil mesofauna are shown in Figs. 3.4, 3.5, 3.6 and 3.7. The x-axis shows fragment size, with each distinct fragment length representing a distinct fungal species. The peak heights on the yaxis show the abundance of that fungal taxon in the mesofauna. Acari at Akita (Fig. 3.5) showed a peak only for Laccaria sp. Acari at Nagano (Fig. 3.7) also showed a dominant peak for Laccaria sp., and Collembola showed a dominant peak for Paxillus sp.

3.2.2.3

Predominance of Acari and Collembola and Their Roles in the Soil Ecosystem

Insects in the order Oribatida (Acari), identified by having fungivorous mouth parts, were selected for further study, and Acari with other types of mouth parts (e.g., chelate or subchelate pedipalps) were not included in this analysis. Most of the Collembola found belonged to the family Entomobrydae (69%) and the rest belonged to the order Poduromorpha (31%). The length of the Acari collected ranged from 0.6 to 0.9 mm, whereas Collembola were 1.3–1.5 mm in length. The litter layer at the Nagano sampling site had the highest average weight, and mesofaunal abundance was also highest at this site (Fig. 3.8). The number of individuals was correlated to the average weight of the litter layer, and as litter weight increased, the abundance of both Acari and Collembola tended to increase. The mean abundance of Acari and Collembola in this study, conducted in temperate deciduous forests, translates to a density of 11,043 individuals/m2 and 10,774 individuals/m2, respectively. These values are lower than those reported for most temperate areas of central Japan (Hijii 1994) and some tropical sites (e.g., Seastedt 1984; Gonzalez et al. 2001). At the Chokai and Iwaki sites, Acari were more abundant. Greater abundance of Acari than Collembola in forests of central Japan has been reported previously, and the authors proposed this trend was related to the heavy rainfall, acidic soil, large amount of litter accumulation, and slow decomposition rate (Takeda and Abe 2001; Lin et al. 2002). At the Akita and Nagano sites, Acari were less abundant than Collembola, in accordance with the findings of Hijii (1994), who reported on the ratio of springtails to mites in coniferous forests of Japan. Using our estimates of the proportion of each type of mesofauna at each site, we estimated the quantity of each fungal species consumed by each group in each area, based on RFLP peak heights (Fig. 3.9). Not only did Acari and Collembola predominate in abundance, but their intake of ectomycorrhizal fungi was highest among all groups of mesofauna. This result shows that Acari and Collembola play a major role in the regulation of ectomycorrhizal fungi in forest soils. We also investigated whether the fungal communities extracted from Acari and Collembola significantly differed from those extracted from other soil mesofauna. Figure 3.10 shows the results of R-analysis with a stress value of 0.0078, suggesting

3 Relationships Between Soil Mesofauna, Ectomycorrhizal Fungi, and Sclerotia in. . .

43

Fig. 3.4 T-RFLP profile of fungal DNA isolated from soil mesofauna at Akita. Hymenoptera were not found in Akita samples (Amasya 2015)

44

A. Amasya and K. Narisawa

Fig. 3.5 T-RFLP profile of fungal DNA isolated from soil mesofauna at Chokai (Amasya et al. 2015)

3 Relationships Between Soil Mesofauna, Ectomycorrhizal Fungi, and Sclerotia in. . .

45

Fig. 3.6 T-RFLP profile of fungal DNA isolated from soil mesofauna at Iwaki (Amasya 2015)

46

A. Amasya and K. Narisawa

Fig. 3.7 T-RFLP profile of fungal DNA isolated from soil mesofauna at Nagano (Amasya 2015)

3 Relationships Between Soil Mesofauna, Ectomycorrhizal Fungi, and Sclerotia in. . .

47

Fig. 3.8 Mean number of Acari and Collembola captured at each study site per gram of soil, as compared to the average litter layer weight. Upper left: Acari; lower left: Collembola (Amasya 2014)

that Acari and Collembola are associated with specific ectomycorrhizal fungi, whereas other soil mesofauna are associated with a broader range of fungal species. This finding also suggests Acari and Collembola may serve as indicators for specific ectomycorrhizal species. Given the tendency of Acari and Collembola to associate with specific ectomycorrhizal fungi and their importance in the uptake of ectomycorrhizae in soil, we focused on identifying which species of ectomycorrhizal fungi are associated with Acari and Collembola at the four study sites. The results are shown in Fig. 3.11. At Akita, Acari showed high peak for Laccaria sp., and Collembola showed high peaks for Paxillus sp. and Tuber sp. At Chokai, Acari-associated fungi were dominated by Phyllactinia sp., Tuber sp., and Laccaria sp., whereas Collembola from the same site were associated with Inocybe sp. and Paxillus sp. In Collembola at Ikari, Trichoderma sp. and Inocybe sp. were detected. At Iwaki, the predominant ectomycorrhizal fungi consumed by Acari were Trichoderma sp. and Tuber sp., whereas at Nagano, Acari were predominantly associated with Laccaria sp. and Collembola with Paxillus sp. Inonotus sp. (Basidiomycota: Hymenochaetales) causes a common fungal disease of birch trees, including Betula ermanii, and Laccaria sp. (Basidiomycota: Agaricales) is a common mycorrhizal symbiont (Martin et al. 2008; Plett et al. 2011) that Acari is reported to prefer for grazing (Schneider et al. 2005). Trichoderma sp. is a fast-growing soil fungus reported to be effective for biocontrol of plant-pathogenic fungi and soil-borne disease (Wells et al. 1972), whereas Phyllactinia sp. is an Ascomycete known to cause powdery mildew on leaves and stems in a broad range of host plants, including Quercus serrata (Homma 1937), which is present at two of our study sites. Tuber species are obligate ectomycorrhizal fungi and form ectomycorrhizal associations 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 mycorrhizal associations with orchids (Bidartondo et al. 2004; De Roman et al. 2005). In Japan, Inocybe

48

A. Amasya and K. Narisawa

Fig. 3.9 Proportions of fungal species consumed by mesofauna of forest soils (Amasya 2015)

sp. (Basidiomycota: Agaricales) is commonly found on roadsides near Abies mariesii, Pinus koraiensis, and B. ermanii forests (Kobayashi et al. 2006). 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 have also been reported to graze on Paxillus sp.,

3 Relationships Between Soil Mesofauna, Ectomycorrhizal Fungi, and Sclerotia in. . .

49

Fig. 3.10 R-analysis for analyzing the fungal community structure of soil mesofauna. The results showed the stress value 0.0078 (Amasya 2015)

and a low density of collembolans induced a shift toward a larger proportion of Paxillus involutus growth (Ek et al. 1994).

3.3

Relationship Between Sclerotia and Mesofauna in Forest Soils Based on Ectomycorrhizal Fungal Community Profiles

3.3.1

Materials and Methods

3.3.1.1

Fungal Community Profiles in Soil

Soil samples were collected from the four study sites described above, all of which are located in cool forest zones with low-pH soils. To compare with areas with higher soil pH outside the cool forest zones, we collected soil samples from temperate deciduous forest dominated by Quercus acutissima and Q. serrata associated with Sasa kurilensis located in Minamiosawa, Tokyo Metropolitan University campus, Tokyo. We collected sclerotia and mesofauna and performed T-RFLP analysis of ectomycorrhizal fungi isolated from mesofauna, using the methods previously described in Sect. 3.2. To survey the soil fungal community, fungal DNA was extracted using ISOIL for Bead Beating Soil DNA Extraction Kit (Nippon Gene, Tokyo, Japan). This extraction solution uses both chemical lysis by a surface-active agent and physical disruption of cells by beads for DNA extraction. Extracted DNA was subjected to

50

A. Amasya and K. Narisawa

Fig. 3.11 Ectomycorrhizal fungi identified in Acari and Collembola samples by study site (Amasya 2015)

3 Relationships Between Soil Mesofauna, Ectomycorrhizal Fungi, and Sclerotia in. . .

51

T-RFLP analysis to survey the ectomycorrhizal fungal community, using the methodology described in Chap. 2.2.1.

3.3.1.2

Soil pH and Exchangeable Aluminum Content

Sclerotia contain a relatively high concentration of aluminum, and the formation of sclerotia has been reported to be regulated by the quantity of exchangeable aluminum and active aluminum in the soil, regardless of soil type (Watanabe et al. 2002). To obtain an index of soil acidity, we measured pH using 1 M KCl and determined the exchangeable aluminum content of soil. Sclerotia grains were isolated from soil samples and then tested for pH (KCl) according to the method of Bertsch and Bloom (1996). Sclerotia-free and sclerotia-containing soil samples were prepared. Isolated sclerotia grains were crushed with a mortar and pestle and then mixed with soil samples prior to KCl extraction. The exchangeable aluminum content was determined for both sclerotia-containing and sclerotia-free soils using a plasma atomic emission spectrometer (ICPE-9000, Shimadzu Corp, Kyoto, Japan).

3.3.2

Results and Discussion

3.3.2.1

Ectomycorrhizal Fungal Community Profiles of Sclerotia, Soil Mesofauna, and Soil Samples

Sclerotia were not detected in soil samples from Minamiosawa, perhaps due to the low soil acidity (pH ¼ 6.11  0.21; Table 3.3), because it has been suggested that low soil pH is important for sclerotia formation (Aycock 1966). Because no sclerotia were collected, the T-RFLP analysis of sclerotia was not conducted for this site. For the remaining study sites, the dominant sclerotia-forming fungi were identified as

Table 3.3 Exchangeable aluminum content in soil with and without sclerotia. Data are mean  standard errors (Amasya 2015)

Akita

Soil pH (KCl) (n ¼ 9) 4.76  0.02

Sclerotia Diameter (mm) (n ¼ 54) 0.98  0.21

Soil AlEx (g kg1) Weight density (mg/g) (n ¼ 54) 0.05  0.01

Sclerotia-free (n ¼ 27) 0.57  0.03

Nagano

4.17  0.03

1.07  0.29

0.07  0.01

0.59  0.03

Chokai

3.88  0.03

1.33  0.62

0.11  0.02

0.69  0.04

Iwaki

3.44  0.03

2.73  1.13

0.29  0.04

0.87  0.02

With added sclerotia 0.61  0.02 (n ¼ 6) 0.64  0.003 (n ¼ 3) 0.76  0.04 (n ¼ 9) 0.97  0.01 (n ¼ 27)

52

A. Amasya and K. Narisawa

Fig. 3.12 Ectomycorrhizal fungi in soil, sclerotia, and mesofauna samples at Akita based on T-RFLP peaks. The peaks surrounded by the red dotted lines in each figure indicate Laccaria sp. (Amasya 2015)

Laccaria sp. at Akita, Arthrinium arundinis at Mt. Chokai, Inonotus sp. at Iwaki, and Tuber sp. at Nagano. The main ectomycorrhizal fungi associated with soil mesofauna were Laccaria laccata for Acari at Akita, Chokai, and Minamiosawa; Inonotus sp. for Acari at Akita; Paxillus obscurosporus for Collembola at Chokai and Minamiosawa; Phyllactinia sp. for Acari at Chokai; Tuber aestivum for Acari at Chokai, Iwaki, and Minamiosawa; Inocybe sp. for Collembola at Chokai and Iwaki; and Trichoderma viride for Acari at Iwaki and for Collembola at Akita and Iwaki. A summary of the ectomycorrhizal fungi identified in soil, sclerotia, and mesofauna samples at the four study sites is shown in Figs. 3.12, 3.13, 3.14, 3.15 and 3.16. The vertical red boxes in these figures highlight ectomycorrhizal species identified across sclerotia, mesofauna, and soil samples. Interestingly, at Chokai, Iwaki, and Nagano, where ectomycorrhizal fungal sclerotia were relatively abundant, the same species were less abundant or undetected in Acari and/or Collembola. The opposite was observed at Akita, where the ectomycorrhizal fungus Laccaria sp. was abundant in Acari, but scarcely identified from the sclerotia sample. A simplified graph of this result is shown in Fig. 3.17. When the relative abundance of ectomycorrhizal fungi was high in mesofauna, it tended to be low in the sclerotia

3 Relationships Between Soil Mesofauna, Ectomycorrhizal Fungi, and Sclerotia in. . .

53

Fig. 3.13 Ectomycorrhizal fungi in soil, sclerotia, and mesofauna samples at Chokai based on T-RFLP peaks. The peaks surrounded by the red dotted lines in each figure indicate Arthrinium arundinis. (Amasya 2015)

sample, and vice versa. Thus, ectomycorrhizal fungal abundance in sclerotia and mesofauna samples tended to be inversely proportional. At Akita, when the abundance of Laccaria sp. was high in Acari, it was low in the sclerotia sample. Schneider et al. (2005) reported that Laccaria laccata is one of the ectomycorrhizal fungi preferred by Acari. Our findings suggest that Laccaria sp. was grazed by Acari before it could form sclerotia. Regarding Arthrinium sp. and Inonotus sp., to our knowledge there are no reports that these species are preferred for grazing by Acari or Collembola. However, for Tuber sp. at Nagano, Queralt et al. (2014), who studied the relationships between T. melanosporum and Oribatid mites, reported that in some cases, mites have been seen with spores attached to their bodies. We surfacesterilized mesofauna with 70% ethanol before performing DNA isolation and T-RFLP analysis. Thus, we may not have detected Tuber sp. in mesofauna from Nagano owing to the surface-sterilization stage in our methodology.

54

A. Amasya and K. Narisawa

Fig. 3.14 Ectomycorrhizal fungi in soil, sclerotia, and mesofauna samples at Iwaki based on T-RFLP peaks. The peaks surrounded by the red dotted lines in each figure indicate Inonotus sp. (Amasya 2015)

3.3.2.2

Environmental Factors Regulating Sclerotia Formation

Apart from relationships with mesofauna, the development of sclerotia by fungi is reported to be promoted by a high content of active aluminum in low-pH soils (Watanabe et al. 2002). Table 3.3 shows the soil pH and exchangeable aluminum data. Soils with a higher exchangeable aluminum (AlEx) content also contained sclerotia with a larger diameter. This is in accordance with reports by Watanabe et al. (2002) and Sakagami (2008), who considered the content of exchangeable aluminum to be an effective factor for accelerating formation of large sclerotia. Formation of large sclerotia can be interpreted as a physiological response of sclerotia-forming ectomycorrhizal fungi to aluminum stress. Sclerotia formation was also affected by exchangeable aluminum content, which was 10.40% higher in soil with sclerotia than in sclerotia-free soil at Iwaki (Fig. 3.18). This result also suggests that sclerotia were formed as a result of high acidity and aluminum stress in soil.

3 Relationships Between Soil Mesofauna, Ectomycorrhizal Fungi, and Sclerotia in. . .

55

Fig. 3.15 Ectomycorrhizal fungi in soil, sclerotia, and mesofauna samples at Nagano based on T-RFLP peaks. The peaks surrounded by the red dotted lines in each figure indicate Tuber sp. (Amasya 2015)

3.4

Model of Relationship Between Sclerotia Formation and Soil Mesofauna Mediated Via Ectomycorrhizal Fungi

One of the focuses of this study was to investigate the role of mesofauna in sclerotia formation by using T-RFLP analysis to determine the abundance of ectomycorrhizal fungi. We propose the following two models. The first model is based on the abundance of ectomycorrhizal fungi in soil, sclerotia, and mesofauna samples. The second model is based on (1) abundance of sclerotia by density (mg/g of soil) and (2) fungivory by mesofauna, measured as the diversity of ectomycorrhizal fungi isolated from mesofauna. For the first model, we categorized soil conditions by pH values as Iwaki < Chokai < Nagano < Akita < Minamiosawa. We further divided soils into three pH categories: pH < 4 at Chokai and Iwaki, where sclerotia were abundant and relatively large in diameter, pH 4–5 at Akita and Nagano, where sclerotia were present but with lower abundance and smaller diameter, and pH > 5 at

56

A. Amasya and K. Narisawa

Fig. 3.16 Ectomycorrhizal fungi in soil, sclerotia (not found), and mesofauna samples at Minamiosawa based on T-RFLP peaks (Amasya 2015)

Minamiosawa, where sclerotia were not detected. Second, we combined this information with ectomycorrhizal fungal abundance, defined as T-RFLP peak height, in soil, sclerotia, and mesofauna samples. In Fig. 3.19, the largest square in the background represents the soil, the yellow box represents the mesofauna, in this case Acari and Collembola, the green oval represents ectomycorrhizal fungi, and the black box represents sclerotia. In the leftmost part of Fig. 3.19, the larger black box indicates that sclerotia are abundant. At these sites, with pH < 4, mesofaunal abundance was relatively low, presumably due to soil acidity. Incorporating the results in Fig. 3.17, ectomycorrhizal fungi were detected in sclerotia samples but not in mesofauna samples such as in Chokai and Iwaki sites; therefore the green oval does not overlap the yellow box. In the center of Fig. 3.19, showing sites where soil pH ranged from 4 to 5, such as in Akita and Nagano, ectomycorrhizal fungi were detected from both sclerotia and mesofauna samples. We inferred that under these conditions, sclerotia had only begun to form because the abundance of sclerotia was relatively low and they were small in diameter. A larger number of mesofauna were collected than at sites

3 Relationships Between Soil Mesofauna, Ectomycorrhizal Fungi, and Sclerotia in. . .

57

Fig. 3.17 Abundance of ectomycorrhizal fungi in Acari, Collembola, soil, and sclerotia samples collected from the study sites (Amasya 2015)

Fig. 3.18 Relationship between soil pH(KCl) and exchangeable aluminum content (AlEx) at the four study sites; Iwaki, Nagano, Akita, and Chokai. Left: Soil including sclerotia grain, Right: Sclerotia grain free soil. Sample size of each item is referred in Table 3.3

where pH was lower. We infer that these soil conditions were more favorable to mesofauna. Peaks of ectomycorrhizal fungal abundance were detected in mesofauna and sclerotia samples as well as in soil, and we observed a decrease in the relative abundance of ectomycorrhizal fungi in mesofauna. We infer that both the lower soil pH and grazing by mesofauna played a role in stimulating sclerotia formation. For the second model, we used density of sclerotia to describe sclerotia abundance, and the Shannon–Wiener diversity index (H0 ) of ectomycorrhizal fungal communities obtained from mesofauna to indicate fungivory by mesofauna. From

58

A. Amasya and K. Narisawa

Fig. 3.19 Model of relationships between sclerotia and soil mesofauna as mediated by ectomycorrhizal (ECM) abundance (Amasya 2015)

the T-RFLP results in Figs. 3.12, 3.13, 3.14, 3.15 and 3.16, sclerotia-forming ectomycorrhizal fungi such as Tuber sp., Laccaria sp., and Paxillus sp. were detected in both soil and mesofauna samples at Minamiosawa, even though no sclerotia were observed. As the soil pH decreased, such as at Akita, H0 (indicating fungivory) increased, and sclerotia were detected. At Nagano, where the pH is even lower, fungivory was highest among the study sites, and sclerotia were also more abundant. Based on these findings, we suggest that fungivory, along with soil acidity, plays a role in triggering sclerotia formation. At Chokai and Iwaki, where soil pH is arabinose ’ galactose ’ mannose ’ rhamnose ’ fucose ’ ribose (’means no significant difference). (3) Average proportion of each monosaccharide of three coniferous trees (Maries fir is excluded); NEH-glucose > EH-glucose ’ arabinose ’ mannose > xylose ’ galactose > rhamnose ’ fucose ’ ribose

98

S. Murayama and Y. Sugiura

Table 6.1 Molar ratio of galactose + mannose (GM) to arabinose + xylose (AX) and that of easily hydrolysable (EH)-glucose to non-easily hydrolysable (NEH)-glucose in the monosaccharide composition of plant materials TotalC (%) Herbaceous plants Pampas grassa 46.1 Bamboo grassa 46.0 Goldenroda 47.2 Paddy riceb 38.7 Barleyb 47.2 Woody plants Coniferous tree Northern Japa- 51.7 nese hemlockc 51.8 Korean pinec Japanese 53.2 thujac 47.0 Maries fir (weathered fallen trunk)c

TotalN (%)

C/N ratio

Saccharide content (g kg
1)

Saccharide-C/ plant-C (%)

Molar ratio of GM/AX

Ratio of EH-Glc/ NEH-Glc

0.62 0.68 0.34 0.57 0.36

75 68 139 69 131

587 571 500 532 621

51.0 49.7 42.4 55.0 52.8

0.042 0.045 0.15 0.058 0.037

0.337 0.202 0.142 0.379 0.129

0.78

66

362

28.1

0.89

0.53

0.61 0.34

85 155

424 252

32.8 19.0

0.78 0.85

0.22 0.30

0.083

566

580

49.4

2.2

0.14

0.52

0.37

0.30

–

0.30

–

0.43

–

0.36

–

0.54

–

0.49

–

0.22

0.31

0.081

0.088

Deciduous tree 1.0 50 259 20.5 Japanese white 50.7 birchc 46.7 2.5 18 – – Beech (green l.)d (n ¼ 25) 1.2 39 – – Beech (leaf l.)d 45.0 (n ¼ 25) Oak (green l.)d 46.3 2.6 18 – – (n ¼ 20) Oak (leaf l.)d 47.6 1.5 33 – – (n ¼ 20) 46.9 2.5 19 – – Chestnut (green l.)d (n ¼ 13) Chestnut (leaf 47.2 1.2 41 – – l.)d (n ¼ 13) Mixture of plant remains and plant roots collected from soil sampleb Kuriyagawa 37.9 0.84 59 233 24.6 forest soil Cyouyou for49.6 0.49 102 382 30.8 est soil

(continued)

6 Origin of Soil Polysaccharides, and Ectomycorrhizal Fungal Sclerotia as Sources . . .

99

Table 6.1 (continued)

Koudonbaru (weed vegetation)

TotalC (%) 41.7

TotalN (%) 0.44

C/N ratio 95

Saccharide content (g kg
1) 371

Saccharide-C/ plant-C (%) 35.7

Molar ratio of GM/AX 0.070

Ratio of EH-Glc/ NEH-Glc 0.21

a

Banno (2010) Murayama (1980) c Sugiura (2010) d Sariyildiz and Anderson (2005). Saccharides were analyzed by 4M TFA hydrolysis and GLC. Green l. green leaves, leaf l. leaf litters. Average data were calculated by the present author from the original data obtained by analysis of samples collected from 13 to 25 sites (n) with different soil types b

the latter were 150 and 190 cm, respectively. Flower petals were separated off from these samples during air-drying. Paddy rice and barley were mature whole top after threshing. Leaf and needle tissue accounts for a great proportion in total above-ground litter fall, 74, 81, and 62% for temperate deciduous broad-leaved forest, temperate coniferous forest, and subalpine coniferous forest of Japan, respectively (Tsutsumi 1973). The contribution of herbaceous vegetation to litter fall amounts to less than 5% in forests of the temperate zone (Kögel-Knabner 2002). The predominant saccharides of herbaceous plant materials sampled were NEH-glucose and xylose (Fig. 6.2). The proportion of EH-glucose was much smaller than xylose. Galactose and mannose constituted only small proportion, 1.3 and 0.98% in average, respectively. In comparison to herbaceous plants, the three coniferous trees sampled contained a larger proportion of galactose, mannose, and arabinose (Fig. 6.2). The fallen trunk of coniferous tree contained a larger proportion of NEH-glucose than the shoots of other coniferous species. Mannose of the tree trunk tended to occupy larger proportion than that of other three coniferous shoots. Some portion of the mannose may originate from microorganisms including wood-rotting fungi, as the trunk had been weathered. Leaf litter of deciduous species, Japanese white birch, contained a much smaller proportion of mannose than shoots of the coniferous trees. The hemicelluloses of broad-leaved trees, grasses, and herbs contained much smaller proportion of mannose across different tissues (leaves, sapwood, bark, roots) than hemicelluloses of needles, sapwood, and bark of conifer (Shädel et al. 2010). The proportion of xylose was vice versa of mannose. The coniferous trees showed higher GM/AX ratio than the herbaceous plant (Table 6.1). The GM/AX ratio of the weathered coniferous trunk was exceptionally high. The EH-glucose/NEH-glucose ratio of plant materials including a mixture of plant remains and plant roots collected from a soil sample was NEH-glucose ’ xylose > arabinose > mannose ’ galactose > rhamnose > fucose > ribose (’means no significant difference). (3) Average proportion of monosaccharide of two groups of forest soil and forest-derived arable soil: EH-glucose > mannose > galactose > xylose > arabinose > NEH-glucose > rhamnose ’ fucose > ribose

In contrast to xylose, the larger proportion of soil arabinose than that of herbaceous plant materials may be attributed in part to difference of decomposability between these pentoses of plant materials. During the second stage after the flush of decomposition in a 36 months agricultural field experiment, arabinose of two herbaceous species, paddy rice and barley straw were decomposed by a slower rate than xylose (Murayama 1984a). For instance, 27% of arabinose in rice straw was decomposed by half-life time of 36.6 month, in contrasting to 19.6 month of 22% of xylose of the same rice straw.

102

S. Murayama and Y. Sugiura

Fig. 6.4 Molar ratio of galactose + mannose (GM) to arabinose + xylose (AX) of soil. Open square: Prairie and grassland soil (non-volcanic ash, n ¼ 2) (Folsom et al. 1974). Filled square: Arable soil developed from prairie and grassland (non-volcanic ash, n ¼ 9) (Oades et al. 1970; Oades and Wagner 1971; Folsom et al. 1974; Whitehead et al. 1975). Open circle: Arable soil developed from non-forest (non-volcanic ash, n ¼ 10) (Murayama 1977b, 1980, 1985, 1988). Filled circle: Arable soil developed from non-forest (riverrine muck, n ¼ 1) (Murayama 1977b). Open diamond: Forest soil (volcanic ash, n ¼ 3) (Murayama 1980; Sugiura et al. 2017). Filled diamond: Arable soil developed from forest (volcanic ash, n ¼ 6) (Murayama 1980, 1985). Open triangle: Forest soil (non-volcanic ash, n ¼ 5) (Folsom et al. 1974). Filled triangle: Arable soil developed from forest (non-volcanic ash, n ¼ 3) (Folsom et al. 1974). Statistical test, excluding soil group of open square and soil of filled circle as datum of the former was two of large difference (0.99 and 1.4), and the number of data of the latter was only one, performed as: open diamond (average ratio; 1.86) ’ filled diamond (1.80) > open triangle (1.50) > filled triangle (1.17) > filled square (0.908) ’ open circle (0.846) (’ means no significant difference)

Decline of radioactivity of xylose of 14C-labeled cereal rye straw incubation for 5 years was fitted to two compartment kinetic model (Cheshire et al. 1988). While, the decline of that of arabinose was fitted to three compartment model, and the third fraction, i.e., the lowest decline rate fraction (16%) declined by slower rate than the second fraction, i.e., the lowest decline rate fraction (19%) of xylose. In addition, it was not herbaceous plants, but content of arabinose of the mixture of deciduous tree litter mixed with brown forest soil was declined only 7.4% by laboratory incubation of 1200 days, in making big contrast to 71.4% decline of xylose (Sowden and Ivarson 1962). An experiment of 56 days incubation which was carried out as a control series (without 13C-labeled glucose) of 13C-glucose transformation experiment showed that arabinose was decomposed by smaller extent than xylose, across three soils of

6 Origin of Soil Polysaccharides, and Ectomycorrhizal Fungal Sclerotia as Sources . . .

103

Fig. 6.5 Relationship between the ratio of easily hydrolysable (EH) glucose to non-easily hydrolysable (NEH) glucose and molar ratio of galactose + mannose (GM) to arabinose + xylose (AX) of soil. Filled square: Arable soil developed from prairie and grassland (non-volcanic, n ¼ 3) (Oades et al. 1970; Oades and Wagner 1971). Open circle: Arable soil developed from non-forest (non-volcanic ash, n ¼ 7) (Murayama 1980, 1985, 1988). Open diamond: Forest soil (volcanic ash, n ¼ 3) Murayama 1980; Sugiura et al. 2017). Filled diamond: Arable soil developed from forest (volcanic ash, n ¼ 6) (Murayama 1980, 1985). Statistical test on the ratio of EH-glucose/NEHglucose; filled diamond (average 4.7) ’ open diamond (4.6) > filled square (1.5) ’ open circle (1.4) (’ means no significant difference) (cf. Fig. 6.4 on molar ratio of GM/AX)

quite different soil properties (Iwami, Hiratsuka and Utsunomiya soils (Murayama 1988). These studies suggest that there might be some essential difference in microbial decomposition process between arabinose and xylose of plant materials. Two classes of enzymes are responsible for release of the xylose residues from xylan structure of plant materials, endoxylanases, and ß-xylosidases (Khosravi et al. 2015). On the other hand, the complete release of arabinose residues from hemicelluloses and pectin requires the concerted action of four arabinanolytic enzymes; endoarabinanase, exoarabinanase, α-L-arabinofuranosidases, and arabinoxylan arabinofuranohydrolase (Seiboth and Metz 2011; Khosravi et al. 2015). Furthermore, microbial catabolic pentose pathway of L-arabinose requires additional composite enzyme system to that of D-xylose (Nevoigt 2008: Khosravi et al. 2015). These suggest that arabinose is less-preferred carbon source than xylose for microroganisms. Derrien et al. (2006, 2007) reported that the turnover of arabinose in particulate organic matter in a cultivated soil is slower than that of xylose and glucose. The more recalcitrant nature to microbial consumption of arabinose than xylose of plant materials may contribute to accumulation of arabinose, hence to a much

104

S. Murayama and Y. Sugiura

larger proportion of arabinose in the monosaccharide composition of non-forest soil than that of herbaceous plant materials. In comparison to 4 woody plant materials except for a fallen trunk of Maries fir (Fig. 6.2), forest soil and forest-derived arable soil contain a significantly much larger proportion of mannose and fucose, and a significantly much smaller proportion of NEH-glucose (Fig. 6.3). Proportion of EH-glucose (average (av); 30.8%) and rhamnose (av; 14.3%) of soil is significantly larger than EH-glucose (av; 5.2%) and rhamnose (av; 2.5%) of woody plant materials at p ¼ 0.065 and p ¼ 0.10, respectively. Other sugars are not significantly different between soil and plant materials. Mannose of needles of coniferous white-pine in Podzol soil was decomposed by much larger rate (51%) than arabinose (26%) and xylose (37%) by 1200 days incubation experiment (Sowden and Ivarson 1962). This suggests that in comparison to pentose, mannose of coniferous litter may not be particularly recalcitrant in soil. Accordingly, the monosaccharide composition suggests that EH-glucose, mannose, fucose, and rhamnose can be synthesized by microorganism in forest soil and forestderived arable soil. Forest soil showed a larger ratio of GM/AX and EH-glucose/NEH-glucose than plant materials except for the fallen trunk of the coniferous tree (Fig. 6.4 vs. Table 6.1, and Fig. 6.5 vs. Table 6.1, respectively). In summary, comparison of the monosaccharide composition between soils and plant materials suggests that EH-glucose, mannose, fucose, and rhamnose might be synthesized by microbial communities in both forest and non-forest soils. Microbial synthesis of galactose is suggested only in non-forest soil. The comparison procedure cannot confirm in principle whether any sugar cannot be synthesized by soil microbial community.

6.3.2

Identification of Neutral Saccharides Synthesized in Soil by Tracer Techniques

Studies in which soil is incubated with 14C- and 13C- labeled compounds such as glucose have shown that the soil microbial community can synthesize all common soil saccharides. However, soil microbes are unable to synthesize arabinose and xylose in comparable amounts to galactose, glucose, and mannose (Cheshire et al. 1969, 1973, 1978; Oades and Wagner 1971; Murayama 1988; Derrien et al. 2007: Basler et al. 2015). The composition of neutral monosaccharides synthesized in soil under the same incubation conditions differ among soils. Figure 6.6 shows the transformation of 13C from 13C-labeled glucose into sugars after 56 days of incubation in the dark at 28  C (Murayama 1988). The relative proportion of arabinose and xylose is smaller than those of galactose, EH-glucose, mannose, fucose, and rhamnose across three soils. This is consistent with the results of many other studies using labeled substrates. However, differences in soil properties can cause some differences. The large

6 Origin of Soil Polysaccharides, and Ectomycorrhizal Fungal Sclerotia as Sources . . .

105

Fig. 6.6 Proportion of amount of 13C of each saccharide in total 13C of all saccharides after incubation with 13C-labeled glucose for 56 days (Murayama 1988) (asterisk C: soil carbon content)

proportion of NEH-glucose synthesis in Utsunomiya soil than in the other two soils is notable. Growth of filamentous fungi was visible to the unaided eye at the soil surface only of Utsunomiya soil (Murayama 1988). Fungal cell wall contains branched ß (1,3) (1,6) glucan to linked chitin as the central core of the cell wall (Latgé 2007; Osherov and Yarden 2010; Fesel and Zuccaro 2016), and pretreatment with strong acid is needed to release glucose (Dallies et al. 1998; Francois 2006; Won et al. 2014). Accordingly, the 13C-labeled glucose having non-easily hydrolysable linkage (NEH-glucose) is considered to be synthesized by the filamentous fungi that thrive in this strongly acidic (pH 5.0) soil. Along with soil properties such as pH, incubation conditions also affect the composition of microbially synthesized polysaccharides in soil. Incubation at low temperature (5  C) of 14C-labeled glucose with air-dried and remoistened soil showed that yeast species synthesize xylose in similar quantities to galactose and mannose (Cheshire et al. 1978). However, incubation at 20  C did not result in such synthesis of xylose, as yeast did not thrive. Incubation at 5  C using with fresh, not pre-dried soil did not result in such synthesis of xylose, either. Polysaccharides that contained xylose at nearly similar proportion to mannose were synthesized in soil by yeast under conditions in which no other microbes could grow (Sparling et al. 1981). Cheshire et al. (1978) summarized that synthesis of xylose-rich polysaccharides by

106

S. Murayama and Y. Sugiura

yeast in soil may not be common in natural environment as the combination of conditions that permit xylose synthesis by yeasts in pre-dried soil are unlikely to occur naturally. In summary, the neutral saccharide composition of microbially synthesized polysaccharides in soil depends on which microbial species thrive with addition of a given substrate, as well as how these microbes are affected by both soil properties such as acidity and incubation conditions such as temperature and pretreatment of soil. Incubation experiments using tracer techniques have shown that the polysaccharides synthesized in soil by microorganisms are dominated by hexoses rather than pentoses.

6.4

Accumulation of Microbial Polysaccharides in Forest Soil

The monosaccharide composition of forest soil and forest-derived arable soil differs from that of non-forest soil (Fig. 6.3). Figure 6.3 does not include forest soil with an origin other than volcanic ash, as no data is available. The proportion of EH-glucose and mannose in forest soil and forest-derived arable soil is significantly larger than in non-forest soil, including prairie and grassland soil. The proportions of NEH-glucose, arabinose, and xylose are the opposite. Proportions of galactose and rhamnose have no significant difference between forest and forest derived arable soil and non-forest soil. The monosaccharide composition is illustrated by the molar ratio of GM/AX (Fig. 6.4). Forest soil and forest-derived arable soil have a higher GM/AX ratio than non-forest soil, irrespective of parent materials. The data for forest soil and forestderived arable soil of volcanic ash origin are biased towards those with a high SOM content, but non-volcanic ash forest soil with a low SOM content has also a significantly higher molar ratio of GM/AX than non-forest soil (Fig. 6.4). Then, monosaccharide composition tends to differ between forest soils including forestderived arable soils and non-forest soils. In comparison to non-forest soil, forest soil and forest-derived arable soil contain more microbial polysaccharide than plant polysaccharides. Forest soil and forest-derived arable soil of volcanic ash have a significantly higher GM/AX ratio than forest soil of non-volcanic ash (Fig. 6.4). This suggests the former soil may have accumulated more abundantly microbial polysaccharides than the latter soil. This difference can be ascribed in part to a larger organic matter accumulative ability of volcanic ash soil than that of non-volcanic ash soil (Murayama 1980). A field experiment on decomposability of indigenous soil saccharides by glass fiber bag method for 2 and/or 3 years showed that galactose, EH-glucose, fucose, and rhamnose in forest-derived arable soil of volcanic ash are more stable (Murayama 1984b) than those components in arable soil of non-volcanic ash (Murayama 1981).

6 Origin of Soil Polysaccharides, and Ectomycorrhizal Fungal Sclerotia as Sources . . .

107

Arable soil developed from prairies and grassland had a significantly lower GM/AX ratio than forest and forest-derived arable soil, irrespective of SOM content (Fig. 6.4). These soils may contain fewer microbial polysaccharides relative to plant polysaccharides than forest and forest-derived arable soils. We observed a positive relationship between the ratio of EH-glucose/NEHglucose and the molar ratio of GM/AX (Fig. 6.5). In both herbaceous and woody plant materials, these ratios are low (Table 6.1), indicating that the higher these ratios, the lower the relative abundance of plant-derived polysaccharides in soil. Woody plant materials contain galactose and mannose by considerable proportion (Fig. 6.2, Shädel et al. 2010). But, contribution of galactose and mannose from woody plants to forest soil saccharides, if any might be small. If the original polysaccharides in woody plant materials are directly, without decomposition by microbes, remaining in soil and make up a large part of forest soil polysaccharides, forest soil should have large proportion of NEH-glucose and small EH-glucose/ NEH-glucose ratio. But, these features were not observed in the monosaccharide composition of forest soils (Figs. 6.3, 6.4 and 6.5). Most of plant litter enters the soil above ground in forest soil, where it forms a humus layer. There, carbohydrates are preferentially mineralized in course of humification, thus organic matter entering mineral soil is depleted in carbohydrates (Kögel-Knabner et al. 1988; Guggenberger et al. 1994). Thus, forest soil and forest-derived arable soil, irrespective of soil parent materials and SOM content, accumulated more microbial polysaccharides than non-forest soil. Though, immature forest soils of sand dune and high elevation forest of creeping pine zone are not included in this conclusion, as SOM of these forest soils are mostly constituted of woody plant debris.

6.5

ECM Fungal Sclerotia as Sources of Forest Soil Polysaccharides

Ectomycorrhizal fungi are ubiquitous in forest ecosystems and form a vital component of the plant-soil interface by forming mutualistic mycorrhizal association with vascular plants, mostly woody plants. A range of ECM genera, including Cenococcum, Cortinarius, Entroma, Hebeloma, Boletus, Leccinum, Gyrodon, Paxillus, Pisolithus, Scleroderma, and Austropaxillus, form hypogenous dormant propagules called sclerotia (Massicotte et al. 1992; Smith et al. 2014) as referred by Sugiura et al. (2017). Cenococcum geophilum forms abundant black spherical sclerotia (Trappe 1969; Grenville et al. 1985; Massicotte et al. 1992).

108

6.5.1

S. Murayama and Y. Sugiura

Monosaccharide Composition of a Forest Soil on Mt. Ontake from Where ECM Fungal Sclerotium Grains Were Collected

ECM fungal sclerotia were collected from a subalpine forest soil in Yunohana pass (2103 m a.s.l.) on Mt. Ontake, a volcano in central Japan at the beginning of August 2008 (Sugiura et al. 2017). The soil is acidic and rich in organic matter (Table 6.2). Soil of all three horizons has a monosaccharide composition characteristic of forest soils, namely a high molar ratio of both GM/AX and EH-glucose/NEH-glucose, indicating that the soil has accumulated microbial polysaccharides (Table 6.2). The O horizon was classified according to the soil taxonomy (Soil Survey Staff 2010) by carbon content, and it is neither a plant litter layer nor an undergrowth layer.

6.5.2

Neutral Saccharides of ECM Fungal Sclerotia

Histochemical analysis has shown that fresh sclerotia produced by pure cultures of ECM fungi contained polysaccharides (Grenville et al. 1985; Massicotte et al. 1992). However, the content and composition of neutral saccharides in ECM fungal sclerotia collected from forest soil were unknown until recently (Sugiura et al. 2017).

6.5.2.1

Collection of Sclerotium Grain from Soil Sample

Sclerotia of different diameter sizes of 0.20–1.0 mm, >1.0–2.0 mm, >2.0 mm were collected by a wet sieving method (Nonoyama 2010) as described previously (Sugiura et al. 2017). The wet sieving method cannot separate viable and dead sclerotium, so the sclerotia collected are designated as sclerotium grains (SG). The SG were tentatively classified as C.geophilum (Sugiura et al. 2017). The weight of SG > 0.2 mm collected from the O, A1, and A2 horizons was 3.3, 0.91, and 0.62 g kg
1 soil, respectively. The 14C ages of SG collected from the O, A1, and A2 horizons were inferred to be approximately 499–831, 1002–1698, and 2121–2798 yr. BP, respectively (Tonosaki et al. (2007, 2008). These SG were collected from the soil profile very close to that of Sugiura et al. (2017).

6.5.2.2

Content and Composition of Neutral Saccharides in Sclerotium Grains

The neutral saccharides of the SG were determined using the same procedure described for soil (Sugiura et al. 2017). Neutral saccharides were substantial organic constituent, accounting for 6.0–16% of SG by carbon content (Table 6.2). The SG from the deepest A2 horizon, with a 14C ages >2000 yr. BP still contained as much as 6.0–6.6% saccharides by carbon content.

6 Origin of Soil Polysaccharides, and Ectomycorrhizal Fungal Sclerotia as Sources . . .

109

Table 6.2 Soil properties and neutral saccharide composition of sclerotium grains (SG) collected from soil in Yunohana pass, Mt. Ontake (adapted from Sugiura et al. 2017) Soil properties Soil horizon O A1 A2 Soil depth 0–13 13
28 28
55 (cm) Soil pH (H2O) 3.8 4.6 4.7 C content 273 127 108 (g kg
1 soil) N content 14.1 4.24 3.38 (g kg
1 soil) Soil saccharides; content and monosaccharide composition by molar ratio Saccharide-C/ 10 8.6 7.8 Soil-C (%) (Gal + Man)/ 1.6 1.6 1.5 (Ara + Xyl) (EH-glucose/ 3.1 3.5 4.9 NEH-glucose) Sclerotium grain (SG) Size of SG >2 >1
2 0.2
1 >2 >1
2 0.2
1 >2 (mm) Content of SG 0.93 1.5 0.90 0.17 0.27 0.47 0.088 (g kg
1 soil) 553 562 550 446 435 462 466 C content (g kg
1 SG) N content 11.1 12.1 16.4 9.18 8.58 9.08 9.18 (g kg
1 SG) Saccharide-C/ 16 14 12 10 7.4 7.1 6.0 SG-C (%) Monosaccharide composition of SG by molar ratio (Gal + Man)/ 15 15 25 10 10 12 9.0 (Ara + Xyl) (17)a (11) (EH-glucose/ 13 14 14 51 39 37 37 NEH-glucose) (14) (41)

>1
2

0.2
1

0.19

0.34

410

440

7.68

9.16

6.3

6.8

7.5 (11) 40 (36)

14 34

(cf. Fig. 6.7) Ara arabinose, Xyl xylose, Gal galactose, Man mannose, EH-glucose easily hydrolysable glucose, NEH-glucose non-easily hydrolysable glucose a Average of each horizon

The SG contained predominantly EH-glucose which account for an average of 79–80% of SG from each horizon, followed by mannose (9.4–12%), NEH-glucose (2.2–5.8%), and galactose (3.2–4.0%). These four components comprised more than 97% of the total (Fig. 6.7). Although they differed in 14C age by approximately 1000 yr., SG from the A1 and A2 horizons showed similar saccharide content and composition (Fig. 6.7, Table 6.2), suggesting that SG polysaccharides persist for a long period of time. In addition, the SG collected from these two subsurface horizons, irrespective of size,

110

S. Murayama and Y. Sugiura

Fig. 6.7 Monosaccharide composition of ectomycorrhizal fungal sclerotium grains (SG) by grain size and soil horizon (Sugiura et al. 2017). EH-glucose easily hydrolysable glucose, NEH-glucose non-easily hydrolysable glucose. Numerical values of major four components are average molar percent of the soil horizon calculated by taking the weight content of SG of different sizes into account

6 Origin of Soil Polysaccharides, and Ectomycorrhizal Fungal Sclerotia as Sources . . .

111

retained all of the major components found in SG from the O horizon, with similar relative abundance. This indicates that SG plays a role as reservoirs of polysaccharides (Sugiura et al. 2017). The differences in the content and composition of SG saccharides between the O and A1 horizons were ascribed in part to the greater relative abundance of viable sclerotia in the O horizon. The molar ratio of GM/AX of SG saccharides ranged from 7.5 to 25 and was much higher than that of matrix soil (Table 6.2). The ratio of EH-glucose/NEHglucose was also much larger than that of matrix soil. Thus accumulation of SG saccharides has contributed to increase these two ratios of forest soil. ECM fungal sclerotia were not observed to incorporate roots or root hairs, although they were formed very close to pine roots (Grenville et al. 1985). Therefore NEH-glucose was inferred to be released not from plant cellulose but from the complex fungal cell wall polysaccharides comprised from ß (1, 3) (1, 6) glucan linked to chitin (cf. Sect. 6.3.2) (Sugiura et al. 2017) The NEH-glucose in soil hydrolysates has been considered to originate from plant cellulose. However, SG is also a source of the NEH-glucose in forest soil hydrolysates. In addition, the saccharides determined in SG may originate also in part from non-ECM fungal saccharides besides SG saccharides (Sugiura et al. 2017). As old and/or dead SG provide a habitat for bacteria (Ohta et al. 2003) and fungi (Obase et al. 2014; Amasya et al. 2015).

6.5.2.3

Quantitative Comparison of Saccharides in ECM Fungal SG to Those in Whole Soil: A Case Study

The total sugar components and EH-glucose in SG from O horizon accounted for 1.9% and 3.6% of those in the soil, respectively (Table 6.3). These results may apply only to the soil at the sampling site in the summer, as the contents of ECM fungal SG differ among forests (Watanabe et al. 2004; Sakagami 2011) and fluctuate with the seasons (Vogt et al. 1981; Lussenhop and Fogel 1999; Sakagami 2011). No comparable data which determined proportion of neutral saccharides of biomass or biomass remains collected from soil as specified microbial species or groups, such as ECM fungal sclerotia in this study, to soil neutral saccharides is known. Although the proportion of SG saccharides to soil ones appears to be small (Table 6.3), we consider it is substantial. As ECM fungal SG are produced by a limited number of species of the vast number of microbial species inhabiting forest soil. It should also be noted that this proportion was obtained only for SG larger than 0.2 mm, although forest soil also contains smaller SG. Trappe (1969) reported live sclerotia vary from 0.05 to 4 or more mm in diameter.

112

S. Murayama and Y. Sugiura

Table 6.3 Proportions of each saccharide component of sclerotium grains (SG) to that of soil, as percent (Sugiura et al. 2017) Soil horizon Galactose EH-glucose NEH-glucose Mannose Arabinose Ribose Xylose Fucose Rhamnose Sum

O 0.30 1.9 0.43 0.55 0.074 0.22 0.022 0.028 0.14 3.6

A1 0.086 0.62 0.081 0.18 0.035 0.087 0.011 0.012 0.053 1.2

A2 0.075 0.45 0.062 0.14 0.023 0.037 0.0091 0.0072 0.031 0.83

(cf. Table 6.2) EH-glucose easily hydrolysable glucose, NEH-glucose non-easily hydrolysable glucose

6.5.3

Production and Stability in Soil of ECM Fungal Sclerotial Polysaccharides

A large proportion of the total net primary production of the host plant allocated to total mycorrhizal fungal components, namely sclerotia, mycorrhizal sheath, and epigenous and hypogenous sporocarps, is distributed to sclerotia (Dahlberg et al. 1997; Vote et al. 1982). Biomass of sclerotia produced by C.geophyum in a Pacific silver fir stand was estimated to be 2700 kg ha
1 year
1 (Vogt et al. 1982). ECM fungal sclerotia persist in soil for several years forming a reservoir of fungal inocula (Lo Buglio 1999), some of which, even in low numbers persist for a long time as coherent organic bodies (Watanabe et al. 2007b: Tonosaki et al. 2007, 2008). ECM fungal sclerotia have a tough melanized rind (Massicotte et al. 1992) and a minute honeycomb structure inside (Watanabe et al. 2004). These features seem to make the grain tough and resistant, thereby hindering decomposition by other microbes. Furthermore, the SG contains considerable amounts of metal elements such as Al and Zn (Watanabe et al. 2001, 2002, 2007a), which may stabilize polysaccharides by forming chemical complexes (Cheshire 1979). Thus, the high rate of production of sclerotia and persistent nature of SG result in accumulation of SG poysaccharides in forest soil. Sclerota-forming ECM fungal species such as C.geophilum may be key sources of forest soil polysccharides (Sugiura et al. 2017).

6 Origin of Soil Polysaccharides, and Ectomycorrhizal Fungal Sclerotia as Sources . . .

6.6

113

Origins of Forest Soil Polysaccharides

Forest soil contains accumulated microbial polysaccharides, largely producted by fungi and bacteria, the two largest functional microbial subgroups in soil. Fungal polysaccharides should form a larger component than bacterial polysaccharides, as the fungal component of the total microbial biomass is greater than the bacterial component in forest soil (Ananyeva et al. 2006). Furthermore, soils from temperate forests are overwhelmingly dominated by taxa belonging to ECM fungi rather than endomycorrhizal or non-mycorrhizal fungi (Shi et al. 2014). Schweigert et al. (2015) reported that ECM fungi comply a large proportion (80%) of fungal biomass, and up to 30% of total soil microbial biomass in forest soil. Consequently, ECM fungal polysaccharides including sclerotial polysaccharides are the predominant sources of microbial polysaccharides in forest soil. Our discussion focused on SG as they persist and accumulate in soil, and because they represent as collectable biomass of ECM fungi. The monosaccharide composition of forest-derived arable soil where woody plants no longer grow suggested an accumulation of microbial polysaccharides. Some portion of the saccharides accumulated in forest-derived arable soil may originate from ECM fungal SG produced when the soil was under forest cover. We have focused on ECM fungal SG as the origin of forest soil polysaccharides. Other functions of this small but many-formed and accumulative organic body in soil processes, such as below-ground carbon dynamics in forest ecosystem might be considerable under global warming.

References Amasya AF, Narisawa K, Watanabe M (2015) Analysis of sclerotia-associated fungal communities in cool-temparate forest soils in North Japan. Microbes Environ 30:113–116. https://doi.org/10. 1264/jsme2.ME:14135 Amelung W, Flach KW, Zech W (1999a) Neutral and acidic sugars in particle-size fractions as influenced by climate. Soil Sci Soc Am J 63:865–873. https://doi.org/10.2136/sssaj1999. 634865x Amelung W, Zhang X, Flach KW, Zech W (1999b) Amino sugars in native grassland soils along a climosequence in North America. Soil Sci Soc Am J 63:86–92. https://doi.org/10.2136/ sssaj1999.03615995006300010014x Ananyeva ND, Susyan EA, Chernova OV, Chernova IY, Makarova OL (2006) The ratio of fungi and bacteria in the biomass of different types of soil determined by selective inhibition. Microbiology 6:702–707. https://doi.org/10.1134/S0026261706060130 Atsumi S (2008) Decomposition kinetics of plant materials in sandy soil and biosynthesis of microbial saccharides under upland field conditions. Master thesis. Graduate School of Agricultural Science, Meijo university. Nagoya, Japan. (in Japanese) Banno A (2010) Decomposition of wild plant residue and production of neutral saccharide in heavy clayley soil under field conditions. Master thesis. Graduate School of Agricultural Science, Meijo university. Nagoya, Japan. (in Japanese)

114

S. Murayama and Y. Sugiura

Basler A, Dippold M, Helfrich M, Dyckmans J (2015) Microbial carbon recycling—an underestimated process controlling soil carbon dynamics—part 1: a long-term laboratory incubation experiment. Biogeosciences 12:5929–5940. https://doi.org/10.5194/bg-12-59292015 Benzing-Purdie L (1984) Amino sugar distribution in four soils as determined by high resolution gas chromatography. Soil Sci Soc Am J 48:219–222. https://doi.org/10.1236/sssaj1984. 03615995004800010039x Benzing-Purdie L, Nikiforuk JH (1988) Determination of glucuronic and galacturonic acids in soils by high-performance gas chromatography. Soil Sci 145:264–269 Cheshire MV (1979) Nature and origin of carbohydrates in soils. Academic Press, London Cheshire MV, Mundie CM, Shepherd H (1969) Transformation of 14C glucose and starch in soil. Soil Biol Biochem 1:117–130. https://doi.org/10.1016/0038-0717(69)90002-9 Cheshire MV, Mundie CM, Sheperd H (1971) The origin of the pentose fraction of soil polysaccharide. J Soil Sci 22:222–236. https://doi.org/10.1111/j.1365-2389.1971.tb01609.x Cheshire MV, Mundie CM, Shepherd H (1973) The origin of soil polysaccharide: transformation of sugars during the decomposition in soil of plant material labelled with 14C. J Soil Sci 24:54–68. https://doi.org/10.1111/j.1365-2389.1973.tb00741.x Cheshire MV, Sparling GP, Mundie CM, Shepherd H, Murayama S (1978) Effect of temperature and soil drying on the transformation of (14C) glucose in soil. J Soil Sci 29:360–366. https://doi. org/10.1111/j.1365-2389.1978.tb00784.x Cheshire MV, Sparling GP, Mundie CM (1983) Effect of periodate treatment of soil on carbohydrate constituents and soil aggregation. J Soil Sci 34:105–112. https://doi.org/10.1111/j.13652389.1983.tb00817.x Cheshire MV, Sparling GP, Mundie CM (1984) Influence of soil type, crop and air drying on residual carbohydrate content and aggregate stability after treatment with periodate and tetraborate. Plant and Soil 76:339–347. https://doi.org/10.1007/BF02205591 Cheshire MV, Inkson RHE, Mundie CM, Sparling GP (1988) Studies on the rate of decomposition of plant residues in soil by following the changes in sugar components. J Soil Sci 39:227–236. https://doi.org/10.1111/j.1365-2389.1988.tb01209.x Dahlberg A, Jonsson L, Nylund J-E (1997) Species diversity and distribution of biomass above and below ground among ectomycorrhizal fungi in an old-growth Norway spruce forest in South Sweden. Can J Bot 75:1323–1335. https://doi.org/10.1139/b97-844 Dallies N, Francois J, Paquet V (1998) A new method for quantitative determination of polysaccharides in yeast call wall. Application to the cell wall defective mutants of Saccharomyces cervesiae. Yeast 14:1297–1306. https://doi.org/10.1002/(SICI)1097-0061(1998100) 14:143.0.CO;2-L Derrien D, Marol C, Balabane M, Balesdent J (2006) The turnover of carbohydrate carbon in a cultivated soil estimated by 13C natural abundances. Eur J Soil Sci 57:547–557. https://doi.org/ 10.1111/j.1365-2389.2006.00811.x Derrien D, Marol C, Balesdent J (2007) Microbial biosyntheses of individual neutral sugars among sets of substrates and soils. Geoderma 139:190–198. https://doi.org/10.1016/j.geoderma.2007. 01.017 Fesel PH, Zuccaro A (2016) β-Glucan: crucial component of the fungal cell wall and elusive MAMP in plants. Fungal Genet Biol 90:53–60. https://doi.org/10.1016/j.fgb.2015.12.004 Folsom BL, Wagner GH, Scrivner CL (1974) Comparison of soil carbohydrate in several prairie and forest soils by gas-liquid chromatography. Soil Sci Soc Am Proc 38:305–309. https://doi. org/10.2136/sssaj1974.03615995003800020027x Francois JM (2006) A simple method of quantitative determination of polysaccharides in fungal cell walls. Nat Protoc 1:2995–3000. https://doi.org/10.1038/nprot.2006.457 Gow NAR, Latgé J-P, Munro CA (2017) The fungal cell wall: structure, biosynthesis, and function. Microbiol Spectrum 5(3):1–25. https://doi.org/10.1128/microbiolspec.FUNK0035-2016

6 Origin of Soil Polysaccharides, and Ectomycorrhizal Fungal Sclerotia as Sources . . .

115

Grenville DJ, Peterson RL, Piché Y (1985) The development, structure, and histochemistry of sclerotia of ectomycorrizhal fungi. II Paxillus involutus. Can J Bot 63:1412–1417. https://doi. org/10.1139/b85-195 Guggenberger G, Christensen BT, Zech W (1994) Land-use effects on the composition of organic matter in particle-size separates of soil: I. Lignin and carbohydrate signature. Eur J Soil Sci 45:449–458. https://doi.org/10.1111/j.1365-2389.1994.tb00530.x Hoch G (2007) Cell wall hemicelluloses as mobile carbon stores in non-reproductive plant tissues. Funct Ecol 21:821–834. https://doi.org/10.1111/j.1365-2435.2007.01305.x Khosravi C, Benocci T, Battaglia E, Benoit I, de Vries RP (2015) Sugar catabolism in Aspergillus and other fungi related to the utilization of plant biomass. Adv Appl Microbiol 90:1–28. https:// doi.org/10.1016/bs.aambs.2014.09.005 Kögel-Knabner I (2002) The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter. Soil Biol Biochem 34:139–162. https://doi.org/10.1016/500380717(01)00158-4 Kögel-Knabner I, Zech W, Hatcher PG (1988) Chemical composition of the organic matter in forest soils: the humus layer. Z Pflanz Bodenkunde 151:331–340. https://doi.org/10.1002/jpln. 19881510512 Latgé J-P (2007) The cell wall: a carbohydrate armour for the fungal cell. Mol Microbiol 66:279– 290. https://doi.org/10.1111/j.1365-2958.2007.05872.x Lo Buglio KF (1999) Cenococcum. In: Cairney JWG, Chambers SM (eds) Ectomycorrhizal Fungi: key genera in profile. Springer, Berlin, Heidelberg, pp 287–309 Lussenhop J, Fogel R (1999) Seasonal change in phosphorus content of Pinus strobes-Cenococcum geophilum ectomycorrhizae. Mycologia 91:742–746. https://doi.org/10.1080/00275514.1999. 12061079 Massicotte HB, Trappe JM, Peterson RL, Melville LH (1992) Studies on Cenococcum geophilum. II. Sclerotium morphology, germination, and formation in pure culture and growth pouches. Can J Bot 70:125–132. https://doi.org/10.1139/b92-017 Murayama S (1977a) An automated anion-exchange chromatographic procedure for the estimation of saccharides in acid hydrolysates of soil. Soil Sci Plant Nutr 23:247–252. https://doi.org/10. 1080/00380768.1977.10433042 Murayama S (1977b) Saccharides in some Japanese paddy soils. Soil Sci Plant Nutr 23:479–489. https://doi.org/10.1080/00380768.1977.10433066 Murayama S (1980) The monosaccharide composition of polysaccharides in Ando soils. J Soil Sci 31:481–490. https://doi.org/10.1111/j.1365-2389.1980.tb02097.x Murayama S (1981) Persistency and monosaccharide composition of polysaccharides of soil which received no plant materials for a certain period under field conditions. Soil Sci Plant Nutr 27:463–475. https://doi.org/10.1080/00380768.1981.10431302 Murayama S (1984a) Decomposition kinetics of straw saccharides and synthesis of microbial saccharides under field conditions. J Soil Sci 35:231–242. https://doi.org/10.1111/j.13652389.1984.tb00279.x Murayama S (1984b) Origins, decomposability and composition of soil saccharides, and soil microorganisms. In: Wada H, Tsuru S (eds) Soil biomass - contents and metabolism of soil organisms. Hakuyusha, Tokyo, pp 65–114. (in Japanese) Murayama S (1985) Studies on soil saccharides, particularly on dynamics of neutral saccharides in soil, Doctoral thesis. Department of Agricultural Science, Tohoku University, Sendai, Japan. (in Japanese) Murayama S (1988) Microbial synthesis of saccharides in soils incubated with 13C-labelled glucose. Soil Biol Biochem 20:193–199. https://doi.org/10.1016/0038-0717(88)90036-3 Nevoigt E (2008) Progress in metabolic engineering of Saccharomyces cerevisiae. Microbiol Mol Biol R 72:379–412. https://doi.org/10.1128/MMBR.00025-07 Nonoyama Y (2010) Studies on the microbial community of sclerotium grain found in forest soil and evaluation of the grain as carrier of microorganisms. Doctoral thesis. Department of

116

S. Murayama and Y. Sugiura

Environmental Science and Technology, interdisciplinary graduate School of Science and Engineering, Tokyo Institute of Technology. Tokyo, Japan. (in Japanese) Oades JM (1984) Soil organic matter and structural stability: mechanisms and implications for management. Plant and Soil 76:319–337. https://doi.org/10.1007/BF02205590 Oades JM, Wagner GH (1971) Biosynthesis of sugars in soils incubated with 14C glucose and 14C dextran. Soil Sci Soc Am Proc 35:914–917. https://doi.org/10.2136/sssaj1971. 03615995003500060021x Oades JM, Kirkman MA, Wagner GH (1970) The use of gas-liquid chromatography for determination of sugars extracted from soils by sulfuric acid. Soil Sci Soc Am Proc 34:230–235. https:// doi.org/10.2136/sssaj1970.03615995003400020017x Obase K, Douhan GW, Matsuda Y, Smith ME (2014) Culturable fungi assemblages growing within Cenococcum sclerotia in forest soils. EFMS Microbiol Ecol 90:708–717. https://doi.org/10. 1111/1574-6941.12428 Ohta H, Yagi M, Suzuki J, Fujitake N, Watanabe M (2003) Characterization of Sphingomonas species found as predominant members in the cultural bacterial community of a green pigmentcontaining sclerotium grain from Mt. Myoko (Japan). Microbes Environ 18:126–132. https:// doi.org/10.1264/jsme2.18.126 Osherov N, Yarden O (2010) The cell wall of filamentous funi. In: Borkovich KA, Ebbole DJ (eds) Cellular and molecular biology of filamentous fungi. ASM Press, Washington, DC, pp 224–237. https://doi.org/10.1128/9781555816636 Puget P, Angers DA, Chenu C (1999) Nature of carbohydrates associated with water stable aggregates of two cultivated soils. Soil Biol Biochem 31:55–63. https://doi.org/10.1016/ s0038-0717/(98)00103-5 Sakagami N (2011) Distributional optimum of sclerotia, resting bodies of Cenococcum geophilum in forest soils. In: Geographical Reports of Tokyo Metropolitan University, vol 46, pp 63–72 Sariyildiz T, Anderson JM (2005) Variation in the chemical composition of green leaves and leaf litters from three deciduous tree species growing on different soil types. Forest Ecol Manag 210:303–319. https://doi.org/10.1016/j.foreco.2005.02.043 Schweigert M, Herrmann S, Miltner A, Fester T, Kästner M (2015) Fate of ectomycorrhizal biomass in a soil bioreactor system and its contribution to soil organic matter formation. Soil Biol Biochem 88:120–127. https://doi.org/10.1016/j.soilbio.2015.05.012 Seiboth B, Metz B (2011) Fungal arabinan and L-arabinose metabolism. Appl Microbiol Biotechnol 89:1665–1673. https://doi.org/10.1007/s00253-010-3071-8 Shädel C, Blöchl A, Richter A, Hoch G (2010) Quantification and monosaccharide composition of hemicelluloses from different plant functional types. Plant Physiol Biochem 48:1–8. https://doi. org/10.1016/j.plaphy.2009.09.008 Shi L-L, Mortimer PE, Ferry Slik JW, Zou X-M, Xu J, Feng W-T, Qiao L (2014) Variation in forest soil fungal diversity along a latitudinal gradient. Fungal Divers 64:305–315. https://doi.org/10. 1007/s13225-013-0270-5 Smith ME, Henkel TW, Rollins JA (2014) How many fungi make sclerotia? Fungal Ecol 13:211–220. https://doi.org/10.1016/j.funeco.2014.08.010 Soil Survey Staff (2010) Keys to soil taxonomy, 11th edn. Natural Resources Conservation Service/ USDA, Washington, DC. https://doi.org/10.1007/978-94-007-7420-9 Sowden FJ, Ivarson KC (1962) Decomposition of forest litters, III. Changes in the carbohydrate constituents. Plant and Soil 16:389–400. https://doi.org/10.1007/BF01381348 Sparling GP, Cheshire MV, Mundie CM, Murayama S (1981) The transformation of 14C-labelled glucose in sterilized soil incubated with selected microorganisms. Rev Ecol Biol Sol 18:447–457 Sugiura Y (2010) Studies on saccharides of sclerotium grain in forest soil, Master thesis. Graduate School of Agricultural Science, Meijo University. Nagoya, Japan. (in Japanese) Sugiura Y, Watanabe M, Nonoyama Y, Sakagami N, Guo Y, Murayama S (2017) Saccharides of ectomycorrhizal fungal sclerotia as sources of forest soil polysaccharides. Soil Sci Plant Nutr 63:426–433. https://doi.org/10.1080/00380768.2017.1381928

6 Origin of Soil Polysaccharides, and Ectomycorrhizal Fungal Sclerotia as Sources . . .

117

Tadaki Y (1996) Japanese forest zone. In: Ohta T, Kitamura M, Kumazaki M, Suzuki K, Sudo S, Tadaki Y, Fujimori T (eds) Encycropedia of forest. Maruzen, Tokyo, pp 16–18, and p 27. https://doi.org/10.11519/jjsk.20.0_79_1 Tonosaki K, Matsuzaki H, Kobayashi T, Fujitake N (2007) Persistency of soil sclerotium grain determined by AMS 14C age. In: Watanabe M (ed) Process of formation, development and extinction of sclerotium grains from 14C dating of sclerotium grain and humic acid. Interdisciplinarly graduate School of Science and Engineering, Tokyo Institute of Technology, Yokohama, pp 66–69. (in Japanese) Tonosaki K, Matsuzaki H, Inoue G, Fujitake N, Watanabe M (2008) Carbon loss rate of sclerotium grains by AMS14C dating. In: The Japanese Society for Accelerator Mass Spectorometry Research (ed) Proceedings of the 10th Symp. Japanese AMS Soc., 7th March. Tokyo University, Tokyo, pp 226–233. (in Japanese) Trappe JM (1969) Studies on Cenococcum graniforme. I An efficient method for isolation from sclerotia. Can J Bot 47:1389–1390. https://doi.org/10.1139/b69-198 Tsutsumi T (1973) Material production of terrestrial plant community; Ib,
material circulation of forest
. In: Kitazawa Y, Kira T, Hougetsu K, Morishita M, Moji M, Yamamoto M (eds) Ecology series 5b. Kyouritsu Publishing, Tokyo, pp 1–69. (in Japanese) Vogt KA, Edmonds RL, Grier CC (1981) Dynamics of ectomycorrhizae in Abies Amabilis stands: the role of Cenococcum graniforme. Holarctic Ecol 4:167–173. https://doi.org/10.1111/j.16000587.1981.tb00994.x Vogt KA, Grier CC, Meier CE, Edmonds RL (1982) Mycorrhizal role in net primary production and nutrient cycling in Abies Amabilis ecosystems in western Washington. Ecology 63:370–380. https://doi.org/10.2307/1938955 Watanabe M, Fujitake N, Ohta H, Yokoyama T (2001) Aluminum concentrations in sclerotia from a buried humic horizon of volcanic ash soils in Mt. Myoko, Central Japan. Soil Sci Plant Nutr 47:411–418. https://doi.org/10.1080/00380768.2001.10408404 Watanabe M, Kado T, Ohta H, Fujitake N (2002) Distribution and development of sclerotium grains as influenced by aluminum status in volcanic ash soils. Soil Sci Plant Nutr 48:569–575. https:// doi.org/10.1080/00380768.2002.10409240 Watanabe M, Ohishi S, Pott A, Hardenbicker U, Aoki K, Sakagami N, Ohta H, Fujitake N (2004) Soil chemical properties and distribution of sclerotium grains in forest soils, Harz Mts., Germany. Soil Sci Plant Nutr 50:863–870. https://doi.org/10.1080/00380768.2004.10408547 Watanabe M, Inoue Y, Sakagami N, Bolormaa O, Kawasaki K, Hiradate S, Fujitake N, Ohta H (2007a) Characterization of major and trace elements in sclerotium grains. Eur J Soil Sci 58:786–793. https://doi.org/10.1111/j.1365-2389.2006.00868.x Watanabe M, Sato H, Matsuzaki H, Kobayashi T, Sakagami N, Maejima Y, Ohta H, Fujitake N, Hiradate S (2007b) 14C ages and δ13C of sclerotium grains found in forest soils. Soil Sci Plant Nutr 53:125–131. https://doi.org/10.1111/j.1747-0765.2007.00121.x Whitehead DC, Buchan H, Hartley RD (1975) Components of soil organic matter under grass and arable cropping. Soil Biol Biochem 7:65–71. https://doi.org/10.1016/0038-0717(75)90033-4 Whitfield C, Szymanski CM, Aebi M (2017) Eubacteria. In: Valki A, Cummings RD, Esko JD, Stanley P, Hart GW, Aebi M, Darvill AG, Kinoshita T, Packer NH, Prestegard JH, Schnaar RL, Seeberger PH (eds) Essentials of glycobiology, 3rd edn. Cold Spring Harbor Laboratory Press, New York, pp 265–282. https://doi.org/10.1101/glycobiology.3e.021 Won CB, Park JH, Park YA, Ka HK (2014) Determination of glucan contents in the fruiting bodies and mycelia of Lentinula edodes cultivars. Microbiology 42:301–304. https://doi.org/10.5941/ MYCO.2014.42.3.301

Chapter 7

Dating of Sclerotia Grains in Andosol Profiles Makiko Watanabe, Nobuo Sakagami, and Kiminori Tonosaki

Abstract Sclerotia grains found from soils are not all active for germination but remain in soil. The age of sclerotia grains existing in soils is able to be estimated by 14 C dating measurement. Sclerotia grains from surface A horizons and buried A horizons collected from Andosol profiles in central to northern Japan were found to have ages of 100–200 year BP and 300–1200 year BP, respectively. The carbon content of the grains tended to decrease as 14C age increased. The 14C age of sclerotia grains indicates the age of individual grain formation and is more likely to indicate the beginning of soil formation than the 14C age of humic acid. The Al/C atomic ratio of the cell wall and the melanin-like spherules of sclerotia grains were observed using SEM-EDS analysis. Carbon content decreased and aluminum content increased from the outer to the inner portions of the grains, probably owing to microbial degradation. Additionally, the Al/C ratio of sclerotia grains showed a significant positive correlation with 14C age. Keywords Sclerotia grain · Humic acid · Humin · 14C age · Andosol profile

M. Watanabe (*) Department of Geography, Graduate School of Urban Environmental Sciences, Tokyo Metropolitan University, Hachioji, Tokyo, Japan e-mail: [email protected] N. Sakagami Department of Biological Production Science, College of Agriculture, Ibaraki University, Ibaraki, Japan e-mail: [email protected] K. Tonosaki JEOL Ltd., Tokyo, Japan e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2021 M. Watanabe (ed.), Sclerotia Grains in Soils, Progress in Soil Science, https://doi.org/10.1007/978-981-33-4252-1_7

119

120

7.1

M. Watanabe et al.

Introduction

Accelerator mass spectrometry (AMS)–14C dating requires at least 1 mg of graphite for measurements. Since Cenococcum geophilum sclerotia grains are composed of approximately 50% carbon and some found in forest soils are approximately 0.5–5 mm in diameter and have a mass as large as 2 mg, 14C dates can be obtained, even for single grains (Watanabe et al. 2004a, b). Sclerotia of C. geophilum are used as decay-resistant markers of the paleoenvironment in paleopedology, geochemistry, and sedimentology. Hormes et al. (2004) examined the C. geophilum spores in paleosols of moraines in Lapland, northern Sweden, and reported that the 14C ages of the spores were 5000–6000 year BP (Note, however, that the “spores” were actually sclerotia; C.geophilum does not produce spores according to LoBuglio 1999). Benedict (2011) reported evidence of a shift in tree limit by studying sediment samples from the Rocky Mountains, Colorado, USA, where charred C. geophilum sclerotia with a median diameter of 1.1 mm were demonstrated to have radiocarbon dates of 4770
25 year BP. Cenococcum geophilum sclerotia can contribute significantly to the fungal biomass of forest environments and thus represent an important source of carbon assimilated from host species (LoBuglio 1999). As extramatrical mycelia represent a considerable biomass component and potential carbon sink in many forest soils, Cairney (2012) reviewed the estimates of mean, median, and maximum longevities of ectomycorrhizal roots, sclerotia, and mycelia in the field to discuss turnover of carbon stored in ectomycorrhizal root and mycelial biomass. Samples of C. geophilum sclerotia grains, humic acid, and humin fractions from three different Andosol profiles soils were examined to measure their AMS-14C dates. We discuss the implications of the AMS-14C ages and carbon turnover in sclerotia.

7.2 7.2.1

14

C Ages of Sclerotia Grains in Three Andosol Profiles Materials and Methods

Soil samples were collected from three Andosol profiles (MYK, ONT, and IWK) for analysis of sclerotia grains and extraction of humic acid and humin. The Myoko profile (MYK) is a Fulvic Andosol, WRB/FAO-Unesco, located in central Japan (36 540 N 138 80 E, elevation: 1330 m, vegetation: Fagus crenata), which is comprised of a surface A horizon and buried humic horizons beneath KG-b: Koyaike ash-b and KG-c: Koyaike ash-c, tephra deposits derived from eruptions of the Yakeyama volcano in 650
100 year BP and 950
80 year BP (Hayatsu et al. 1994), respectively. The ONT profile is an Andic Podzol on Mt. Ontake in Gifu Prefecture, beneath Abies veitchii and Tsuga diversifolia forests (35 550 1100 N, 137 270 5300 E; elevation: 2100 m). Soil samples were taken from each horizon of

7 Dating of Sclerotia Grains in Andosol Profiles

121

the profile (Ah1/Ah2/E/Bs). The IWK profile is a Fulvic Andosol located on Mt. Iwaki, Aomori Prefecture, at an elevation of 800 m (40 390 N, 140 160 E; F. crenata forest, Ah1/Ah2/BA/Bw). Sclerotia grains were collected from each horizon of the soil profiles. Sclerotia grains larger than 1 mm in diameter were collected from air-dried soils using sterilized tweezers. Grains smaller than 1 mm in diameter were collected by using the floatation method in distilled water. Sclerotia grain samples were exposed to ultrasonic treatment for 5 min to remove soil from their surface and then dried at room temperature (20–25  C). The sclerotia grains were weighed using an electronic balance and their morphological features were observed using a digital high-density video microscope (VH-7000, Keyence, Osaka) and a scanning electron microscope (JSM 6490 LA, JEOL, Tokyo). Samples were fractionated into three grain sizes: large (>2 mm), medium (1–2 mm), and small (2 mm), medium (1–2 mm), and small (2 mm) frequently found in horizon 3A of MYK soil were likely to have a distorted shape. However,

Fig. 7.1 Diameter and 14C ages of sclerotia grains by horizon in the MYK profile. (Reproduced from Watanabe et al. 2007b, Taylor & Francis Ltd, http://www.tandfonline.com)

124

M. Watanabe et al.

several small grains may have merged to form a single large grain, and this might explain why the relationship between 14C age and grain size was somewhat unclear for larger grains in buried A horizons. Although difficulties remain in comparing 14C ages between humus extracts and sclerotia grains, the average age of organic materials incorporated during formation of sclerotia grains is thought to be older than that of humic substances (Watanabe et al. 2007b). Most of the sclerotia grains examined here are thought to be “dead” sclerotia of C. geophilum, which persist in the soil and can be differentiated from “live” sclerotia by being hard and brittle, as described by Trappe (1969) and Massicotte et al. (1992). In in vitro studies of sclerotium germination and formation on agar, initial growth of a white hyphal tip from field-collected “live” sclerotia occurred within 10 days (Trappe 1969) or 20 days at the most (Massicotte et al. 1992). In most sclerotia that proved to be viable, hyphal growth was detected within 30 days, but some sclerotia remained on the agar for up to 75 days before germinating, and at maturity, after formation of a dark rind, they resembled sclerotia formed in soil (Massicotte et al. 1992). It therefore seems plausible to conclude that sclerotium formation in soils may also terminate within several months. Humic acids, continuously exposed to metabolism by microbes, are composed of heterogeneous material. On the other hand, sclerotia grains, as spore structures derived from fungal cell walls, are likely to be composed of homogeneous material. Consequently, the AMS-14C ages of sclerotia grains are considered to indicate the age of individual grains and may be older than the soil profile they occupy, consistent with the age of the beginning of soil formation, compared with humic acids, which may indicate the average age of soil humus. As the cell wall of ectomycorrhizae contains a high concentration of aluminum (Brunner and Frey 2000; Brunner 2001), the homogeneous distribution of aluminum in the transverse wall of ignited grains observed by Watanabe et al. (2004a) suggests that the grains may be a biosynthetic product of the ectomycorrhizae, responsible for aluminum retention. Furthermore, the mean weight of sclerotia grains is regulated by the amount of exchangeable aluminum in soils, regardless of soil type (Watanabe et al. 2002, 2004a). Watanabe et al. (2007a) performed a quantitative elemental analysis of MYK sclerotia, which clarified that the concentrations of carbon and oxygen, the major elements regulating the weight of grains, were 48 wt% and 30 wt %, respectively, and that of aluminum was 1.4 wt%, with no significant differences in element concentrations between grains of different size. Extending the investigation to various forest soils and comparing grain turnover in light of the relationship between soil chemical properties and 14C age of grains would be warranted. Carbon content was measured by using a CO2 pressure gauge during the process of preparing graphite. The carbon content of sclerotia grains in surface A horizons was 40–50%, whereas that of sclerotia grains in buried A soils was 38–46%. Older grains showed a slight decrease in carbon content (Watanabe et al. 2007b). The results from Myoko profile may indicate that sclerotia grains in buried humic soils are undergoing decomposition.

7 Dating of Sclerotia Grains in Andosol Profiles

7.2.2.2

125

ONT and IWK Profiles

Table 7.2 shows AMS-14C dates for sclerotia, humic acid, and humin from the ONT soil samples. Comparisons of the AMS-14C dates of sclerotia based on grain size and horizon are shown in Fig. 7.2. 14C ages for sclerotia in the ONT Ah1 layer were 500–830 year BP. Large sclerotia tended to be older than small sclerotia when different-sized sclerotia from each layer were individually compared. When 14C dates were compared between horizons and grain sizes, larger sclerotia were again found to be older. The 14C dates for humin were older than those of sclerotia in the E/A and Bs horizons, and the 14C dates for sclerotia were closer to the dates for humic acid than those for humin. Figure 7.3 shows the results for sclerotia in Iwaki profile. In the Ah1 layer (Iw-1), 14 C dating of sclerotia indicated that they were modern. Sclerotia from lower horizons were older, regardless of grain size. Moreover, sclerotia in the B horizon were dated at almost 3000 year BP (Table 7.3).

7.2.2.3

Changes in Total Carbon Content of Soil, Humic Acid, and Sclerotia by 14C Date

Table 7.4 shows total carbon content TC (%) of sclerotia grains, humic acid, and soil in ONT profile, and Table 7.5 shows TC (%) of sclerotia grains and soil in IWK profile. Figure 7.4 shows the relationship between the 14C dates and TC content of soil samples, humic acid, and sclerotia of medium size in ONT profile. Figure 7.5 shows the 14C dates and TC content of soil samples and sclerotia of medium size in IWK profile. The dates of soil samples from the ONT soils were determined based on the humin 14C date and those of the IWK soils were based on sclerotia 14C dates. In the A horizons, the values for carbon content of sclerotia were about 50 wt%, whereas in the B layer they were approximately 40 wt%. The carbon content of sclerotia decreased linearly with time, whereas that of soil decreased logarithmically. These results clearly indicated that sclerotia, structural bodies, undergo a ubiquitous decomposition and denaturation process.

7.3

Relationship Between 14C Age and Al/C Ratio of Sclerotia Grains

7.3.1

Materials and Methods

7.3.1.1

Sclerotia Samples

Sclerotia, formed by C. geophilum or related species, can easily be identified in soil samples based on the morphological characteristics reported by Trappe (1969). Cenococcum geophilum sclerotia were obtained from soil samples collected from

Others

Sample Sclerotia

Table 7.2

15–24

24–34

34–44

Ah2

E/A

Bs

Bs

E/A

Ah2

Ah1

Depth (cm) 0–15

Horizon Ah1

Grain size/or fraction S M L S M L S M L S M L Humic acid Humin Humic acid Humin Humic acid Humin Humic acid Humin

Diameter (mm) 0.6 1.9 2.4 0.6 1.6 2.9 0.6 1.9 2.5 0.6 1.6 2.1

Sample amount (mg) 3.3 4.0 1.8 3.9 3.1 4.4 3.5 4.0 2.0 2.8 3.5 1.8 3.8 18.0 3.9 47.0 3.6 31.7 4.0 119.7

C ages of sclerotia, humic acid, and humin in the ONT profile

14

Number of grains 25 3 1 34 4 1 25 3 1 30 5 1 C age (year BP) 499
37 687
30 831
44 1134
40 1450
34 1688
42 1590
44 1620
33 1897
46 2411
35 2121
35 2720
41 444
32 980
33 962
53 1589
33 1851
44 2977
37 2505
44 3754
42

14

Labo code MTC-08472 MTC-08043 MTC-07789 MTC-09442 MTC-08044 MTC-07792 MTC-08478 MTC-08045 MTC-07790 MTC-08481 MTC-08046 MTC-08485 MTC-09431 MTC-09435 MTC-09432 MTC-09436 MTC-09433 MTC-09437 MTC-09434 MTC-09438 2417

1702

1424

Average 14C age (year BP) 672

126 M. Watanabe et al.

7 Dating of Sclerotia Grains in Andosol Profiles

127

Fig. 7.2 AMS 14C ages for sclerotia, humic acid, and humin in the ONT soil by horizon. Open circle: sclerotia (large, medium, small), open square: Humin, open triangle: Humic acid

Fig. 7.3 AMS 14C ages of sclerotia grains by horizon in the IWK profile

three locations in central and northern Japan: Mount Ontake, Gifu Prefecture (ONT; 35 550 N, 137 270 E; 2100 m asl; A. veitchii and T. diversifolia forests; Andic Podzol); Mount Iwaki, Aomori Prefecture (IWK; 40 390 N, 140 160 E; 800 m asl;

3–10

10–20

20–44

Horizon Ah1

Ah2

BA

Bw

Grain size S M S M S M S M

Diameter (mm) 0.6 1.4 0.6 1.3 0.6 1.4 0.6 1.1

C ages of sclerotia in the IWK profile

14

Depth (cm) 0–3

Table 7.3 Sample amount (mg) 2.9 2.4 2.4 2.8 3.9 2.4 2.7 3.3

Number of grains 28 6 31 6 36 4 32 12 C age (year BP) Modern Modern 228
40 125
35 951
34 1340
37 2798
40 2620
42

14

Labo code MTC-08482 MTC-08172 MTC-08484 MTC-08173 MTC-08477 MTC-08174 MTC-08476 MTC-08175

2709

1146

177

Average 14C age (year BP) 50%), was identified (Fig. 8.2f). Figure 8.2g–i show TEM micrographs of ignited samples. Because the samples were treated to remove carbon, the EDX analysis excluded the carbon content. The relatively large plate-shaped structure with a hole was assumed to be a fragment of

144

M. Watanabe and A. Genseki

Fig. 8.3 Micromorphological features observed by transmission electron microscopy showing the round particles with attached fibrous fragments (a, d), broken needle-like particles (b, e) denoted by an arrow in (a), and needle structure at high magnification (c). Electron diffraction pattern obtained for the broken needle-like structure (f) at camera length of 0.3 m. (Reproduced from Watanabe et al. 2004, Taylor & Francis Ltd, http://www.tandfonline.com)

the transverse wall associated with a septal pore (Fig. 8.2g). At a higher magnification, the micromorphology of the matrix had a honeycomb structure with a chemical composition of 2:1 O:Al (Fig. 8.2h, i). Metal elements such as nickel and lead were frequently detected as roughly spherical structures intermingled with the matrix (Fig. 8.2i). Figure 8.3 shows the round structures with fibrous fragments found in the ignited samples. Because their diameter was approximately 5 μm, these structures were considered to correspond to the acicular structures observed in the SEM micrographs. The round structures were composed of Si (56%), O (41%), and Al (3%), based on the EDX spectrum (Fig. 8.3a), whereas Si (38%), O (33%), Al (26%), and C (1%) were the predominant elements in the fragment structures, as shown in Fig. 8.3b. A characteristic polymorph was found near the round structure, as shown in Fig. 8.3. This polymorph resembled a broken needle and appeared to be hollow. EDX analysis indicated that its chemical composition was Al:O ¼ 77:23 (at.%). At a higher magnification, lattice bands were observed in small areas (Fig. 8.3e). These characteristics suggest that the needle-shaped structure is a species of aluminum hydroxide characterized by aggregate microcrystalline structures with a random arrangement. Figure 8.3f shows the electron diffraction pattern of this aluminum polymorph. The lattice distances were as follows: d1 ¼ 0.228, d2 ¼ 0.161,

8 Micromorphological Features of Sclerotia Grains

145

Fig. 8.4 Morphology of structures in the interior of Myoko-3A sclerotia determined by scanning electron microscopy energy-dispersive X-ray analysis: (a) transverse wall, (b) detail of boehmite structures, and (c) detail of rectangular structures (with elemental composition)

d3 ¼ 0.133, and d4 ¼ 0.114 for the diffraction points located on the four readable rings. Based on the relative brightness of the points, equivalent to the relative intensity of the X-rays (I), these four lattice distances are concordant with X-ray powder diffraction data for boehmite, γ-AlOOH. Nevertheless, the lattice distance expected for hkl ¼ 120 (I ¼ 65) was not confirmed here, probably due to the absence of a specific diffraction point behind the aggregate of microcrystalline structures. In general, boehmite consists of aluminum oxyhydroxides present in many types of bauxites and is normally the ultimate product of intensive weathering of primary aluminum silicates in soils (Hsu 1989). Experimental alteration of obsidian (volcanic glass) conducted by Kawano and Tomita (1993) confirmed that boehmite has a fibrous structure corresponding to alteration products associated with spherical kaolinite and precursors of smectite in Al3+-enriched solution. On the other hand, the number of ectomycorrhizal tips formed by Cg has been reported to increase in soils exposed to simulated rain with a pH of 2.5 by Meira et al. (1989). Consequently, formation of Al oxyhydroxide polymorphs in sclerotia grains might result from Al dissolution–precipitation inside the grains. A mycelium-like structure was observed in the central part of the sclerotia grains, and a biochemical process performed by fungi colonizing the sclerotia might be involved in the dissolution of aluminum from the matrix of the grain, in the form of aluminum–carbon complexes. This might induce aluminum saturation and precipitation under acidic conditions, which could lead to formation of boehmite-like aluminum oxyhydroxides. Characteristic acicular and rectangular structures were also observed in the transverse wall of the MYK-3A sample (Fig. 8.4). The C/O/Na/Al/Si/S atomic ratio of one of the rectangular structures was 13:53:1:19:1:13, suggesting the presence of aluminum sulfate or sodium alum.

8.4

Carbonaceous Granular Particles and Nanoparticles

SEM images of the powdered samples taken from the interior of the Myoko and Ontake sclerotia are shown in Fig. 8.5. In the Ontake sample, the edge of the cell wall had a granular structure (Fig. 8.5a). EDX analysis indicated that the C/O/Al atomic

146

M. Watanabe and A. Genseki

Fig. 8.5 Morphology and elemental compositions (atomic ratios) of the cell wall structure of (a) Ontake-A1 and (b, c) Myoko-3A sclerotia samples, determined by scanning electron microscopy energy-dispersive X-ray spectrometer analysis

ratio of this granular structure was 77:21:2 and that of the cell wall area was 78:20:2; thus, the C/O ratios of these two areas were almost identical. Similar granular structures were observed on the cell wall surface of the Myoko sample (Fig. 8.5b), and the C/O/Al atomic ratios were 66:31:3 for the granular structure and 76:22:2 for the cell wall (Fig. 8.5c). Fungal cell wall melanin may appear to be granular or fibrillar; microsclerotia of Verticillium spp. are covered with a layer of granular melanin (Wheeler et al. 1976). Figure 8.5 in the present study resembles SEM imagery of cell wall melanin in microsclerotium of V. dahlia presented in Wheeler et al. (1976), which shows heavy deposition of melanin granules. Although Cg sclerotia have not been reported to contain melanin, Fig. 8.5 may show granular melanin structures in the cell wall of Cg sclerotia. Fungal melanin is known to bind metals such as copper, aluminum, zinc, and iron (Gadd and De Rome 1988; Rizzo et al. 1992). Aluminum may be present in the substrate of the Cg mycelium cell wall. From our previous study, the cell walls of Cg sclerotia from low-pH forest soils had a high aluminum content (Watanabe et al. 2001, 2002, 2004). Nevertheless, Cg sclerotia have a hard rind penetrated only by labyrinthine septal pores 1 μm or less in diameter, making them a semi-closed system. SEM observations in the present study did not identify morphological characteristics of aluminum-rich layers on sclerotia cell walls, which would indicate successive aluminum coating of sclerotia cell walls in soil. Figure 8.6 shows images of a powder sample from the interior of a Myoko sclerotium scattered on carbon tape. The sample contained cell wall structures, soil clay-silt aggregate, and nanoparticles, some bright and some dull. The C/O atomic ratio of the carbon tape was 97:3 (Fig. 8.6a). The nanoparticles were composed of carbon particles and titanium oxide particles (Fig. 8.6a). The dull rectangular particles were 200–500 nm in size and had a C/O atomic ratio of 96:4, suggesting graphene-like particles. The bright particles had a C/O/Ti ratio of 44:35:21. Graphene-like particles (200–600 nm) were also observed in powder samples from Ontake sclerotia, and small amounts of calcium, magnesium, or iron adhered to the particles (Fig. 8.6c).

8 Micromorphological Features of Sclerotia Grains

147

Fig. 8.6 (a) Scanning electron microscopy image of powder samples collected from the interior of a Myoko-1A sclerotium, including cell wall structures, soil clay-silt aggregate, and nanoparticles. Scanning electron microscopy images and elemental compositions of nanoparticles obtained from (b) Myoko-1A and (c) Ontake-A1 sclerotia samples

8.5

Spherical Silica Particles

Spherical silica particles of 3–4 μm in diameter are frequently observed inside sclerotia grains found from volcanic ash soils (Watanabe et al. 2004). Figure 8.7 shows the small spherical silicon particles (diameter 200 nm, O/Si ¼ 6:1) observed in the cell wall structure of the Myoko surface soil sample (1A), and the larger spherical particles (10 μm, O/Si ¼ 4:1) observed in the cell wall structure (C/O/ Al ¼ 68:30:2) of sclerotia collected from the Myoko buried soil horizon, 3A (Chap. 7).

148

M. Watanabe and A. Genseki

Fig. 8.7 Scanning electron microscopy images of spherical Si structures observed in (a) MYK-1A and (b) MYK-3A sclerotia samples

Fig. 8.8 Sclerotium sample placed in the equipment container (a) and 3D imagery of the interior of the Iwaki sclerotium (b)

8.6

Micro-CT Analysis

Figure 8.8 (left) shows the sample grain placed in the equipment container and Fig. 8.8 (right) shows 3D images of the sclerotium interior. The striped patterns on the exterior are X-ray scattering noise; X-ray scattering images superimposed between vertically adjacent 2D slice images are slightly shifted and interfere with each other due to the roughness of the surface and the inorganic components on the surface of the grain. Contrasting bright and dark areas indicate differences in elemental composition. The mycelial strand is clearly visible in the center of the grain. Compared with SEM images from sliced samples of sclerotia, collected from the same soil horizon (Fig. 8.9), which reveal the presence of mycelia, micro-CT scanning may reveal the distribution of mycelial strands and metals in a whole, undisturbed grain.

8 Micromorphological Features of Sclerotia Grains

149

Fig. 8.9 Scanning electron micrographs of the interior of a sclerotium section, collected from the same Iwaki A horizon soil used for micro computed tomography analysis (Fig. 8.8). The box in part (a) indicates the area of part (b), which shows the fungal hyphae-like structures

8.7

Conclusion

The morphology and elemental composition of Cg sclerotia collected from low-pH forest soils in central and northern Japan were analyzed by SEM-EDX. The cell wall structure and melanin-like spherules consisted mainly of carbon, oxygen, and aluminum. Graphite and graphene, products or by-products of substrate degradation, were observed in the interior of sclerotia, and the graphene was adjacent to titanium oxides or small amounts of adhering calcium, magnesium, or iron oxides. Precise observation of the micromorphology of the interior of sclerotia grains suggests the transformation of aluminum–carbon complexes from aluminum-rich melanin spherules to aluminum accumulated layers. This hypothesis would explain how Cg sclerotia can resist degradation in soil and retain their structure, but it requires further investigation. The crystalline aluminum structures, including boehmite and aluminum sulfates, observed in the interior of sclerotia indicate the possibility of aluminum enrichment and mineralization. Spherical silicon particles were also observed, although their origin (formed inside the sclerotia or contaminants) remains unclear. The activities of microorganisms inside sclerotia could drive degradation of the sclerotia and mineralization of their internal structure, but this too will require further investigation. Acknowledgements MW thanks Mr. T. Hatano, Mr. T. Hattori, and Mr. H. Terashima of JEOL Co. Ltd., Tokyo, Japan, for technical support with SEM-EDS and micro-CT analyses.

150

M. Watanabe and A. Genseki

References Butler MJ, Day AW (1998) Fungal melanins: a review. Can J Microbiol 44:1115–1136 Cessna SG, Sears VE, Dickman MB, Low PS (2000) Oxalic acid, a pathogenicity factor for Sclerotinia sclerotiorum, suppresses the oxidative burst of the host plant. Plant Cell 12:2191–2199 Chet I, Henis Y, Mitchell R (1967) Chemical composition of hyphal and sclerotial walls of Sclerotium rolfsii Sacc. Can J Microbiol 13:137–141 Dahlberg A, Jonsson L, Nylund JE (1997) Species diversity and distribution of biomass above and below ground among ectomycorrhizal fungi in an old-growth Norway spruce forest in south Sweden. Can J Bot 75:1323–1335 Dallies N, Francois J, Paquet V (1998) A new method for quantitative determination of polysaccharides in yeast cell wall. Application to the cell wall defective mutants of Saccharomyces cerevisiae. Yeast 14:1297–1306 Daulton TL, Amari S, Scott AC, Hardiman M, Pinter N, Anderson RS (2016) Comprehensive analysis of nanodiamond evidence relating to the Younger Dryas Impact Hypothesis. J Quatern Sci 32:7–34 Fernandez CW, Koide RT (2013) The function of melanin in the ectomycorrhizal fungus Cenococcum geophilum under water stress. Fungal Ecol 6:479–486 Gadd GM, De Rome L (1988) Biosorption of copper by fungal melanins. Appl Microbiol Biotechnol 29:610–617 Hausner G, Reid J (1999) Factors influencing the production of sclerotia in the wild rice (Zizania aquatica) pathogen Sclerotium hydrophilum. Mycoscience 40:393–400 Hodson MJ, Wilkins DA (1991) Localization of aluminum in the roots of Norway spruce (Picea abies (L.) Karst) inoculated with Paxillus involutus Fr. New Phytol 118:273–278 Hormes A, Karlen W, Possnert G (2004) Radiocarbon dating of palaeosol components in moraines in Lapland, northern Sweden. Quatern Sci Rev 23:2031–2043 Hsu PH (1989) Aluminum hydroxides and oxyhydroxides. In: Dixon JB, Weed SB (eds) SSSA Book series: 1 Minerals and soil environments. Soil Science Society of America, Madison, WI, pp 331–373 Jentschke G, Schlegel H, Godbold DL (1991) The effect of aluminum on uptake and distribution of magnesium and calcium in roots of mycorrhizal Norway spruce seedlings. Physiol Plant 82:266–270 Kawano M, Tomita K (1993) Formation of clay minerals during low temperature hydrothermal alteration of obsidian (part1): effect of addition of Al ions. Nendo Kagaku (J Clay Sci Soc Jpn) 33:59–71. (in Japanese with English abstract) Kinzie CR, Hee SSQ, Stich A, Tague KA, Mercer C, Razink JJ, Kennett DJ, DeCarli PS, Bunch TE, Wittke JH, Israde-Alcántara I, Bischoff JL, Goodyear AC, Tankersley KB, Kimbel DR, Culleton BJ, Erlandson JM, Stafford TW, Kloosterman JB, Moore AMT, Firestone RB, Aura Tortosa JE, Jordá Pardo JF, West A, Kennett JP, Wolbach WS (2014) Nanodiamond-rich layer across three continents consistent with major cosmic impact at 12,800 Cal BP. J Geol 122:5. https://www.journals.uchicago.edu/doi/abs/10.1086/677046 LoBuglio KF (1999) Cenococcum. In: Cairney JWG, Chambers SM (eds) Ectomycorrhizal fungi: key genera in profile. Springer, Berlin Malik K, Haider K (1982) Decomposition of 14C-labeled melanoid fungal residues in a marginally sodic soil. Soil Biol Biochem 14:457–460 Massicotte HB, Trappe JM, Peterson RL, Melville LH (1992) Studies on Cenococcum geophilum. II. Sclerotium morphology, germination, and formation in pure culture and growth pouches. Can J Bot 70:125–132 Meira S, Robargeb WP, Brucka RI, Granda LF (1989) Effects of simulated rain acidity on ectomycorrhizae of red spruce seedlings potted in natural soil. Environ Pollut 59:315–324 Obase K, Douhan GW, Matsuda Y, Smith ME (2014) Culturable fungal assemblages growing within Cenococcum sclerotia in forest soils. FEMS Microb Ecol 90:708–717

8 Micromorphological Features of Sclerotia Grains

151

Rizzo DM, Blanchette RA, Palmer MA (1992) Biosorption of metal compounds by Armillaria rhizomorphs. Can J Bot 70:1515–1520 Scott AC, Pinter N, Collinson ME, Hardiman M, Anderson RS, Brain AP, Smith SY, Marone F, Stampanoni M (2010) Fungus, not comet or catastrophe, accounts for carbonaceous spherules in the Younger Dryas “impact layer”. Geophys Res Lett 37:L14302 Sugiura Y, Watanabe M, Nonoyama Y, Sakagami N, Guo Y, Murayama S (2017) Saccharides of ectomycorrhizal fungal sclerotia as sources of forest soil polysaccharides. Soil Sci Plant Nutr 63:426–433 Takahashi T, Nanzyo M, Hiradate S (2007) Aluminum status of synthetic Al-humic substance complexes and their influence on plant root growth. Soil Sci Plant Nutr 53:115–124 Trappe JM (1969) Studies on Cenococcum graniforme, I. An efficient method for isolation from sclerotia. Can J Bot 47:1389–1390 Watanabe M, Fujitake N, Ohta H, Yokoyama T (2001) Aluminum concentrations in sclerotia from a buried humic horizon of volcanic ash soils in Mt. Myoko, central Japan. Soil Sci Plant Nutr 47:411–418 Watanabe M, Kado T, Ohta H, Fujitake N (2002) Distribution and development of sclerotium grain as influenced by aluminum status in volcanic ash soils. Soil Sci Plant Nutr 48:569–575 Watanabe M, Genseki A, Sakagami N, Inoue Y, Ohta H, Fujitake N (2004) Aluminum oxyhydroxide polymorphs and some micromorphogical characteristics in sclerotium grains. Soil Sci Plant Nutr 50:1205–1210 Wheeler MH, Tolmstoff WJ, Meola S (1976) Ultrastructure of melanin formation in Verticillium dahlia with (+)-scytalone as a biosynthetic intermediate. Can J Microbiol 22:702–711 Willetts HJ (1971) The survival of fungal sclerotia under adverse environmental conditions. Biol Rev 46:387–407 Willetts HJ (1972) The morphogenesis and possible evolutionary origins of fungal sclerotia. Biol Rev 47:515–536

Chapter 9

Spatial Distribution of Sclerotia Grains in Forest Soils, Northern and Central Japan Nobuo Sakagami and Sayuri Kato

Abstract Cenococcum geophilum is known for its vast habitat range in temperate and arctic-alpine climatic zones. The distribution of Cg sclerotia, resting bodies which persist for a long term as a structural organic component in soils, is studied from soil science and geographical aspects in this chapter. The objective of this study is to understand the nature of their distribution as an organic component of forest soils in Japan. Distributional properties of sclerotia were examined in terms of weight density (mg/g), count density (grain/g), and mean weight per grain (mg/grain). Harmonizing with previous studies, sclerotia tended to distribute in acidic soils which have an Alp/Alo ratio larger than 0.5. Although these relationships are not clear among surface soils, relationships between soil chemical properties and sclerotia formation were suggested. Furthermore, sclerotia of Cg were assumed to be one of the sources of “Pg,” the green fraction of humic acid. In a study on the distribution of sclerotia in surface soils along an altitudinal gradient, the distributional peak was likely to be regulated by a balance of sclerotia formation and decomposition. Formation of sclerotia may have a close relationship to the activities of Cg (i.e., formation of its mycorrhizae) and thus it would be strongly affected by dominant plants. Keywords Cenococcum geophilum · Distribution of sclerotia · Exchangeable aluminum · Non-allophanic andosols · Sclerotia · Soil profile · Soil properties

N. Sakagami (*) College of Agriculture, Ibaraki University, Ibaraki, Japan e-mail: [email protected] S. Kato University Education Centre, Tokyo Metropolitan University, Tokyo, Japan e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2021 M. Watanabe (ed.), Sclerotia Grains in Soils, Progress in Soil Science, https://doi.org/10.1007/978-981-33-4252-1_9

153

154

9.1

N. Sakagami and S. Kato

Introduction

Cenococcum geophilum Fr. (Cg) is known for its vast habitat range (LoBuglio 1999). Ectomycorrhiza of Cg have a worldwide distribution from temperate to arctic-alpine climatic zones, and have even been observed above the Arctic Circle in Alaska and the Canadian High Arctic (75
330 N, 84
400 W) and at timber line in the Washington and Oregon Cascade mountain range as an important symbiont of trees (Trappe 1964, 1988; Haselwandter and Read 1982; Bledsoe et al. 1989). Such wide distribution of Cg suggests the adaptability of it against severe environments. The experiment, which demonstrated that Cg still grew in soils under exposure of simulated rain at pH 2.5 (Meier et al. 1989), suggests one of the excel ability in soil ecosystem. According to Trappe (1969), sclerotia of Cg tend to be particularly abundant near Cg mycorrhizae. It is also well known that mycorrhizal root tips and sclerotia have their maximum production in autumn (e.g., Vogt et al. 1981, 1982; Lussenhop and Fogel 1999). Watanabe et al. (2002) reported that Cg sclerotia distribute in acidic soils which are non-allophanic (Alp/Alo > 0.5), and tend to form larger grains in soils with a higher content of exchangeable aluminum (AlEx) which is potentially phyto-toxic for plant roots. Watanabe et al. (2007b) reported the 14C ages of sclerotia collected from buried A horizons of Fulvic Andosol in Mt. Myoko, central Japan, as ca. 300–1200year BP and thus they exhibited its persistence as a structural organic component in soils. In this chapter, we examined the distribution of sclerotia in surface and subsurface soils in eight sites (Fig. 9.1) in central, north-eastern, and north Japan in order to better understand both their roles as organic components in forest soils and their interactions with soil chemical properties. Fig. 9.1 Study sites in this chapter

9 Spatial Distribution of Sclerotia Grains in Forest Soils, Northern and Central. . .

9.2

155

Distribution of Sclerotia in Soil Profile

In this part, the distribution of sclerotia in 12 soil profiles at six sites were examined (Fig. 9.2). Sclerotia in the air-dried soil samples were floated in pure water, carefully picked up from soils using tweezers and then kept in air-dried conditions. The contents of sclerotia in soils were obtained based on the weight (mg/g) and the

Fig. 9.2 Soil profiles in Myoko, Ontake, Jumonji, Tazawa, Sasamori, and Rishiri. Jumonji and Sasamori profiles are courtesy of T. Nozawa

156

N. Sakagami and S. Kato

count (grain/g). Almost all sclerotia from air-dried soils floated in water in this experiment. These floated sclerotia were considered to include both “live” and “dead” sclerotia, noted by Trappe (1969). The brief explanation of soil profile (horizon, depth, soil color, and texture) and the sclerotia contents are summarized in Table 9.1. The total C (T-C) and N (T-N) contents of soils were measured by the dry combustion method using an NC-analyzer (NC-80, SCAS Ltd., Tokyo). The value of the soil pH (H2O and KCl) was measured by the glass electrode method in a suspension mixture of soil and a 2.5 times greater volume of either H2O or 1 M KCl. Quantitative analysis of dithionite-citrate, acid oxalate, and pyrophosphate extractable Al and Fe (Ald, Alo, Alp, Fed, and Feo) was carried out by the selective dissolution method (Blakemore et al. 1987). The content of AlEx was obtained on the extract with 1 M KCl according to the method of Blakemore et al. (1987). The soil profile data, sclerotia contents and soil analyses data for each profile are summarized in Table 9.1, and Fig. 9.3. The first profile, Myoko Tsubame (Fig. 9.2 (1)), is from Fulvic Andosol beneath a F. crenata forest (36
540 0900 N, 138
080 1600 E; 1320 m asl.) on Mt. Myoko, Niigata Prefecture. The mean temperature and the annual precipitation in this area are 5.8
C and 2280 mm, respectively. Floor vegetation of the site was characterized by the presence of Sasa kurilensis. In Japan, in the context of soil science, Kumada (1987) first noted the abundance of large sized sclerotia (over 7 mm in diameter) in this site. The sclerotia showed abundant distribution in surface A and buried A horizons. As opposed to Watanabe et al. (2002), the sclerotia contents did not correlate with AlEx nor Alp/Alo. The second profile, Myoko Town (Fig. 9.2 (2)), is located in the pediment area of Mt. Myoko (700 m asl.). Although the soil pH was lower than that of Tsubame soil, the sclerotia content was lower in this profile. This supposedly results from the lack of F. crenata, one of the important symbionts of Cg. Figure 9.2 (3) is a Haplic Podzol beneath a mixed forest of Abies veitchii and Tsuga diversifolia (35
550 1100 N, 137
270 5300 E; 2100 m asl.) on Mt. Ontake, Gifu Prefecture. This profile showed lower pH, higher AlEx content, and higher sclerotia content comparing to Myoko profiles. Sugiura et al. (2017) reported detailed distribution of sclerotia in this profile, examined their saccharides and discussed their importance as an origin of forest soil polysaccharides. Jumonji (Fig. 9.2 (4)) is one of the most famous Podzolic soils in Japan. Jumonji pass, in Mts. Oku-Chichibu, Saitama Prefecture, is under subalpine coniferous forest (Kitagawa et al. 2001). Its surface soil showed extremely low pH, and high AlEx content. Although the contents of sclerotia based on their weight and count are low, large sclerotia (0.8 mg/grain) distributed in this profile. In Tazawa study site (39
470 3000 N, 140
460 2100 E; 620 m asl.) on the Lake Tazawa plateau, Akita Prefecture, sclerotia were collected from four different points: two under Cryptomeria japonica forest (Fig. 9.2 (5, 6)) and two under F. crenata forest (Fig. 9.2 (7, 8)). Sclerotia showed its distributional peak in the surface A horizons. Although C. japonica is known as a non-ectomycorrhizal tree species, the contents of sclerotia were not small under afforested cedars. Those sclerotia might be the remains associated with ectomycorrhizal trees in the past.

Horizon Depth (cm) Myoko Tsubame 1A 0 1C 12 2A 14 2C 21 3A 27 3Bw 37 Myoko Town A 0 Bs 10 Bir 13 Ontake Yunohana Ah 0 A 8 A/E 14 B 26 Bw 29 Jumonji E 0 Bh 11 Bs1 20

Soil texture

SiL SiL SiCL SiCL SiCL SiCL

SCL SL SL

SiC SC SiC SiCL SiC

CL LiC HC

Soil color

7.5YR2/2 7.5YR4/3 7.5YR2/2 7.5YR4/2 7.5YR2/2 10YR4/4

10YR2/2 10YR4/4 10YR4/6

7.5YR2/1 7.5YR2/2 7.5YR2/3 7.5YR3/2 7.5YR3/4

10YR3/4 5YR2/2 5YR2/3

0.15 0.08 0.06

2.08 1.25 1.96 1.59 0.11

0.04 0.31 0.13

1.33 0.22 1.67 0.05 2.45 0.41

0.17 0.14 0.11

12.01 9.31 16.74 13.98 1.55

0.12 2.29 1.29

0.81 0.05 0.79 0.25 2.18 0.75

Sclerotia content Weight Count (mg/g) (grain/g) Horizon Depth (cm) Tazawa Fagus 1 Aa(1) 2 Ab(2) 10 Bw(1) 21 Bw(2) 34 BC 46 Tazawa Fagus 2 1A1 0 1A2 5 1CB 16 2B 21 Sasamori 1 1A 0 1AB 19 2A 23 2BC 27 Sasamori 2 A 0 AB 6 B1 10 B2 16 SiCl SCL SL SL CL SL CL SL

7.5YR2/1 7.5YR3/2 7.5YR3/3 7.5YR3/2

SiC LiC L CL

10YR1.7/1 7.5YR2/2 10YR4/4 7.5YR3/4 7.5YR2/1 7.5YR3/2 2.5YR3/2 10YR3/4

SiL SiL LiC LiC SL

Soil texture

7.5YR2/2 7.5YR2/2 10YR4/6 10YR4/6 10YR4/4

Soil color

4.39 0.77 0 0

1.27 0 0 0

0.36 1.48 0.83 0.27

0.19 0.38 0.31 0.07 0.02

(continued)

3.44 0.38 0 0

1.71 0 0 0

0.72 6.84 5.75 1.94

0.62 1.57 1.62 0.58 0.38

Sclerotia content Weight Count (mg/g) (grain/g)

Table 9.1 Soil profile data and sclerotia contents in Myoko, Ontake, Jumonji, Tazawa, Sasamori, and Rishiri. Data of Jumonji and Sasamori were taken from Nozawa (2003)

9 Spatial Distribution of Sclerotia Grains in Forest Soils, Northern and Central. . . 157

Horizon Depth (cm) Bs2 28 Bs3 50 BC 67 Tazawa Cedar 1 A 1 Bw 13 CB 23 Tazawa Cedar 2 A 0 B 11 BC 35

Table 9.1 (continued)

Soil texture HC LiC SCL

SiL SL SL

SiC SL LS

Soil color 7.5YR3/4 10YR3.5/4 10YR3/3

10YR2/3 10YR4/6 10YR4/6

10YR2/2 10YR4/3 10YR5/2

0.82 0.01 0.00

0.87 0.06 0.01 0.95 0.09 0.04

2.28 0.17 0.18

Sclerotia content Weight Count (mg/g) (grain/g) 0.04 0.04 0 0 0 0 Horizon Rishiri Pd Ah AB Bw1 Bw2 BC Rishiri Mt Ah AB Bw BC

Soil color 7.5YR2/1 7.5YR3/2 10YR3/4 10YR4/3 10YR4/3 7.5YR1.7/1 7.5YR2/2 7.5YR2/2.5 7.5YR3/3

Depth (cm) 0 4 11 24 44 0 8 14 38

LiC HC HC SL

HC HC HC HC LiC

Soil texture

0.17 0.15 0.06 0.03

0.62 0.22 0.09 0.01 0

0.30 0.23 0.09 0.05

1.34 0.54 0.25 0.04 0

Sclerotia content Weight Count (mg/g) (grain/g)

158 N. Sakagami and S. Kato

9 Spatial Distribution of Sclerotia Grains in Forest Soils, Northern and Central. . . Soil pH(H2O) 4

5

6

1A 1C 2A 2C 3A

Tsubame

3Bw

Myoko

A

Yunohana

Alp/Alo ratio 0

0.5

1

1.5

Sclerotia Content Weight (mg/g) Count (grain/g) 0 2 4 0 5 10 15

Bs Bir

Town

Ontake

AlEx Content (g/kg) 0 0.5 1 1.5

Depth 50cm

3

Myoko

T-C Content (g/kg) 0 150 300

159

Ah A A/E B Bw

Jumonji

E Bh Bs1 Bs2 Bs3 BC

Tazawa

A Bw CB

Cedar 1

Tazawa

A

Cedar 2

B CB

Tazawa

A

Fagus 1 Bw

BC

Tazawa Fagus 2

1A1 1A2 1CB 2B 2C

3C 1A 1AB 2A 2BC A AB B1

Sasamori 1 Sasamori 2 Rishiri Pd

Ah AB Bw1

B2 C

Bw2 BC

Rishiri Mt

Ah AB Bw

BC

Fig. 9.3 Soil pH (H2O), T-C content, AlEx content, Alp/Alo ratio, and sclerotia contents in each soil profile

160

N. Sakagami and S. Kato

Fig. 9.4 Holistic relationship between soil pH (KCl), Alp/Alo, and sclerotia content by weight

Mt. Sasamori profiles (Fig. 9.2 (9, 10)) are podzolic soils near Mt. AkitaKomagatake (39
450 4000 N, 140
470 5800 E; 1637 m asl.). In this site only surface soils showed strong acidity and the sclerotia only distributed in their thin surface layers. In Mt. Rishiri (Fig. 9.2 (11, 12)), sclerotia were collected from two different Fulvic Andosols under Pinus pumila (Rishiri Pd.: 500 m asl.; and Rishiri Mt.: 1000 m asl.). Sakagami et al. (2007) reported that the Fe/Al ratios of internal parts of sclerotia tend to be high (Fe/Al ratio > 1) in sclerotia in Rishiri Mt. comparing to them in Rishiri Pd., and concluded that the chemical composition of sclerotia can strongly be influenced by soil-environmental conditions. Watanabe et al. (2001) noticed that C was the major element in sclerotia associated with a relatively large concentration of octahedral Al, which suggested an Al-humus complex, and Watanabe et al. (2002, 2004) concluded that formation of sclerotia was regulated by the content of exchangeable Al and the status of active Al in the soil, regardless of soil type. Figure 9.4 depicts the holistic relationship between soil pH (KCl), Alp/Alo ratio and weight-based sclerotia content in this study (including Mt. Chokai data in the next part). Harmonizing with Watanabe et al. (2002), sclerotia tended to distribute in acidic soils which have an Alp/Alo ratio larger than 0.5. However, as shown in Fig. 9.5, these relationships are not clear among surface soils. However, from studies on the spatial distribution of Cg sclerotia in Picea abies forests in Germany, severe conditions such as low pH and high AlEx content were undoubtedly assumed to be regulating factors in forming large sized sclerotia (Sakagami 2009).

9 Spatial Distribution of Sclerotia Grains in Forest Soils, Northern and Central. . .

161

Fig. 9.5 Relationship between soil pH (KCl), Alp/Alo, and sclerotia content by weight among surface soils

9.3

Variation of Sclerotia Distribution in Mt. Chokai

In this part, sclerotia distributions in soil profiles were examined at mesoscale by conducting soil surveys and analyses along a transitional soil sequence developed under Quercus serrata and F. crenata trees in Mt. Chokai, northern Japan. Soil profiles were examined at 7 points on the western slope of Mt. Chokai (39
050 5700 N, 140
130 1000 E; 2236 m asl.), in Yamagata Prefecture (Fig. 9.6). The annual mean temperature and total precipitation at the summit of Mt. Chokai are 0.5
C and 3285 mm, respectively. The parent materials of the soils were weathered andesite associated with continental aeolian dust. Extremely high precipitation (mainly snowfall) and the non-andic mineralogical properties regulate the development of the investigated soils. The soil profile data, sclerotia contents and soil analyses data for each profile are summarized in Table 9.2, and Fig. 9.7. Sclerotia were observed in all investigated soil profiles, where the content by weight and count in A horizons were 0.23–2.53 mg/g and 2.5–7.3 grain/g, respectively. The Chokai 3 profile was characterized by a high abundance of sclerotia, especially in count density. A further particularity of the Chokai 3 profile was recognized by its high Alp content. It can be suggested that the formation process of Al-hums complex is specific in this site. As shown in Fig. 9.7, the soil pH of Chokai 3 was relatively high (pH (H2O) > 4) compared to other profiles. Furthermore, from results of the content of free Al in profiles (Fig. 9.8a), the relatively high amount of Al colloids, mainly in amorphous and complex Al states, also suggests the exceptional property of Chokai 3. The reason for this exception may be explained by vegetation (lack of F. crenata), and/or by parent materials such as the absence of 2:1 layered silicates. Figure 9.8b shows

162

N. Sakagami and S. Kato

Fig. 9.6 Soil profiles and landscapes in Chokai

the distribution of free Fe oxides in profiles. It is understandable that Fe leaching gradually becomes intensive in profiles at higher elevations. From the viewpoint of soil genesis and soil organic chemistry, sclerotia of Cg were assumed to be one of the sources of “Pg,” the green fraction of humic acid by Kumada and Hurst (1967). They examined Cg sclerotia in British and Swedish Podzols and showed that the distribution of sclerotia was larger in A or B horizon. Among Mt. Chokai soils, there was a positive relationship between count of sclerotia and Pg index (Fig. 9.9), although the count of sclerotia in the Chokai 3 profile was exceptionally high. Kobayashi et al. (2005, 2006) reported on the chloroformextractable green fraction (CEGF), whose color is green in alkaline solution, as one of the components of, or a closely related substance to Pg, and considered Cg sclerotia as one of the origins of CEGF. Although the causes of variance of distribution (i.e., decomposition and formation) of sclerotia are not clear, a large variance of formation and decomposition of sclerotia may result in P-type humic acids accumulating in forest soils. Quantitative analyses on relationships between

9 Spatial Distribution of Sclerotia Grains in Forest Soils, Northern and Central. . .

163

Table 9.2 Soil profile data and sclerotia contents in Chokai

Horizon Mt. Chokai 1 A AB Bw Mt. Chokai 2 A1 A2 Bw Mt. Chokai 3 A1 A2 A3 Bs Mt. Chokai 4 A1 A2 Bs Mt. Chokai 5 A1 A2 Bs

Depth (cm)

Soil color

Soil texture

Sclerotia content Weight Count (mg/g) (grain/g)

0 7 15

5YR2/2 5YR3/4 5YR4/6

LiC CL CL

0.30 0.27 0.07

5.29 6.63 1.59

0 2 5

7.5YR2/1 7.5YR3/4 7.5YR4/6

LiC LiC CL

0.23 0.44 0.07

2.80 3.47 1.61

0 5 9 17

7.5YR2/1 7.5YR2/2 7.5YR3/2 7.5YR3/4

LiC LiC CL LC

0.51 1.11 1.15 0.11

4.51 17.60 22.91 2.59

0 5 14

7.5YR1.7/1 7.5YR3/2 7.5YR4/4

CL CL LiC

2.53 1.17 0.06

3.95 5.90 1.29

0 6 10

7.5YR2/1 7.5YR3/1 7.5YR4/4

LiC LiC LiC

0.46 0.37 0.10

2.48 7.51 1.95

sclerotia, CEGF, and Pg contents in soils from the aspect of comparative humus chemistry will help clarify the implications of sclerotial formation in forest soils.

9.4

Altitudinal Distribution of Sclerotia in Surface Soils

Finally, detailed sclerotia distributions in surface soils were examined along an altitudinal gradient in two mountains (Sakagami 2011). The objective of this study is to obtain further knowledge of the function of sclerotia as an organic component of soil by examinations of geographical distribution of sclerotia along altitudinal gradient in temperate and arctic-alpine climatic zones in central and northern Japan. Examinations were carried out at Mt. Iwaki (Aomori prefecture) and Mt. Ontake along altitudinal gradients. Soil samples were collected from surface A horizon (approximately 0–5 cm in depth). Twenty-two points were examined at Mt Iwaki along the driveway and the trail: line 1 (Mt. Iwaki 1), from point 1 (40
370 4100 N, 140
150 1500 E; 400 m asl.) to point 10 (40
390 1400 N, 140
180 0200 E; 1480 m asl.); line 2 (Mt. Iwaki 2), from point 11 (40
370 5600 N, 140
160 0400 E; 484 m asl.) to point

164

N. Sakagami and S. Kato

Fig. 9.7 Soil pH (H2O), T-C content, AlEx content, Alp/Alo ratio, and sclerotia contents in each soil profile of Mt. Chokai

22 (40
390 0700 N, 140
170 2600 E; 1156 m asl.) on July and September 2006 (Fig. 9.10a). The vegetation of the pediment area of Mt. Iwaki was secondary forest (mainly Q. serrata, Fig. 9.10b) and points 4–7 and 14–21 were F. crenata forest (Fig. 9.10c), and higher points were occupied with Betula ermanii, Alnus maximowiczii, or S. kurilensis (Fig. 9.10d). As for Mt. Ontake, soil samples were taken from 16 points (35
550 2900 N, 137
260 4100 E; 1715 m asl.–35
540 3700 N, 137
280 5900 E; 2770 m asl.) along a trail on June 2006 (Fig. 9.11a). The main vegetations were B. ermanii at point 1, and A. veitchii or T. diversifolia at points 2–11 (Fig. 9.11b, c). The timber line was around point 12, and higher regions were covered with P. pumila (Fig. 9.11d). In addition, Warmth Index (WI) for all points was calculated according to Kira (1976), using the Mesh Climatic Data 2000 (Japan Meteorological Agency 2002). The distribution of sclerotia at Mt. Iwaki and Mt. Ontake are summarized in Figs. 9.12 and 9.13. The averages of weight density of sclerotia for Mt. Iwaki and Mt. Ontake were 0.80 mg/g and 0.81 mg/g, respectively. There was a clear peak of sclerotia content at point 5 for the weight density (2.5 mg/g) and at point 6 for the count density (25 grain/g) in F. crenata forest of Mt. Iwaki 1. As for Mt. Iwaki 2, two peaks of weight density were observed around the boundary area of F. crenata forest (points 13, 14 and 20, 21). A relatively low weight and low count of sclerotia was observed at point 16, where C. japonica locally dominated, compared to the surrounding area. Sclerotia of Mt. Ontake tended to be large compared to those of Mt. Iwaki. Three distributional peaks were recognized at points 1, 8, and 15 on

9 Spatial Distribution of Sclerotia Grains in Forest Soils, Northern and Central. . .

165

Fig. 9.8 Contents of (a) free aluminum by their status, and (b) free iron oxides in each profile

Mt. Ontake and extremely large values for both weight (2.9 mg/g) and count (4.7 grain/g) were observed at point 1. As point 1 was occupied with B. ermanii, planted as a part of development of a lodging area in Nigorigo-Onsen village, sclerotia at this location might be formed under anthropogenic conditions. Figure 9.14 illustrates the relationship between pH (KCl) and T-C under different WI values. Besides several exceptions, there was a notable tendency that pH (KCl) descended and T-C ascended when WI shifted from 64 to 46 at Mt. Iwaki (WI > 45 area, Fig. 9.14a). On the contrary, pH increased and T-C decreased when WI shifted from 44 to 30 (Fig. 9.14b). As the same, pH decreased and T-C increased when WI changed from 42 to 35 (Fig. 9.14c), and the opposite trend was observed for points with WI from 33 to 15 (Fig. 9.14d) at Mt. Ontake. The weight density of sclerotia was high in areas of pH (KCl) < 3.5 and T-C > 200, which is represented by a dotted circle in Fig. 9.14.

166

N. Sakagami and S. Kato

Fig. 9.9 Relationship between Pg index and count of sclerotia

Fig. 9.10 Sampling area at Mt. Iwaki, (a) sampling points and (b–d) landscapes

9 Spatial Distribution of Sclerotia Grains in Forest Soils, Northern and Central. . .

167

Fig. 9.11 Sampling area at Mt. Ontake, (a) sampling points and (b–d) landscapes

Figure 9.15 shows a strong positive relationship between the C/N ratio and mean weight of sclerotia. According to Littke et al. (1984) and LoBuglio (1999), Cg was observed to grow more rapidly at low N concentrations, but at the expense of biomass production. It is suggested that sclerotia may enlarge their sizes in soil of low N concentrations with association of preservation of their species. As it is known that approximately 50 wt% of sclerotia consists of C (Watanabe et al. 2007a), the contribution of carbon in sclerotia to soil T-C can be estimated from the following equation “Sclerotial C contribution (%) ¼ (Weight density of sclerotia  0.5)/ (T-C)  100.” The average of their contribution in the studied area was 0.17% and the maximum contribution was 0.96% in mineral soil under P. pumila at Mt. Ontake (Point 15). Sclerotia had an optimum distribution in cool-temperate and subalpine vegetation zones with a distinct peak at the boundary of these two zones. Sclerotia content showed larger seasonal variance compared to T-C, presumably due to activities of micro-organisms. Soils of which large amounts of sclerotia were accumulated, such as Mt. Iwaki and Mt. Ontake, are likely to be resulted from lower decomposing rates of soil organics due to the lower temperatures. Although the contribution of sclerotial carbon to total soil carbon (T-C) is small (0.6) of organic bonding aluminum to amorphous aluminum, and with high contents of exchangeable aluminum (Al3+) (>0.54 g kg
1). The content and state of active aluminum appeared to be the factor responsible for the development of sclerotia, because sclerotia grains were not detected in acid soils with a low content of free colloidal aluminum. The intensive clay destruction associated with past lessivage, or clay particle leaching, may have depleted the free colloid aluminum in these forest soils. Although there was no clear relationship between sclerotia distribution and microtopography, field examination of sclerotia grains in soil showed that the presence of sclerotia grains is an indicator of soil chemical properties such as acidity and the degree of soil humification. Keywords Albic luvisols · Sclerotia grain density · Dystric cambisols · Ergosterol · Exchangeable aluminum · Haplic podzols · Microtopography · pH value

M. Watanabe (*) Department of Geography, Graduate School of Urban Environmental Sciences, Tokyo Metropolitan University, Hachioji, Tokyo, Japan e-mail: [email protected] N. Sakagami College of Agriculture, Ibaraki University, Ibaraki, Japan e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2021 M. Watanabe (ed.), Sclerotia Grains in Soils, Progress in Soil Science, https://doi.org/10.1007/978-981-33-4252-1_10

173

174

10.1

M. Watanabe and N. Sakagami

Distribution of Sclerotia Grains in Forest Soil Profiles in the Harz Mountains

10.1.1 Introduction Kumada and Hurst (1967) examined sclerotia grains of Cenococcum graniforme (currently Cenococcum geophilum) in Podzols in Britain and Sweden. They showed that the density of sclerotia grains was higher in the B horizon than in the A horizon. Studies of sclerotia grains in Andosols in Japan showed that sclerotia grains collected from acidic forest soils contain aluminum in the form of aluminum–humus complexes. Furthermore, the distribution of sclerotia grains in several Andosol profiles in Japan indicated that sclerotia grains are present in surface A and buried A horizons of nonallophanic (organic bonding aluminum/amorphous aluminum [Alp/Alo] > 0.5) soils, and that the mean weight of individual sclerotia grains was highly correlated with the content of exchangeable aluminum (AlEx) (Watanabe et al. 2001, 2002). These findings suggest that the development of sclerotia grains in Andosols is dependent on the status of active aluminum in the soil and may be part of a symbiotic relationship, protecting plant roots from toxic aluminum ions. We performed a comparative study in the Harz Mountains of central Germany, examining the distribution of sclerotia grains and the chemical properties of non-volcanic ash soils to determine whether aluminum status in soils is an important factor for sclerotium development at low pH.

10.1.2 Materials and Methods 10.1.2.1

Geography and Soil Profiles of the Study Area

The Harz range is 90 km long, 30 km wide, and extends NW–SE in central Germany. Mt. Brocken is the highest peak in the Harz range, with an elevation of 1142 m a.s.l. (Fig. 10.1). Paleozoic rock is present near the surface and is covered with detrital accumulation, which was developed during the Pleistocene under periglacial conditions. This cover of debris contains weathered bedrock and wind-borne sediment

Fig. 10.1 Diagram of the transect layout in the study area on the eastern slope of the Harz Mountains, Germany

10

Spatial Distribution of Sclerotia Grains in Low-pH Forest Soils, Central Germany 㻜 㻭㼔 㻜㻚㻤㻝

㻰㼑㼜㼠㼔䠄㼏㼙䠅

㻝㻜

㻞㻜

㻱 㻜㻚㻝㻝 㻮㼟 㻜

㻭㼔 㻝㻚㻝㻢 㻱 㻜㻚㻥㻟

㻭䠍 㻜㻚㻢㻝

㻮㻝 㻝㻚㻠㻞

㻭㻞 㻠㻚㻥㻥

㻮㻞 㻜㻚㻝㻢

㻮㼣 㻜

㻭㼔 㻝㻚㻢㻥 㻮㻝 㻜㻚㻜㻢

㻭㻟 㻝㻚㻟㻢

㻮 㻜㻚㻟㻝

㻮㻞 㻜

㻭㻝 㻜㻚㻡㻟

㻭㼔 㻜㻚㻝㻟

㻭㻞 㻜㻚㻜㻢 㻱 㻜

㻮㻝 㻜㻚㻜㻤

㻮㻛㻯 㻺㼛 㻰㼍㼠㼍

㻭㼔 㻜㻚㻝㻟 㻮㻝 㻜㻚㻜㻞 㻮㻞 㻺㼛 㻰㼍㼠㼍

㻭㼔 㻜㻚㻤㻢 㻮㻝 㻝㻚㻝㻠

㻭㼔 㻜 㻮㻝 㻜

㻮㻞 㻜㻚㻞㻡 㻮㻛㻯 㻺㼛 㻰㼍㼠㼍

㻮㻞 㻺㼛 㻰㼍㼠㼍

㻭㼔 㻜㻚㻝㻠 㻱 㻜㻚㻟㻟

㻭㼔 㻜 㻮㻝 㻜㻚㻜㻝 㻮㻞 㻜

㻭㼔 㻜 㻮㻝 㻜㻚㻜㻝 㻮㻞 㻜

㻮㼟 㻜㻚㻜㻠 㻮㻟 㻜

㻮㻞 㻜

㻯 㻺㼛 㻰㼍㼠㼍

㻟㻜

175

㻮㻛㻯 㻺㼛 㻰㼍㼠㼍

HoheWarte

Heinrichsburg

Ramberg

Allrode

Galgenberg

Mosebach

Tanne

Elend

Sorge

Brandhai

㻡㻜

Oderteich

㻠㻜

Guentersberg

㻮㻛㻯 㻜 㻮㻛㻯 㻜

Fig. 10.2 Distribution of sclerotium grains by soil profiles in Harz Mts. The density (g kg
1) of grains in each layer is shown at the corresponding position in the figure. (Reproduced from Watanabe et al. 2004, Taylor & Francis Ltd, http://www.tandfonline.com)

such as loess (Haasse et al. 1989). The climate is sub-Atlantic to subcontinental. The western slope of the Harz range is more strongly influenced by the oceanic climate, whereas the eastern side shows a typical montane pattern of precipitation and temperature. Mainly due to the Luv–Lee effect, annual total precipitation decreases from west (>1000 mm) to east (0.5 mm in diameter) were collected by using tweezers and air-dried. Sclerotia grain content was determined based on the count or the weight of oven-dried sclerotia grains per gram of oven-dried soil.

10.1.2.3

Scanning Electron Microscopy and Energy Dispersive X-ray Spectroscopy of Sclerotia Grains

Sclerotia grains were sliced in half with a sterilized knife and the internal morphological features were observed using a scanning electron microscope (SEM) (S-800: Hitachi Ltd., Tokyo, Japan). The elemental composition of the internal structures (targeted area: 100–200 μm) was determined by using energy dispersive X-ray spectrometry (EDS) (PV9900I, EDAX Mahwah, NJ, USA).

10.1.2.4

Chemical Analyses

All soil samples were air-dried and passed through a 0.5-mm sieve for elemental analysis or through a 2.0-mm sieve for determination of humus composition, cation exchange capacity (CEC), content of exchangeable bases (Na, K, Mg, Ca), selective dissolution, AlEx, and pH value. Analyses of element composition, humus composition, CEC, exchangeable bases, and mineral composition were performed only for

10

Spatial Distribution of Sclerotia Grains in Low-pH Forest Soils, Central Germany

177

the three representative profiles (Oderteich, Elend, and Guentersberg); all other chemical analyses were conducted for all profiles. An energy dispersion X-ray fluorescence micro-analyzer, EDX (JSX-3201; JEOL, Tokyo, Japan) was used to determine soil chemical composition. The total carbon and nitrogen contents were measured by the dry combustion method using a NC-analyzer (NC-80: Sumica Bunseki Center, Osaka, Japan). Soil pH values in H2O, KCl, and NaF were measured by using the glass electrode method on soil suspended in 2.5 (H2O, KCl) or 50 (NaF) times volume of H2O, 1 mol L
1 KCl, and 1 mol L
1 NaF, respectively. The CEC was determined according to the semi-micro Scholenberger method. Exchangeable bases, expressed in cmolc kg
1 soil, were determined using an atomic absorbance spectrophotometer (AA-6400, Shimadzu Corp., Kyoto, Japan) on a soil sample extracted with 1 mol L
1 ammonium acetate (CH3COONH4) at pH 7. Base saturation (%) was calculated from CEC values and total amount of exchangeable bases. Quantitative analysis of free oxides (Al, Fe, Si) was carried out by using the selective dissolution method (Blakemore et al. 1987). Contents of Al, Fe, and Si, extracted by shaking for 4 h in the dark in acid oxalate reagent (pH 3; Alo, Feo, Sio), and contents of Al and Fe, extracted by shaking for 16 h in sodium pyrophosphate (Alp, Fep), were measured using an atomic absorbance spectrophotometer (AA-6400, Shimadzu Corp.). The AlEx content was determined using an extract prepared with 1 mol L
1 KCl, according to the method of Blakemore et al. (1987). Humus composition was determined by using the procedure proposed by Kumada et al. (1967). Free and combined humic acids (HA) were extracted using 1 M NaOH or 1 M Na4P2O7, respectively, and their colorimetric properties were measured by using a UV spectrophotometer (UV 2400, Shimadzu Corp.).

10.1.2.5

Mineral Analysis

Mineral composition was determined by using an X-ray diffractometer (Rigaku miniflex, Tokyo, Japan) under CuKα radiation conditions (30 kV, 15 mA). X-ray diffraction patterns for the clay fraction were obtained on untreated, dithionitecitrate-bicarbonate-treated, magnesium-treated, ethylene glycol-treated, potassiumtreated, and K-saturated samples heated to 550  C from 3 to 30 at 2θ.

10.1.2.6

Soil Ergosterol Analysis

Ergosterol is used as a marker of living fungal biomass (Grant and West 1986; Montgomery et al. 2000). The ergosterol content of each soil sample was determined to examine the relationship between fungal biomass and sclerotium grain density. Soil ergosterol was extracted by using the microwave-assisted method of Montgomery et al. (2000) and determined by high-performance liquid chromatography, using a system composed of a pump (CCPE-II, Tosoh, Tokyo, Japan), an injector (7125, Rheodyne, Bensheim, Germany), a UV detector (UV-8000, Tosoh, Tokyo, Japan) operating at 282 nm, and an octadecylsilyl column (100  4.6 mm,

178

M. Watanabe and N. Sakagami

Tosoh Super ODS, Tosoh, Tokyo, Japan). The mobile phase consisted of acetonitrile and methanol (8:2, vol/vol) and the flow rate was 1.3 mL min
1. Ergosterol content was determined by comparing peak areas of samples with those of an external standard (Sigma-Aldrich, St. Louis, MO, USA).

10.1.3 Results and Discussion 10.1.3.1

Properties of Representative Soil Profiles

The properties of Oderteich, Elend, and Guentersberg soil profiles are shown in Tables 10.1, 10.2, and 10.3. Oderteich soil (Podsole): Ah/E/Bs/BC, Haplic Podzol—The basic mineralogical properties of this soil were weathered mica, quartz, and feldspar derived from granite bedrock. Characteristic layered silicates were not detected in the Ah horizon, whereas the dominant clay minerals in the E and Bs horizons were determined to be smectite and hydroxy-interlayered vermiculite, respectively. The SiO2/R2O3 ratio and the value of the Alo + ½ Feo element composition obtained from total analysis and selective dissolution analysis, respectively, defined the spodic horizon (Bs) in this profile. The basic chemical properties of this profile were extremely low pH values (pH (H2O) < 4, pH (KCl) < 3), relatively high CEC values due to the high carbon content, and low base saturation (9.5) of the E and B horizons indicated the influence of tephric materials. The pH (H2O, KCl) values, content of exchangeable bases, and base saturation of the E horizon were slightly higher than those of the Oderteich E horizon. The CEC and total carbon content of the Elend E horizon were slightly lower than those of the Oderteich E horizon. The humification degree of the Ah horizon was higher than that of Oderteich soil for both free and combined HA. The free and combined HA were determined to be P and

a

not determined

Guentersberg

Elend

Soil profile Oderteich

Horizon Ah E Bs Bw Ah E B B/C Ah B1 B2 B/C

Depth (cm) 0–5 5–10 10–15 15– 0–6 6–12 12–34 34– 0–2 2–10 10–35 35–

Color 10YR2/2 2.5YR3/1 10YR4/4 2.5Y6/4 10YR2/2 10YR4/4 2.5YR6/4 5YR7/3 2.5YR3/2 10YR6/3 2.5YR7/3 2.5YR7/3

Texture SL SL SCL LS SiC SiL SC SCL SiC LiC LiC SiC 14.6 10.9 7.1

a

19.1 20.5

a

a

a

9.2 22.1

a

Clay content (%)

pH H2O 3.81 3.57 3.91 4.65 4.39 4.30 4.60 5.10 3.55 4.09 4.20 4.20 3.02 3.39 4.24 3.89 2.90 3.37 3.78 3.90

a

KCl 2.01 2.08 2.78

9.71 11.32 9.34 6.66 8.10 9.26 9.18

a

a

6.48 8.30

a

NaF

TC (%) 44.25 12.82 8.23 1.35 26.73 12.06 4.00 0.50 16.87 2.98 1.01 0.37

TN (%) 1.98 0.59 0.37 0.08 1.15 0.59 0.24 0.05 0.68 0.19 0.07 0.02

C/N ratio 22.3 21.7 22.3 16.3 23.3 20.5 16.7 9.7 24.9 16.0 14.3 17.1

Table 10.1 Horizon, color, texture, clay content (%), pH values, TC (%), TN (%), CN ratio of Oderteich, Elend, and Guentersberg soil (Revised from Watanabe et al. 2004)

10 Spatial Distribution of Sclerotia Grains in Low-pH Forest Soils, Central Germany 179

a

not determined

Guentersberg

Elend

Soil profile Oderteich

Horizon Ah E Bs Bw Ah E B B/C Ah B1 B2 B/C

3.61 3.41 2.86

a

a

a

10.10 11.37 11.22

a

a

79.69 80.14 79.68

a

a

5.88 5.56 5.56

a

Elemental composition by EDX (wt%) Al2O3 Fe2O3 SiO2 SiO2/R2O3 9.50 13.35 58.80 2.56 12.27 2.61 78.68 5.26 13.80 9.57 71.40 3.03 17.84 4.33 70.76 3.23 13.58 8.75 68.63 3.03 19.54 9.69 63.98 2.17 23.24 7.47 62.92 2.04

Extractable Al, Fe, Si by selective dissolution (g kg
1) Ald Alo Alp Sio Fed Feo 1.81 3.47 2.34 0.63 1.86 3.71 2.22 3.82 2.75 0.36 3.07 4.43 6.77 7.99 7.62 2.01 29.89 40.62 5.11 10.83 3.99 1.56 6.11 6.12 2.71 4.28 4.50 1.16 12.03 12.17 11.69 12.45 10.26 2.11 16.31 20.08 13.71 17.87 8.97 4.54 9.30 8.00 1.14 3.24 1.84 0.64 4.03 3.92 0.79 2.31 1.67 0.56 5.82 5.20 1.03 3.30 2.28 0.94 6.30 6.84 0.46 3.00 1.96 0.81 4.08 3.89 0.00 2.13 1.42 0.79 2.53 2.47

Alo + ½ Feo 5.33 6.04 28.30 13.88 10.37 22.49 21.87 5.20 4.91 6.72 4.94 3.37

Table 10.2 Element composition by EDX analysis and Al, Fe, Si content by selective dissolution analysis of Oderteich, Elend, and Guentersberg soil (Revised from Watanabe et al. 2004)

180 M. Watanabe and N. Sakagami

10

Spatial Distribution of Sclerotia Grains in Low-pH Forest Soils, Central Germany

181

Table 10.3 Mineral composition and content of exchangeable aluminum (cmol(+) kg
1 soil) of Oderteich, Elend, and Guentersberg (Revised from Watanabe et al. 2004)

Soil profile Oderteich

Elend

Guentersberg

Horizon Ah E Bs Bw Ah E B B/C Ah B1 B2 B/C

Clay mineral composition Sm/ AlVt Ch Sm Vt Vt





++




++

Mc   

Kt   

Qz + + +

Fd   

Exchangeable Al (cmol (+) kg
1 soil) 7.95 11.06 13.46





  

   +   


+


+ +

+ + + + ++ ++ ++ +

 

  

8.11 10.88 3.11 3.56 5.31 5.86 3.53


  
 





+



++


+





+





2.58

Vt vermiculite, Ch chlorite, Sm smectite Sm/Vt interstratified smectite–vermiculite, Al-Vt hydroxyinterlayered vermiculite, Mc mica, Kt kaolinite, Qz quartz, Fd feldspars

A/B, respectively. More than 90% of the extracted HA was in free form for all horizons except the C horizon (Watanabe et al. 2004). Guentersberg soil (Braunerde-Podsole): Ah/B1/B2/BC, Dystric Cambisol—The primary minerals in the Guentersberg soil were chlorite, mica, quartz, and feldspar. Smectite and interstratified smectite-vermiculite were found to be the dominant silicates in the Ah horizon, whereas kaolinite was detected in the B horizons, along with abundant quartz. The mineral composition and relatively low clay content of this soil indicate a strong weathering process, compared with the Podsole and Braunfahlerde profiles. Element composition analysis showed a relatively large SiO2/R2O3 ratio, and the AlEx content (Ald, Alo, Alp) was relatively low throughout the profile. These characteristics suggested the clay deconstruction process is stronger in this profile than in the former two profiles. The pH values in H2O and KCl (3.55–4.20 and 2.90–3.90, respectively) were lower than the values in Elend soil (4.39–5.10 and 3.02–3.89, respectively). Nevertheless, base saturation values were relatively high (>100) due to the low CEC and high content of exchangeable bases. The field texture of the B2 and B/C horizons, described as LiC, demonstrated that the silt fraction, not the humus, might be responsible for the high content of exchangeable bases in these soils. The humus properties of Guentersberg soil were similar to those of the former two soils, except for the low RF2 value (30–40).

182

10.1.3.2

M. Watanabe and N. Sakagami

Content of Sclerotia Grains and Soil Ergosterol

Figure 10.2 shows the spatial distribution of sclerotia grains along an eastward transect from Mt. Brocken. Sclerotia grain content is represented as grain weight per soil weight (g kg
1). In the A, E, and B horizons, the values were 0–4.99 g kg
1, 0–0.93 g kg
1, and 0–0.31 g kg
1, respectively. The largest content was observed in the A2 horizon of the Sorge soil. Across the transect, content decreased in the leeward (eastward) direction. The boundary between sclerotia-containing soil and sclerotia-free soil was near Allrode, about 30 km east of Mt. Brocken.

10.1.3.3

Relationship Between Soil Properties and Sclerotia Grains

Soil ergosterol content tended to harmonize with sclerotia grain content in the ergosterol-rich soil profiles at Brandhai, Sorge, and Allrode (Fig. 10.4). However, sclerotia content was small in the ergosterol-rich Heinrichsburg soil profile. The pH (KCl) values and AlEx content for Horizons A and E for all 12 profiles differed by soil types. Haplic Podzols (Podsole) and Dystric Cambisols (BraunerdePodsole) both had low pH values, but the exchangeable Al (Al3+) content was higher in Haplic Podzols. Albic Luvisols (Braunfahlerde) had higher pH, with a wide range of AlEx content. It was recognized that the relationship between AlEx content and pH (KCl) value is not clear for soils that with pH (KCl) < 4. The free oxide composition and clay mineral composition of the three representative profiles (Tables 10.2 and 10.3) indicate that a strong leaching process migrated aluminum colloids from the surface layers of both Podsole and Braunerde-Podsole. Furthermore, past lessivage likely induced clay destruction in the Braunerde-Podsole. This process may also be responsible for the properties of the Hohe Warte soil (Braunfahlerde), having low pH and low AlEx content. The pH (KCl), Alo, Alp, and Al3+ content of Hohe Warte soil were 3.85 g kg
1, 3.09 g kg
1, 1.88 g kg
1, and 0.21 g kg
1, respectively, indicating that the low AlEx content under low pH conditions is related to the low content of active aluminum in the form of humus-Al complexes (Alp). Volcanic glass particles were observed by microscopy in Elend soil, and the pH (NaF) value was >9.5. Further investigation is required to determine whether tephric material is the origin of the free aluminum in the Elend soil, which had a relatively large sclerotia grain content. Figure 10.5 shows the relationship of Alp/Alo to AlEx content, combined with sclerotia grain density. With the exception of the Bs horizons of the Podzols (Podsole), which were less than 10 cm from the surface, the content of sclerotia grains was relatively high in soil samples with an Alp/Alo value >0.7 and an Al3+ content >0.54 g kg
1 (6 cmolc kg
1). Soil samples from Guentersberg (Dystric Cambisols/Braunerde-Podsole), Heinrichsburg (Dystric Cambisols/BraunerdePodsole), and Hohe Warte (Albic Luvisols/Braunfahlerde), in which few sclerotia grains were detected, were clearly excluded from the area (Alp/Alo ratio > 0.7, Al3+ content > 0.54 g kg
1) described in Fig. 10.5.

10

Spatial Distribution of Sclerotia Grains in Low-pH Forest Soils, Central Germany

Density of sclerotium grain, (mg g–1) 1

0

2

3

4

5

Density of sclerotium grain, (mg g–1) Density of sclerotium grain, (mg g–1) 4 5 0 2 4 0 1 2 3 Ah

A1

Ae

A2

Bh

A3

Ah E (1) Oderteich

Bir

B

Bw

0

1

2

4

3

Density of sclerotium grain, (mg g–1)

5

1

0

Ah

0

15

5 10 Ergosterol, (µg g–1) 2

3

4

(3) Sorge

B

(2) Brandhai 0

Density of sclerotium grain, (mg g–1)

183

5 10 15 Ergosterol, (µg g–1)

Density of sclerotium grain, (mg g–1)

5

0

Ah

1

2

3

4

5

Ah

Bw Bh C1

(4) Elend

B1

(5) Tanne

(6) Mosebach

Bg

C2

15 5 10 Ergosterol, (µg g–1)

0 Density of sclerotium grain, (mg g–1)

Density of sclerotium grain, (mg g–1) 0

1

2

3

4

0

5

1

2

3

4

Density of sclerotium grain, (mg g–1)

5

Ah

0

1

2

3

4

5

Ah

Ah Bw

Bh (8) Allrode

BC 0

5 10 Ergosterol, (µg g–1)

1

2

3

4

0

15

Density of sclerotium grain, (mg g–1) 0

5 10 Ergosterol, (µg g–1)

C2 15

Density of sclerotium grain, (mg g–1)

5

0

1

2

3

4

Density of sclerotium grain, (mg g–1)

5

0

Ah

Ah

Ah

Ae

Bh

Bh B1

(10) Ramberg

B 0

5 10 Ergosterol, (µg g–1)

(9) Guntersberge

C1

(7) Galgenberg

B1

(11) Heinrichburg

B

15

1

2

3

4

5

(12) HoheWarte

B2 0

5 10 Ergosterol, (µg g–1)

15

Ergosterol, µg g–1 Density of sclerotium grain, mg g–1

Fig. 10.4 Content of sclerotia grains (g kg
1) and ergosterol in soil. (Data source: Watanabe et al. 2004)

10.2

Micro-Topographical Distribution in Single Stand Forest Soil

10.2.1 Field Sites and Methodology To determine fine-scale distribution of sclerotia, we focused on the area of maximum sclerotia density in the Harz Mts. Topographical measurement, identification of

184

M. Watanabe and N. Sakagami

Magnitude of the sclerotium grain density 0mg/g soil 0-0.1 0.1-0.2 0.2-0.5 0.5-1.0 1.04.99

-1

Exchangeable-Al (g kg soil)

2

1

0.54

0

0

0.6

1.2

Alp/Alo Fig. 10.5 The relationship of organic bonding aluminum to amorphous aluminum (Alp/Alo) and exchangeable aluminum content (AlEx, g kg
1), showing sclerotia grain density. (Reproduced from Watanabe et al. 2004, Taylor & Francis Ltd, http://www.tandfonline.com)

understory vegetation, and surface soil sampling were performed in a single stand of Picea abies located in Elend. Soil samples were collected at five sites (A–E) along seven transects (A, B, C, D1–3, and E), to capture differences in microtopography. The slope was measured at 1-m intervals along the seven transects, ranging in length from 10 to 114 m, and surface soil samples (approximately 0–5 cm in depth) were collected at 86 points along the transects, followed by vegetation surveys and soil analyses. Understory vegetation was classified as (1): Dicotyledoneae; (2): Monocotyledoneae; (3): Lichens and Bryophyta and was evaluated qualitatively by intensity (+ to +++). Analyses of soil properties were carried out on air-dried soil samples that had been passed through a 0.5-mm sieve for determination of total carbon and nitrogen contents and a 2.0-mm sieve for other analyses. Soil pH values in H2O and KCl were measured by using the glass electrode method, using soil suspended in 5 volume of H2O and 1 M KCl, respectively. The content of AlEx was determined using soil extracted with 1 M KCl, according to the method of Blakemore et al. (1987). The extract was titrated with 0.01 M NaOH, 4 mL of NaF was added, and the content of AlEx was estimated by titration with 0.01 M HCl. To measure Alp, soil samples were shaken in sodium pyrophosphate for 16 h and then analyzed using inductively coupled plasma spectrometry (ICPS-1000IV, Shimadzu Corp.). The total carbon (T-C) and nitrogen (T-N) contents were measured by the dry combustion method using a NC-analyzer (NC-80, Sumica Chemical Analysis Service Ltd., Tokyo, Japan). The Melanic Index (MI) and Pg index (PI) were determined by the procedure

10

Spatial Distribution of Sclerotia Grains in Low-pH Forest Soils, Central Germany

185

proposed by Honna et al. (1988) and Yamamoto et al. (2000). The colorimetric properties of free HA extracted with 1 M NaOH were measured by using a UV spectrophotometer (UV 2400, Shimadzu Corp.). Absorbance values were measured at 450, 520, 600, and 610 nm, and K450/K520 and K610/KA600 were calculated to obtain the MI and PI, respectively. A lower MI value indicates a higher degree of humification, and a higher PI value indicates a higher content of “Pg.” Using these indices, the HA type can be classified into the following five categories: P+ to +++; P and Rp(1); B and P0; Rp(2); and A (Yamamoto et al. 2000). For sclerotia collection, 500 mL of distilled water was added to 10–30 g air-dried soil samples and then the floated sclerotia were collected using tweezers. Sclerotia were classified by diameter: 0.2–0.5 mm, 0.6–1.0 mm, 1.1–1.5 mm, 1.6–2.0 mm, 2.1–2.5 mm, and 2.6–3.0 mm. Sclerotia content was determined by weight (SGw: mg g
1) and count (SGc: count g
1) of sclerotia per gram of oven-dried soil. Air-dried sclerotia were sliced in half using a sterilized knife to observe the morphological features and chemical composition of the transverse wall. For this analysis, large grains were selected from three sites (B-5, C-5, and E-8) that differed in soil pH (KCl) and AlEx content. The chemical composition of these samples was determined using SEM-EDS analysis system (JSM-6490LA, JEOL Ltd., Tokyo, Japan), accelerating voltage of 15 eV.

10.2.2 Results and Discussion Figures 10.6 and 10.7 show the distribution of sclerotia (SGw and SGc) along the transects at sites A–C and D–E, respectively. The overall averages of SGw and SGc were 0.54 mg g
1 and 1.3 count g
1, respectively. Sites A and B are a side slope and a crest slope, respectively. At site A, sclerotia were most abundant at point A-2 (2.0 mg g
1 and 2.1 count g
1). At site B, sclerotia were most abundant at point B-2, near the bottom of the crest slope (1.1 mg g
1 and 3.0 count g
1). No sclerotia were detected at the bottom of the opposite side slope at site B, at point B-12. This point lacked P. abies but had grass coverage. Site C is a transverse section of the lower side slope and bottomland, encompassing a river. Sclerotia were evenly distributed across the bottomland. At site D, we established three transects (D1–D3), three parallel transverse lines that crossed the watercourse at the head of the channel hollow. Point D2-7 showed the maximum SGc and D3-9 had the maximum SGw, at 7.8 count g
1 and 2.8 mg g
1, respectively. Five points at site D contained no sclerotia. These points were relatively flat and had high levels of crude organics. Site E is a channel hollow tangential to the side-slope line. At site E, sclerotia were most abundant at point E-2 (5.4 mg g
1 and 5.8 count g
1). Point E-9, near the edge of a cliff, showed a higher content of sclerotia than adjacent points. The small peak in sclerotia at point E-14 might have been caused by transportation of sclerotia down the cliff wall. Exceptionally, no sclerotia were present at the top of the cliff at point E-17, but sclerotia were present on the cliff wall (E-15).

186

M. Watanabe and N. Sakagami

Fig. 10.6 Microtopography and vegetation of sites A–C and the distribution of sclerotial biomass. The upper diagram for each site shows the microtopography, with sampling points indicated by . The bar graphs below each topographical diagram show understory vegetation (FV) (solid bars: Dicotyledoneae; gray bars: Monocotyledoneae; open bars: lichens and Bryophyta). The third and fourth graphs show sclerotial biomass by weight (filled circle: SGw, mg g
1) and count (filled diamond: SGc, count g
1) of air-dried sclerotia in air-dried soil. Open circles and diamonds indicate that no sclerotia were collected at that point. (Reproduced from Sakagami 2009)

pH values (H2O and KCl) of the study sites (A–E) ranged from 3.0 to 4.5 and from 2.5 to 4.0, respectively. The average pH (KCl) at site B was 3.7; this value was the highest among the five sites, which had an overall average of 3.1. The AlEx content was higher in lower pH (KCl) soils, with a maximum of 2.5 g kg
1. The average AlEx content across all sites (A–E) was 0.58 g kg
1. T-C was approximately 150–300 g kg
1. Since the content of crude organics in soil was high at sites A and E, the high T-C contents (>400 g kg
1) observed at sites A and E may have contained crude organics. MI and PI values were 1.9–2.3 and 0.91–1.0, respectively. Sclerotia were present at every site under P. abies forest canopy, but sclerotia presence was not correlated with microtopography. At most points, SGw was

10

Spatial Distribution of Sclerotia Grains in Low-pH Forest Soils, Central Germany

187

Fig. 10.7 Microtopography of sites D and E and the distribution of sclerotial biomass. The upper diagram for each site shows the microtopography, with sampling points indicated by . The bar graphs below each topographical diagram show understory vegetation (FV) (solid bars: Dicotyledoneae; gray bars: Monocotyledoneae; open bars: lichens and Bryophyta). The third and fourth graphs show sclerotial biomass by weight (filled circle: SGw, mg g
1) and count (filled diamond: SGc, count g
1) of air-dried sclerotia in air-dried soil. Open circles and diamonds indicate that no sclerotia were collected at that point. (Reproduced from Sakagami 2009)