Fungi in Polar Regions [1 ed.] 9781138089709, 9781315109084, 9781351611985, 9781351611978, 9781351611992

Table of contents :

TABLE OF CONTENTS



An Index of Non-lichenized Fungi Recorded in the Vicinity of Syowa Station, East Antarctica


Masaharu Tsuji



Diversity and Ecology of Fungi in Polar Region: Comparisons between Arctic and Antarctic Plant Remains


Takashi Osono, Shunsuke Matsuoka, Satoru Hobara, Dai Hirose and Masaki Uchida



Snow Molds and their Antagonistic Microbes in Polar Regions


Tamotsu Hoshino, Hisahiro Morita, Yuka Yajima, Masaharu Tsuji, Motoaki Tojo and Oleg B. Tkacehnko



Pathogenic Fungi on Vascular Plants in the Arctic: Diversity, Adaptation, Effect on Host and Ecosystem, and Response to Climate Change


Shota Masumoto



DNA Metabarcoding for Fungal Diversity Investigation in Polar Regions


Shunsuke Matsuoka, Yoriko Sugiyama and Hideyuki Doi



Oomycetes in Polar Regions


Motoaki Tojo



Biotechnological Potentials of Arctic Fungi


Purnima Singh and R. Kanchana



Dairy Wastewater Treatment under Low-Temperature condition by an Antarctic Basidiomycetous Yeast


Masaharu Tsuji, Sakae Kudoh and Tamotsu Hoshino



Ethanol Fermentation by the Basidiomycetous Yeast Mrakia blollopis Under Low Temperature Conditions


Masaharu Tsuji and Tamotsu Hoshino

Citation preview

Fungi in Polar Regions

Editors

Masaharu Tsuji National Institute of Polar Research (NIPR) Tachikawa, Tokyo Japan

Tamotsu Hoshino

Bio-production Research Institute National Institute of Advanced Industrial Science and Technology (AIST) Tsukisamu-higashi, Toyohira-ku, Sapporo Hokkaido, Japan

p, p,

A SCIENCE PUBLISHERS BOOK A SCIENCE PUBLISHERS BOOK

Cover illustration: Figure of Antarctic Yeasts. Reproduced by kind courtesy of Dr. Masaharu Tsuji (first editor)

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2019 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed on acid-free paper Version Date: 20190205 International Standard Book Number-13: 978-1-138-08970-9 (Hardback)

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Names: Tsuji, Masaharu, editor. Title: Fungi in Polar regions / editors, Masaharu Tsuji, National Institute     of Polar Research (NIPR), Tachikawa, Tokyo, Japan, Tamotsu Hoshino,     Bio-production Research Institute, National Institute of Advanced     Industrial Science and Technology (AIST), Tsukisamu-higashi, Toyohira-ku,     Sapporo, Hokkaido, Japan. Description: Boca Raton, FL : Taylor & Francis Group, [2019] | “A science     publishers book.” | Includes bibliographical references and index. Identifiers: LCCN 2019000258 | ISBN 9781138089709 (hardback) Subjects:  LCSH: Fungi--Polar regions. Classification: LCC QK615 .F86 2019 | DDC 579.50911--dc23 LC record available at https://lccn.loc.gov/2019000258

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Preface The polar regions consist of the Arctic and Antarctica, which make up around 14% of the Earth’s surface. Antarctica is essentially a continent, and about 1,900 m of ice covers the continent’s ground on average. Approximately 98% of Antarctica is covered in ice and snow, and temperatures around the coastal areas usually range from 5°C to –35°C. Temperatures on the Antarctic plateaus are much more extreme, approximately ranging from –25°C in the summer to –70°C in the winter. On the other hand, the Arctic is mostly ocean, and approximately 10 m of ice covers the sea surface. Temperatures in the North Pole are usually around –18°C. Many people consider Antarctica and the Arctic to have the same environments; however, as mentioned above, they are quite different. True Fungi without Blastocladiomycota, Chytridiomycota, Cryptomycota, Microsporidia are microorganisms that have the following characteristics: (1) they are eukaryotes, (2) they lack plastids, (3) they are heterotrophic organisms, and (4) they lack an amoeba-like stage. There are several reports that discuss about the estimation of fungal diversity. Currently, over 100,000 species of fungi have been reported, and they are known to be the second-most diverse organisms on the planet, next to insects. According to Hawksworth (1991), about 1.5 million species of fungi are estimated to exist in the world. Based on this estimation, most fungal species still remain undiscovered and are yet to be reported. Fungi that inhabit the polar regions not only grow here, but also decompose organic compounds under sub-zero temperatures. Out of these, cold-adapted fungi work as decomposers and play crucial roles in the nutrient cycle of polar-region ecosystems. Fungal numbers in the polar regions are being shown to increase year after year, regardless of extreme environments such as low temperatures, dry ecosystems, and low water and nutrient availabilities. The Japanese Antarctic Research Expedition (JARE) was started in 1957, and marked its 60th anniversary in 2017. Furthermore, the base station at Ny-Ålesund (Spitsbergen, Norway) was established in 1991 and marked its 25th anniversary in 2016. This book was planned and written to commemorate both of these anniversaries. In this book, we focus on fungi that inhabit the polar regions i.e., the Arctic and Antarctica. It provides an up-to-date overview of several areas related to fungi and fungal ecology and physiology. Each chapter is written by experts in their respective field. The chapters

iv  Fungi in Polar Regions in this book approach the fungal biodiversity of different polar region areas, such as the Canadian Arctic, Nordic Arctic, and Antarctica, via culture base techniques and next-generation sequencing. Separate chapters are devoted to pathogenic fungi in the Arctic, along with a checklist of fungi isolated near the Syowa station, which is a JARE base. Some chapters also review application studies of fungi in polar regions for biotechnological advancements. Several previously published books generally focused on cold-adapted microorganisms and extremophiles. However, to best of our knowledge, this is the first book that specifically focuses on fungi inhabiting the polar regions. We gratefully acknowledge all the people who contributed to this book. We hope that it will provide a useful overview regarding the biodiversity and ecophysiology of fungi in polar regions. We also hope that this book will cultivate an interest about polar region fungi in the upcoming generations, and that they will study and contribute to this field in the future. January 2019

Masaharu Tsuji Tachikawa, Tokyo, Japan Tamotsu Hoshino Tsukuba, Ibaraki, Japan

Reference Hawksworth, D.L. 1991. The fungal dimension of biodiversity: Magnitude, significance, and conservation. Mycol. Res. 95: 641–655.

Contents Preface 1. An Index of Non-Lichenized Fungi Recorded in the Vicinity of Syowa Station, East Antarctica Masaharu Tsuji

iii 1

2. Diversity and Ecology of Fungi in Polar Region: Comparisons Between Arctic and Antarctic Plant Remains Takashi Osono, Shunsuke Matsuoka, Satoru Hobara, Dai Hirose and Masaki Uchida

17

3. Snow Molds and Their Antagonistic Microbes in Polar Regions Tamotsu Hoshino, Hisahiro Morita, Yuka Yajima, Masaharu Tsuji, Motoaki Tojo and Oleg B. Tkacehnko

30

4. Pathogenic Fungi on Vascular Plants in the Arctic: Diversity, Adaptation, Effect on Host and Ecosystem, and Response to Climate Change Shota Masumoto

44

5. DNA Metabarcoding for Fungal Diversity Investigation in Polar Regions Shunsuke Matsuoka, Yoriko Sugiyama and Hideyuki Doi

67

6. Oomycetes in Polar Regions Motoaki Tojo

83

7. Biotechnological Potentials of Arctic Fungi Purnima Singh and R. Kanchana

92

8. Dairy Wastewater Treatment Under Low-Temperature Condition by an Antarctic Basidiomycetous Yeast Masaharu Tsuji, Sakae Kudoh and Tamotsu Hoshino

110

9. Ethanol Fermentation by the Basidiomycetous Yeast Mrakia blollopis Under Low Temperature Conditions Masaharu Tsuji and Tamotsu Hoshino

120

Index

133

Color Plate Section 135

1 An Index of Non-Lichenized Fungi Recorded in the Vicinity of Syowa Station, East Antarctica Masaharu Tsuji

Introduction Antarctica is the southernmost landmass on Earth, and has an area of approximately 14 million km2, making it the fifth-largest continent in the world. Approximately 98% of Antarctica is covered by ice and snow, and temperatures in coastal areas usually range from 5°C to –35°C (Ravindra and Chaturvedi 2011). Ice and snowfree areas, present during the austral summer, are located around the coastal area of the continent. Most life forms in the continental Antarctic region are known to inhabit the ice and snow-free areas (Onofri et al. 2007). East Ongul Island (69° 1′ S, 39° 35′ E) is located at the east side of Lützow Holm Bay, East Antarctica. Syowa station was established on this island in 1957 as the base of the Japanese Antarctic Research Expedition (JARE). The island is an ice-free area during the austral summer. To the best of my knowledge, over 1,000 fungal species belonging to 421 genera have been isolated and recorded from Antarctica (Bridge and Spooner 2012); the list of known species from culturing and collection includes 68% Ascomycetes, 23% Basidiomycetes, and 5% Zygomycetes, with the final 4% comprising various other lineages. In addition, 12 Ascomycetous and four Basidiomycetous species National Institute of Polar Research (NIPR), 10-3 Midori-cho, Tachikawa, Tokyo 190-8518, Japan. Email: [email protected]

2  Fungi in Polar Regions have been reported near Syowa station (Soneda 1961, Tubaki 1961a,b, Tubaki and Asano 1965). More recently, 14 genera of microfungi were isolated from willow wood located near the station (Hirose et al. 2013), and four genera of Ascomycetous fungi were newly recorded from moss stem (Hirose et al. 2016). Furthermore, I have reported 10 species of Basidiomycetous fungi and five species of Ascomycetous fungi from the Skarvsnes ice-free area and two new Basidiomycetous yeast species—Cystobasidium tubakii and C. ongulense, and 10 Basidiomycetous yeasts have recently been reported in East Ongul Island (Tsuji et al. 2013a, Tsuji et al. 2017, Tsuji 2018a). The JARE has been active for over 60 years since 1957. As there have been several fungal reports, I have compiled an overview of the fungal species recorded in previous studies. In this chapter, I present a catalog of fungi recorded in the area surrounding Syowa station, including the Breidvagnipa area, Langhovde area, Ongul Islands, Skallen area, and Skarvsnes ice-free area (Fig. 1.1) (Tsuji 2018b).

Fig. 1.1.  Location of the Lützow Holm Bay area, East Antarctica.

Checklist of Fungal Species Recorded Near Syowa Station The following checklist includes both filamentous fungi and yeasts reported in the area surrounding Syowa Station, East Antarctica. As there have been no reports of Chytridiomycota or Zygomycota near the station, this checklist covers members of the Ascomycota and Basidiomycota in the kingdom Fungi. A description of each species is presented alphabetically, starting with Ascomycota and followed by Basidiomycota. Each species name has been verified and corrected using the Index Fungorum (http://www.indexfungorum.org). In addition, for each species, I have listed the major synonyms and recorded collection information near Syowa

An Index of Fungi from Syowa Station 3

station using the references. Of note, a number of isolates or strains were not identified to the species level in the original articles; I have attempted to reclassify these isolates/strains if their DNA sequence was deposited with a DNA data bank. When reclassification to the species level was not possible, the isolates or strains are presented at the genus level. In cases of multiple unclassified isolates or strains of the same genus, the isolates or strains are grouped together.

Ascomycetous fungi Alternaria embellisia Woudenb and Crous 2013, Stud. Mycol. 75: 191 (2013). = Embellisia allii (Campan.) E.G. Simmons, Mycologia 63: 382 (1971). Position in classification: Pleosporaceae, Pleosporales, Pleosporomycetidae, Dothideomycetes, Pezizomycotina. Record near Syowa Station: Surrounding soil of Mago Ike corrected by 48th JARE (Tsuji et al. 2013a). Blodgettia bornetii E.P. Wright, Trans. R. Ir. Acad. 28: 25 (1881). = Blodgettiomyces bornetii (E.P. Wright) Feldmann, Rev. Bryol. Lichénol., N.S. 11: 157 (1939). Position in classification: Incertae sedis, Incertae sedis, Incertae sedis, Incertae sedis, Pezizomycotina. Record near Syowa Station: soil in East and West Ongul Island corrected by 3rd and 4th JARE (Tubaki 1961a). Cadophora luteo-olivacea (J.F.H. Beyma) T.C. Harr. & McNew, Mycotaxon 87: 147 (2003). = Phialophora luteo-olivacea J.F.H. Beyma, Antonie van Leeuwenhoek 6: 281 (1940) [1939–40]. Position in classification: Incertae sedis, Helotiales, Leotiomycetidae, Leotiomycetes, Pezizomycotina. Record near Syowa Station: withering willows in East Ongul Island corrected by 51st JARE (Hirose et al. 2013). The withering willow was brought from Japan. Cadophora malorum (Kidd & Beaumont) W. Gams, Stud. Mycol. 45: 188 (2000). = Phialophora malorum (Kidd & Beaumont) McColloch, Phytopathology 32: 1094 (1942). = Sporotrichum carpogenum Ruehle, Phytopathology 21: 1144 (1931). = Sporotrichum malorum Kidd & Beaumont, Trans. Br. mycol. Soc. 10: 111 (1924). Position in classification: Incertae sedis, Helotiales, Leotiomycetidae, Leotiomycetes, Pezizomycotina.

4  Fungi in Polar Regions Record near Syowa Station: moss stem and withering willows in Lützow-Holm Bay area corrected by 51st JARE (Hirose et al. 2013, Hirose et al. 2016). The withering willow was brought from Japan. Candida albicans (C.P. Robin) Berkhout, De Schimmelgesl. Monilia, Oidium, Oospora en Torula, Disset. Ultrecht: 44 (1923). = Cryptococcus albus (Queyrat & Laroche) Castell. & Chalm., Man. trop. med., 3rd Edn (London): 1080 (1919). Position in classification: Incertae sedis, Saccharomycetales, Saccharomycetidae, Saccharomycetes, Saccharomycotina. Record near Syowa Station: soil in East Ongul Island corrected by 3rd JARE (Tubaki 1961a). Chrysosporium sp., without detailed identification, has been isolated from soil of West Ongul Island corrected by 3rd and 4th JARE (Tubaki 1961a). Cladophialophora minutissima M.L. Davey & Currah, Mycol. Res. 111: 111 (2007). Position in classification: Herpotrichiellaceae, Chaetothyriales, Chaetothyriomycetidae, Eurotiomycetes, Pezizomycotina. Record near Syowa Station: soil, moss stems and withering willows in LützowHolm Bay area corrected by 51st JARE (Hirose et al. 2016). The withering willow was brought from Japan. Clathrosporium intricatum Nawawi & Kuthub., Trans. Br. mycol. Soc. 89: 408 (1987). Position in classification: Incertae sedis, Helotiales, Leotiomycetidae, Leotiomycetes, Pezizomycotina. Record near Syowa Station: withering willows East Ongul Island corrected by 51st JARE (Hirose et al. 2013). The withering willow was brought from Japan. Cladosporium sp., without detailed identification, has been isolated from soil of East Ongul Island corrected by 4th JARE (Tubaki 1961a), and moss stems in Lützow-Holm Bay area corrected by 51st JARE (Hirose et al. 2016). Coniochaeta ligniaria (Grev.) Cooke, Grevillea 16: 16 (1887). = Helminthosphaeria ligniaria (Grev.) Kirschst., Trans. Br. mycol. Soc. 18: 305 (1934) [1933]. Position in classification: Coniochaetaceae, Coniochaetales, Sordariomycetidae, Sordariomycetes, Pezizomycotina. Record near Syowa Station: withering willows East Ongul Island corrected by 51st JARE (Hirose et al. 2013). The withering willow was brought from Japan.

An Index of Fungi from Syowa Station 5

Cosmospora sp. without detailed identification has been isolated from moss stems in Lützow-Holm Bay area or Mt. Riiser-Larsen region corrected by 51st JARE (Hirose et al. 2016). Debaryomyces hansenii (Zopf) Lodder & Kreger-van Rij, in Kreger-van Rij, Yeasts, a taxonomic study, 3rd Edn (Amsterdam): 130 (1984). = Candida famata (F.C. Harrison) E.K. Novák & Zsolt, Acta microbiol. hung. 7: 135 (1961). = Torulopsis famata (F.C. Harrison) Lodder & Kreger-van Rij, Yeasts, a taxonomic study, [Edn 1] (Amsterdam): 417 (1952). Position in classification: Saccharomycetaceae, Saccharomycetales, Saccharomycetidae, Saccharomycetes, Saccharomycotina. Record near Syowa Station: soil in East Ongul Island corrected by 3rd JARE (Soneda 1961, Tubaki 1961a). Exophiala salmonis J.W. Carmich., Sabouraudia 5: 122 (1966). = Aureobasidium salmonis (J.W. Carmich.) Borelli, Medicina Cutánea, Ibera, Latino Americana 3(6): 588 (1969). Position in classification: Herpotrichiellaceae, Chaetothyriales, Chaetothyriomycetidae, Eurotiomycetes, Pezizomycotina. Record near Syowa Station: withering willows in East Ongul Island corrected by 51st JARE (Hirose et al. 2013). The withering willow was brought from Japan. Fusarium sp., without detailed identification, has been isolated from soil of East Ongul Island corrected by 3rd JARE (Tubaki 1961b). Fusidium griseum Ditmar ex Link, Mag. Gesell. naturf. Freunde, Berlin 3: 8 (1809). = Cylindrium griseum (Ditmar ex Link) Bonord., Abh. naturforsch. Ges. Halle 14: 88 (1870). Position in classification: Nectriaceae, Hypocreales, Hypocreomycetidae, Sordariomycetes, Pezizomycotina. Record near Syowa Station: soil of East Ongul Island corrected by 3rd JARE (Tubaki 1961b). Geomyces vinaceus Dal Vesco, Allionia 3(2): 14 (1957). = Geomyces pannorum var. vinaceus (Dal Vesco) Oorschot, Stud. Mycol. 20: 72 (1980). Position in classification: Incertae sedis, Incertae sedis, Incertae sedis, Leotiomycetes, Pezizomycotina. Record near Syowa Station: withering willows in East Ongul Island corrected by 51st JARE (Hirose et al. 2013). The withering willow was brought from Japan.

6  Fungi in Polar Regions Ilyonectria robusta (A.A. Hildebr.) A. Cabral & Crous, Mycol. Progr. 11: 680 (2012). = Ramularia robusta A.A. Hildebr., Canadian Journal of Research, Section C 12: 102 (1935). Position in classification: Incertae sedis, Hypocreales, Hypocreomycetidae, Sordariomycetes, Pezizomycotina. Record near Syowa Station: withering willow in East Ongul Island corrected by 51st JARE (Hirose et al. 2013). The withering willow was brought from Japan. Leptosphaeria sclerotioides (Preuss ex Sacc.) Gruyter, Aveskamp & Verkley, in Gruyter, Woudenberg, Aveskamp, Verkley, Groenewald & Crous, Stud. Mycol. 75: 19 (2012). = Phoma sclerotioides Preuss ex Sacc., Fungi herb. Bruxell.: no. 21 (1892). Position in classification: Leptosphaeriaceae, Pleosporales, Pleosporomycetidae, Dothideomycetes, Pezizomycotina. Record near Syowa Station: withering willows East Ongul Island corrected by 51st JARE (Hirose et al. 2013). The withering willow was brought from Japan. Paradendryphiella salina (G.K. Sutherl.) Woudenb. & Crous, Stud. Mycol. 75: 207 (2013). = Cercospora salina G.K. Sutherl., New Phytol. 15: 43 (1916). = Dendryphiella salina (G.K. Sutherl.) Pugh & Nicot, Trans. Br. mycol. Soc. 47: 266 (1964). = Scolecobasidium salinum (G.K. Sutherl.) M.B. Ellis, More Dematiaceous Hyphomycetes (Kew): 192 (1976). Position in classification: Pleosporaceae, Pleosporales, Pleosporomycetidae, Dothideomycetes, Pezizomycotina. Record near Syowa Station: soil in East and West Ongul Island corrected by H. Fukushima and Y. Kobayasi in 1962 (Japanese government did not carry out Antarctic expedition during 1962–1964) (Tubaki and Asano 1965). Penicillium canescens Sopp, Skr. VidenskSelsk. Christiania, Kl. I, Math.-Natur. 11: 181 (1912). = Penicillium kapuscinskii K.M. Zalessky, Bull. Acad. Polon. Sci., Math. et Nat., Sér. B: 484 (1927). = Penicillium raciborskii K.M. Zalessky, Bull. Acad. Polon. Sci., Math. et Nat., Sér. B: 454 (1927). = Penicillium yarmokense Baghd., Nov. sist. Niz. Rast., 1968 5: 99 (1968).

An Index of Fungi from Syowa Station 7

Position in classification: Trichocomaceae, Eurotiales, Eurotiomycetidae, Eurotiomycetes, Pezizomycotina. Record near Syowa Station: soil in East Ongul Island corrected by H. Fukushima and Y. Kobayasi in 1962 (Japanese government did not carry out Antarctic expedition during 1962–1964) (Tubaki and Asano 1965). Penicillium corylophilum Dierckx, Ann. Soc. Sci. Bruxelles 25: 6 (1901). = Penicillium barcinonense C. Ramírez & A.T. Martínez. Position in classification: Trichocomaceae, Eurotiales, Eurotiomycetidae, Eurotiomycetes, Pezizomycotina. Record near Syowa Station: soil in East Ongul Island corrected by H. Fukushima and Y. Kobayasi in 1962 (Japanese government did not carry out Antarctic expedition during 1962–1964) (Tubaki and Asano 1965). Penicillium dierckxii Biourge, La Cellule 33: 313 (1923). = Penicillium atrovirens G. Sm., Trans. Br. mycol. Soc. 46: 334 (1963). = Penicillium charlesii G. Sm., Trans. Br. mycol. Soc. 18: 90 (1933). = Penicillium fellutanum Biourge, La Cellule 33: 262 (1923). Position in classification: Trichocomaceae, Eurotiales, Eurotiomycetidae, Eurotiomycetes, Pezizomycotina. Record near Syowa Station: soil in East Ongul Island corrected by H. Fukushima and Y. Kobayasi in 1962 (Japanese government did not carry out Antarctic expedition during 1962–1964) (Tubaki and Asano 1965). Penicillium turbatum Westling, Ark. Bot. 11: 54, 128–130 (1911). Position in classification: Trichocomaceae, Eurotiales, Eurotiomycetidae, Eurotiomycetes, Pezizomycotina. Record near Syowa Station: withering willows in East Ongul Island corrected by 51st JARE (Hirose et al. 2013). The withering willow was brought from Japan. Phaeosphaeria sp. without detailed identification has been isolated from moss stems in Lützow-Holm Bay area or Mt. Riiser-Larsen region corrected by 51st JARE (Hirose et al. 2016). Phialocephala fortinii C.J.K. Wang & H.E. Wilcox, Mycologia 77: 954 (1986) [1985]. Position in classification: Vibrisseaceae, Helotiales, Leotiomycetidae, Leotiomycetes, Pezizomycotina. Record near Syowa Station: withering willows in East Ongul Island corrected by 51st JARE (Hirose et al. 2013). The withering willow was brought from Japan.

8  Fungi in Polar Regions Phialocephala lagerbergii (Melin & Nannf.) Grünig & T.N. Sieber, Mycol. Res. 113: 217 (2009). = Cadophora lagerbergii Melin & Nannf., Svensk Skogsvårdsförening Tidskr. 3-4: 416 (1934). = Phialophora lagerbergii (Melin & Nannf.) Conant, Mycologia 29: 598 (1937). Position in classification: Vibrisseaceae, Helotiales, Leotiomycetidae, Leotiomycetes, Pezizomycotina. Record near Syowa Station: withering willows in East Ongul Island corrected by 51st JARE (Hirose et al. 2013). The withering willow was brought from Japan. Phoma herbarum Westend., Bull. Acad. R. Sci. Belg., Cl. Sci. 19: 118 (1852). = Phoma tageticola (Schwein.) Ellis & Everh., Proc. Acad. nat. Sci. Philad. 47: 28 (1896). = Phoma tetragoniae (Sacc. & Berl.) Mussat, in Saccardo, Syll. fung. (Abellini) 15: 286 (1901). = Phyllosticta glycyrrhizae (Hollós) Y.S. Paul & L.N. Bhardwaj, Indian Forester 118: 301 (1992). Position in classification: Didymellaceae, Pleosporales, Pleosporomycetidae, Dothideomycetes, Pezizomycotina. Record near Syowa Station: soil surrounding Ohgi ike and Naga Ike in Skarvsnes ice-free area corrected by 48th JARE (Tsuji et al. 2013a). Phoma sp. without detailed identification has been isolated from withering willows in East Ongul Island corrected by 51st JARE (Hirose et al. 2013). The withering willow was brought from Japan. Pseudogymnoascus carnis (F.T. Brooks & Hansf.) Minnis & D.L. Lindner, Fungal Biology 117: 645 (2013). = Aleurisma carnis (F.T. Brooks & Hansf.) Bisby, Trans. Br. mycol. Soc. 27: 11 (1945). = Sporotrichum carnis F.T. Brooks & Hansf., Trans. Br. mycol. Soc. 11: 124 (1923). Position in classification: Myxotrichaceae, Incertae sedis, Incertae sedis, Leotiomycetes, Pezizomycotina. Record near Syowa Station: soil of West Ongul Island corrected by 3rd JARE (Tsubaki 1961b). Pseudogymnoascus pannorum (Link) Minnis & D.L. Lindner, Fungal Biology 117: 646 (2013). = Chrysosporium pannorum (Link) S. Hughes, Can. J. Bot. 36: 749 (1958).

An Index of Fungi from Syowa Station 9

= Chrysosporium verrucosum Tubaki, Neue Denkschr. Allg. Schweiz. Ges. Gesammten Naturwiss. 14: 6 (1961). = Geomyces pannorum (Link) Sigler & J.W. Carmich. [as ‘pannorus’], Mycotaxon 4: 377 (1976). Position in classification: Myxotrichaceae, Incertae sedis, Incertae sedis, Leotiomycetes, Pezizomycotina. Record near Syowa Station: soil of West Ongul Island corrected by 3rd JARE (Tubaki 1961a), soil surrounding Hamagiku Ike and Bosatsu Ike corrected by 48th JARE (Tsuji et al. 2013a), and Lützow-Holm Bay area or Mt. RiiserLarsen region corrected by 51st JARE (Hirose et al. 2016). Racodium sp. without detailed identification has been isolated from soil of East and West Ongul Island corrected by 3rd JARE (Tubaki 1961a). Tetracladium sp. without detailed identification has been isolated from soil surrounding Shimai Ike, Ageha Ike, and Kuwai Ike, and lake sediment of Jizou Ike, Kuwai Ike, and Naga Ike corrected by 48th JARE (Tsuji et al. 2013a), and moss stems in Lützow-Holm Bay area or Mt. Riiser-Larsen region corrected by 51st JARE (Hirose et al. 2016). Thelebolus microsporus (Berk. & Broome) Kimbr., in Kobayasi et al., Annual Report Institute Fermentation, 1965–66 3: 50 (1967). = Ascobolus microsporus Berk. & Broome, Ann. Mag. nat. Hist., Ser. 3 15: 449 (1865). = Ascophanus microsporus (Berk. & Broome) W. Phillips, Man. Brit. Discomyc. (London): 307 (1877) [1876]. Position in classification: Thelebolaceae, Thelebolales, Leotiomycetidae, Leotiomycetes, Pezizomycotina. Record near Syowa Station: soil surrounding Ebine Ike, Kuwai Ike, Mago Ike, and Ohgi Ike, and lake sediment of Naga Ike and Tokkuri Ike corrected by 48th JARE (Tsuji et al. 2013a). Basidiomycetous fungi Cutaneotrichosporon cutaneum (Beurm., Gougerot & Vaucher bis) X.Z. Liu, F.Y. Bai, M. Groenew. & Boekhout, in Liu, Wang, Göker, Groenewald, Kachalkin, Lumbsch, Millanes, Wedin, Yurkov, Boekhout & Bai, Stud. Mycol. 81: 140 (2015). = Trichosporon cutaneum (Beurm., Gougerot & Vaucher bis) M. Ota, Annls Parasit. hum. comp. 4: 12 (1926). Position in classification: Trichosporonaceae, Trichosporonales, Incertae sedis, Tremellomycetes, Agaricomycotina. Record near Syowa Station: soil of near the Syowa Base corrected by 3rd JARE (Soneda 1961, Tubaki 1961b).

10  Fungi in Polar Regions Cystobasidium laryngis (Reiersöl) A. Yurkov, A. Kachalkin, H.M. Daniel, M. Groenewald, D. Libkind, V. de Garcia, P. Zalar, D. Gouliamova, T. Boekhout & D. Begerow, Antonie van Leeuwenhoek 107: 181 (2014). = Rhodotorula laryngis Reiersöl, Antonie van Leeuwenhoek 21: 287 (1955). Position in classification: Cystobasidiaceae, Cystobasidiales, Incertae sedis, Cystobasidiomycetes, Pucciniomycotina. Record near Syowa Station: soil surrounding Hamagiku Ike corrected by 48th JARE (Tsuji et a. 2013a). Cystobasidium lysinophilum (Nagah., Hamam., Nakase & Horikoshi) A.M. Yurkov, Kachalkin, H.M. Daniel, M. Groenew., Libkind, V. de Garcia, Zalar, Gouliamova, Boekhout & Begerow, Antonie van Leeuwenhoek 107(1): 181 (2014). = Rhodotorula lysinophila Position in classification: Cystobasidiaceae, Cystobasidiales, Incertae sedis, Cystobasidiomycetes, Pucciniomycotina. Record near Syowa Station: soil in East Ongul Island corrected by 49th JARE (Tsuji 2018a). Cystobasidium ongulense M. Tsuji, M. Tsujimoto & S. Imura, Mycoscience 58: 109 (2017). Position in classification: Cystobasidiaceae, Cystobasidiales, Incertae sedis, Cystobasidiomycetes, Pucciniomycotina. Record near Syowa Station: soil in East Ongul Island corrected by 49th JARE (Tsuji et al. 2017). Cystobasidium tubakii M. Tsuji, M. Tsujimoto & S. Imura, Mycoscience 58: 108–109. Position in classification: Cystobasidiaceae, Cystobasidiales, Incertae sedis, Cystobasidiomycetes, Pucciniomycotina. Record near Syowa Station: soil in East Ongul Island corrected by 49th JARE (Tsuji et al. 2017). Dioszegia fristingensis Á. Fonseca, J. Inácio & J.P. Samp., in Inácio, Portugal, Spencer-Martins & Fonseca, FEMS Yeast Res. 5: 1180 (2005). Position in classification: Tremellaceae, Tremellales, Incertae sedis, Tremellomycetes, Agaricomycotina. Record near Syowa Station: soil surrounding Mago Ike and Hyotan Ike, Skarvsnes ice-free area, corrected by 48th JARE (Tsuji et al. 2013a). Glaciozyma antarctica (Fell, Statzell, I.L. Hunter & Phaff) Turchetti, L.B. Connell, Thomas-Hall & Boekhout, in Turchetti, Thomas Hall, Connell, Branda, Buzzini, Theelen & Müller, Extremophiles 15(5): 579 (2011).

An Index of Fungi from Syowa Station 11

Record near Syowa Station: soil in East Ongul Island corrected by 49th JARE (Tsuji et al. 2017). Glaciozyma martinii Turchetti, L.B. Connell, Thomas-Hall & Boekhout, in Turchetti, Thomas Hall, Connell, Branda, Buzzini, Theelen & Müller, Extremophiles 15(5): 579 (2011). Record near Syowa Station: soil in East Ongul Island corrected by 49th JARE (Tsuji 2018a). Glaciozyma watsonii Turchetti, L.B. Connell, Thomas-Hall & Boekhout, in Turchetti, Thomas Hall, Connell, Branda, Buzzini, Theelen & Müller, Extremophiles 15: 582 (2011). Position in classification: Incertae sedis, Incertae sedis, Incertae sedis, Microbotryomycetes, Pucciniomycotina. Record near Syowa Station: soil surrounding Kumogata Ike, lake sediment of Bosatsu Ike, Skarvsnes ice-free area, corrected by 48th JARE (Tsuji et al. 2013a). Goffeauzyma gastrica (Reiersöl & di Menna) X.Z. Liu, F.Y. Bai, M. Groenew. & Boekhout, Stud. Mycol. 81: 119 (2015). = Cryptococcus gastricus Reiersöl & di Menna, Antonie van Leeuwenhoek 24: 28 (1958). Position in classification: Tremellaceae, Tremellales, Incertae sedis, Tremellomycetes, Agaricomycotina. Record near Syowa Station: soil surrounding Oyako Ike and Tokkuri Ike, and lake sediment of Bosatsu Ike corrected by 48th JARE (Tsuji et al. 2013a). Goffeauzyma gilvescens (Chernov & Babeva) Xin Zhan Liu, F.Y. Bai, M. Groenew. & Boekhout, Stud. Mycol. 81: 119 (2015). = Cryptococcus gilvescens Chernov and Babeva 1988. Position in classification: Tremellaceae, Tremellales, Incertae sedis, Tremellomycetes, Agaricomycotina. Record near Syowa Station: soil in East Ongul Island corrected by 49th JARE (Tsuji 2018a). Holtermanniella wattica (Guffogg, Thomas-Hall, P. Holloway & K. Watson) Libkind, Wuczk., Turchetti & Boekhout, in Wuczkowski, Passoth, Turchetti, Andersson, Olstorpe, Laitila, Theelen, Broock, Buzzini, Prillinger & Sterflinge, Int. J. Syst. Evol. Microbiol. 61(3): 685 (2011). = Cryptococcus watticus Guffogg, Thomas-Hall, P. Holloway & K. Watson, in Guffogg, Thomas-Hall, Holloway and Watson (2004). Position in classification: Tremellomycetes, Incertae sedis, Holtermanniales, Holtermanniaceae, Agaricomycotina.

12  Fungi in Polar Regions Record near Syowa Station: soil in East Ongul Island corrected by 49th JARE (Tsuji 2018a). Mrakia blollopis Thomas-Hall, in Thomas-Hall, Turchetti, Buzzini, Branda, Boekhout, Theelen & Watson, Extremophiles 14: 56 (2010). Position in classification: Mrakiaceae, Cystofilobasidiales, Incertae sedis, Tremellomycetes, Agaricomycotina. Record near Syowa Station: soil and lake sediment of Skarvsnes ice-free area corrected by 48th JARE (Tsuji et al. 2013a,b, 2015, 2016, Tsuji 2016). Mrakia gelida (Fell, Statzell, I.L. Hunter & Phaff) Y. Yamada & Komag., J. gen. appl. Microbiol., Tokyo 33: 457 (1987). = Candida gelida Di Menna, Antonie van Leeuwenhoek 32: 26 (1966). = Leucosporidium gelidum Fell, Statzell, I.L. Hunter & Phaff, Antonie van Leeuwenhoek 35: 452 (1970) [1969]. Position in classification: Mrakiaceae, Cystofilobasidiales, Incertae sedis, Tremellomycetes, Agaricomycotina. Record near Syowa Station: soil and lake sediment of Skarvsnes ice-free area corrected by 48th JARE (Tsuji et al. 2013a, 2015, 2016). Mrakia robertii Thomas-Hall & Turchetti, in Thomas-Hall, Turchetti, Buzzini, Branda, Boekhout, Theelen & Watson, Extremophiles 14: 54 (2010). Position in classification: Mrakiaceae, Cystofilobasidiales, Incertae sedis, Tremellomycetes, Agaricomycotina. Record near Syowa Station: soil and lake sediment of Skarvsnes ice-free area corrected by 48th JARE (Tsuji et al. 2013a, 2015, 2016). Naganishia adeliensis (Scorzetti, I. Petrescu, Yarrow & Fell) Xin Zhan Liu, F.Y. Bai, M. Groenew. & Boekhout, Stud. Mycol. 81: 118 (2015). = Cryptococcus adeliensis Scorzetti, I. Petrescu, Yarrow & Fell, Antonie van Leeuwenhoek 77(2): 155 (2000). Position in classification: Tremellaceae, Tremellales, Incertae sedis, Tremellomycetes, Agaricomycotina. Record near Syowa Station: soil in East Ongul Island corrected by 49th JARE (Tsuji 2018a). Naganishia albida (Saito) X.Z. Liu, F.Y. Bai, M. Groenew. & Boekhout, Stud. Mycol. 81: 118 (2015). = Cryptococcus albidus (Saito) C.E. Skinner, Am. Midl. Nat. 43: 249 (1950). = Rhodotorula albida (Saito) Galgoczy & E.K. Novák, Acta microbiol. hung. 12: 155 (1965).

An Index of Fungi from Syowa Station 13

= Torulopsis albida (Saito) Lodder, Verh. K. Akad. Wet., tweede sect. 32: 163 (1934). Position in classification: Tremellaceae, Tremellales, Incertae sedis, Tremellomycetes, Agaricomycotina. Record near Syowa Station: soil in West Ongul Island corrected by 4th JARE (Soneda 1961). Naganishia albidosimilis (Vishniac & Kurtzman) Xin Zhan Liu, F.Y. Bai, M. Groenew. & Boekhout, Stud. Mycol. 81: 118 (2015). = Cryptococcus albidosimilis Vishniac & Kurtzman, Int. J. Syst. Bacteriol. 42(4): 550 (1992). Position in classification: Tremellaceae, Tremellales, Incertae sedis, Tremellomycetes, Agaricomycotina. Record near Syowa Station: soil in East Ongul Island corrected by 49th JARE (Tsuji 2018a). Naganishia friedmannii (Vishniac) X.Z. Liu, F.Y. Bai, M. Groenew. & Boekhout, Stud. Mycol. 81: 119 (2015). = Cryptococcus friedmannii Vishniac, Mycologia 77: 150 (1985). Position in classification: Tremellaceae, Tremellales, Incertae sedis, Tremellomycetes, Agaricomycotina. Record near Syowa Station: soil surrounding Hechima Ike corrected by 48th JARE (Tsuji et al. 2013a). Papiliotrema laurentii (Kuff.) X.Z. Liu, F.Y. Bai, M. Groenew. & Boekhout, Stud. Mycol. 81: 126 (2015). = Cryptococcus laurentii (Kuff.) C.E. Skinner, Am. Midl. Nat. 43: 249 (1950). = Rhodotorula laurentii (Kuff.) T. Haseg., I. Banno & Yamauchi, J. gen. appl. Microbiol., Tokyo 6: 212 (1960). Position in classification: Tremellaceae, Tremellales, Incertae sedis, Tremellomycetes, Agaricomycotina. Record near Syowa Station: soil of near the Syowa Base and West Ongul Island corrected by 3rd and 4th JARE (Soneda 1961, Tubaki 1961a). Phenoliferia glacialis (Margesin & J.P. Samp.) Q.M. Wang, F.Y. Bai, M. Groenew. & Boekhout, Stud. Mycol. 81: 178 (2015). = Rhodotorula glacialis Margesin & J.P. Samp., Int. J. Syst. Evol. Microbiol. 57: 2183 (2007). Position in classification: Kriegeriaceae, Kriegeriales, Incertae sedis, Microbotryomycetes, Pucciniomycotina.

14  Fungi in Polar Regions Record near Syowa Station: soil surrounding Hechima Ike, Skarvsnes ice-free area, corrected by 48th JARE (Tsuji et al. 2013a). Rhodotorula mucilaginosa (A. Jörg.) F.C. Harrison, Proc. & Trans. Roy. Soc. Canada, ser. 3 21(5): 349 (1928). = Rhodotorula rubra (Demme) Lodder, Verh. K. Akad. Wet., tweede sect. 32: 69 (1934). = Rhodotorula sanguinea (Schimon) F.C. Harrison, Trans. Roy. Soc. Canad. 22: 187 (1928). Position in classification: Incertae sedis, Sporidiobolales, Incertae sedis, Microbotryomycetes, Pucciniomycotina. Record near Syowa Station: soil of West Ongul Island corrected by 4th JARE (Soneda 1961, Tubaki 1961b). Tausonia pullulans (Lindner) Xin Zhan Liu, F.Y. Bai, J.Z. Groenew. & Boekhout, in Liu, Wang, Göker, Groenewald, Kachalkin, Lumbsch, Millanes, Wedin, Yurkov, Boekhout & Bai, Stud. Mycol. 81: 116 (2015). = Trichosporon pullulans (Lindner) Diddens & Lodder, Nachr. Ges. Wiss. Göttingen, Math.-Phys. Kl., Fachgr. 6, Nachr. Biol. 9: 10 (1942). Position in classification: Mrakiaceae, Cystofilobasidiales, Incertae sedis, Tremellomycetes, Agaricomycotina. Record near Syowa Station: soil in East Ongul Island corrected by 49th JARE (Tsuji 2018a). Udeniomyces puniceus (Nakase & Komag.) Nakase & Takem., FEMS Microbiol. Lett. 100(1-3): 499 (1992). = Candida punicea Nakase & Komag., J. gen. appl. Microbiol., Tokyo 11: 259 (1965). = Sporobolomyces puniceus (Nakase & Komag.) Ahearn & Yarrow, in Yarrow, Antonie van Leeuwenhoek 38(3): 359 (1972). Position in classification: Mrakiaceae, Cystofilobasidiales, Incertae sedis, Tremellomycetes, Agaricomycotina. Record near Syowa Station: soil in East Ongul Island corrected by 49th JARE (Tsuji 2018a). Vishniacozyma carnescens (Verona & Luchetti) Xin Zhan Liu, F.Y. Bai, M. Groenew. & Boekhout, Stud. Mycol. 81: 124 (2015). = Cryptococcus carnescens (Verona & Luchetti) M. Takash., Sugita, Shinoda & Nakase, Int. J. Syst. Evol. Microbiol. 53(4): 1192 (2003). Position in classification: Bulleribasidiaceae, Tremellales, Incertae sedis, Tremellomycetes, Agaricomycotina. Record near Syowa Station: soil in East Ongul Island corrected by 49th JARE (Tsuji 2018a).

An Index of Fungi from Syowa Station 15

Vishniacozyma victoriae (M.J. Montes, Belloch, Galiana, M.D. García, C. Andrés, S. Ferrer, Torr.-Rodr. & J. Guinea) X.Z. Liu, F.Y. Bai, M. Groenew. & Boekhout, Stud. Mycol. 81: 124 (2015). = Cryptococcus victoriae M.J. Montes, Belloch, Galiana, M.D. García, C. Andrés, S. Ferrer, Torr.-Rodr. & J. Guinea, Syst. Appl. Microbiol. 22: 104 (1999). Position in classification: Tremellaceae, Tremellales, Incertae sedis, Tremellomycetes, Agaricomycotina. Record near Syowa Station: soils and lake sediment in Skrarvsnes ice-free area corrected by 48th JARE (Tsuji et al. 2013a).

Acknowledgments This work was carried out as part of the Science Program of JARE. It was supported by NIPR under MEXT, Japan. This work was supported by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for C Young Scientists (A) [grant numbers 16H06211].

References Bridge, P.D. and B.M. Spooner. 2012. Non-lichenized Antarctic fungi: transient visitors or members of a cryptic ecosystem? Fungal Ecol. 5: 381–394. Hirose, D., Y. Tanabe, M. Uchida, S. Kudoh and T. Osono. 2013. Microfungi associated with withering willow wood in ground contact near Syowa Station, East Antarctica for 40 years. Polar Biol. 36: 919–924. Hirose, D., S. Hobara, S. Matsuoka, K. Kato, Y. Tanabe, M. Uchida et al. 2016. Diversity and community assembly of moss-associated fungi in ice-free coastal outcrops of continental Antarctica. Fungal Ecol. 24: 94–101. Onofri, S., L. Zucconi and S. Tosi. 2007. Continental Antarctic fungi. IHW Verlag, Munich. Ravindra, R. and A. Chaturvedi. 2011. Antarctica. pp. 45–53. In: V.P. Singh, P. Singh and U.K. Haritashya (eds.). Encyclopedia of Snow, Ice and Glaciers. Springer, Berlin. Soneda, M. 1961. On some yeasts from the Antarctic region. Special Publ. Seto. Marine Biol. Lab. 1: 3–10. Tsuji, M., S. Fujiu, N. Xiao, Y. Hanada, S. Kudoh, H. Kondo et al. 2013a. Cold adaptation of fungi obtained from soil and lake sediment in the Skarvsnes ice-free area, Antarctic. FEMS Microbiol. Lett. 346: 121–130. Tsuji, M., Y. Yokota, K. Shimohara, S. Kudoh and T. Hoshino. 2013b. An application of wastewater treatment in a cold environment and stable lipase production of Antarctic basidiomycetous yeast Mrakia blollopis. PLoS One 8: e59376. Tsuji, M., Y. Yokota, S. Kudoh and T. Hoshino. 2015. Comparative analysis of milk fat decomposition activity by Mrakia spp. isolated from Skarvsnes ice-free area, East Antarctica. Cryobiology 70: 293–296. Tsuji, M., S. Kudoh and T. Hoshino. 2016. Ethanol productivity of cryophilic basidiomycetous yeast Mrakia spp. correlates with ethanol tolerance. Mycoscience 57: 42–50. Tsuji, M. 2016. Cold-stress responses in the Antarctic basidiomycetous yeast Mrakia blollopis. R. Soc. Open Sci. 3: 160106. Tsuji, M., M. Tsujimoto and S. Imura. 2017. Cystobasidium tubakii and Cystobasidium ongulense, new basidiomycetous yeast species isolated from East Ongul Island, East Antarctica. Mycoscience 58: 103–110.

16  Fungi in Polar Regions Tsuji, M. 2018a. Genetic diversity of yeasts from East Ongul Island, East Antarctica and their extracellular enzymes secretion. Polar Biol. 42: 249–258. Tsuji, M. 2018b. A catalog of fungi recorded from the vicinity of Syowa Station. Mycoscience 59: 319–324. Tubaki, K. 1961a. On some fungi isolated from the Antarctic materials. Special Publ. Seto. Marine Biol Lab. 14: 3–9. Tubaki, K. 1961b. Note on some fungi and yeasts from Antarctica. Antarc. Rec. (Tokyo). 11: 161–162. Tubaki, K. and I. Asano. 1965. Additional species of fungi isolated from the Antarctic materials. Japan Antarct. Exp. Rep. E. 27: 1–12.

2 Diversity and Ecology of Fungi in Polar Region Comparisons Between Arctic and Antarctic Plant Remains Takashi Osono,1,* Shunsuke Matsuoka,2 Satoru Hobara,3 Dai Hirose4 and Masaki Uchida5

Introduction Fungi are major components of the diversity of soil organisms in polar regions, and they play central roles in the decomposition of plant remains that control carbon and nutrient cycling in soil (van der Wal et al. 2013). Decomposition of plant remains is carried out by a suite of fungal species that differ in the activity of their extracellular enzymes, such as cellulases and ligninases, leading to changes in the species composition as the plant remains decompose, in the process of substrate succession of fungi (Hudson 1968, Osono 2007). The seral succession of fungi, that is, the series of changes in fungal assemblages during ecosystem succession and soil development, is also an important aspect of polar fungal ecology, because glacier forelands at which primary succession of vegetation occurs are a common feature Department of Environmental Systems Science, Faculty of Science and Engineering, Doshisha University, Kyoto 610-0394, Japan. 2 Graduate School of Simulation Studies, University of Hyogo, Kobe, Hyogo 650-0047, Japan. 3 Department of Environmental Symbiotic Sciences, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501, Japan. 4 Faculty of Pharmacy, Nihon University, Funabashi, Chiba 274-8555, Japan. 5 National Institute of Polar Research, Tokyo 173-8515, Japan. * Corresponding author: [email protected] 1

18  Fungi in Polar Regions in polar regions (Svoboda and Henry 1987). Those saprobic fungi encountered in polar regions generally are cosmopolitan and have great dispersal potential (Arenz et al. 2014) and are physiologically adapted to extreme environments, such as cold temperatures, wide temperature fluctuations, strong desiccation, freeze-thaw cycles, and short growing seasons (Robinson 2001). Recent increases in human expeditions and activities in polar regions have provided a new opportunity to explore fungi associated with exotic plant materials of anthropogenic origin, such as wood of historic huts in Antarctica (Arenz et al. 2011). Previous studies have examined the diversity and ecology of either Arctic or Antarctic fungi in relation to these various aspects, but few studies have compared fungal assemblages and their functioning between Arctic and Antarctic plant remains. In the present chapter, we summarize the results of a series of investigations on the ecology and diversity of fungi associated with the decomposition of Arctic and Antarctic plant remains, with special reference to dead materials of moss and willow that are major components of cold regions. Comparing the fungal diversity, substrate succession, and decomposition between the Arctic and Antarctica will provide useful insight into the diversity and ecology of fungi in polar regions. The study areas where Arctic and Antarctic fungal surveys were performed are summarized in Table 2.1 and Fig. 2.1. Studies on the terrestrial vegetation of a polar oasis have been intensively carried out in the high Arctic study area (Mori et al. 2006, 2008, 2017, Osono et al. 2006, 2016, Ueno et al. 2009). In contrast, the ice-free regions of the Antarctic study area have been extensively examined for limnological research on lakes and lakebed vegetation (Tanabe et al. 2012a,b, 2017). Table 2.1.  Study areas.

1

2 3 4

Arctic

Antarctica

Location

Oobloyah Bay area, Nunavut, Canada

Lützow-Holm Bay area, east Antarctica

Latitude, Longitude

80°50’ N, 82°49’ W

69°00’ S, 39°34’ E

Mean annual temperature

–19.7°C1

–10 to –12°C2

Ecosystem

Polar oasis

Terrestrial vegetation

Vascular plants, bryophytes

Bryophytes4

Fieldwork

July to August 2003 and 2004

December 2009 to February 2010

Polar desert 3

Eureka, Nunavut, Canada (80°00’ N, 85°56’ W), located 130 km south of the study area. Atmospheric Environment Service (1982). Syowa station, East Antarctica (69°00’ S, 39°34’ E). Kudo et al. (2015). Mori et al. (2006, 2008, 2017), Osono et al. (2006, 2016), Ueno et al. (2009). Kanda and Inoue (1994).

Diversity and Ecology of Fungi in Polar Region 19 (b)

(a)

Fig. 2.1.  Photographs of study areas. (a) Oobloyah Bay area in the Arctic, (b) Lützow-Holm Bay area in Antarctica. Locations are given in Table 2.1.

Color version at the end of the book

Fungi Associated with Moss Tissues Moss in Polar Regions Mosses are major components of terrestrial ecosystems of the Arctic and Antarctica, and contribute importantly to the crucial budgets of carbon and nutrients via primary production and concomitant decomposition and accumulation of moss tissues in soil (Ochyra et al. 2008). The accumulated live, senescent, and dead moss tissues, termed the bryosphere (Lindo and Gonzalez 2010), harbor a variety of fungi and other organisms that drive carbon and nutrient dynamics in the bryosphere (Osono and Trofymow 2012). Examining vertical patterns of fungal assemblages within moss profiles consisting of live, senescent, and dead tissues in different stages of decomposition sheds light on the functional roles of fungi in the decomposition of moss tissues. Hence, the abundance, diversity, and succession of fungi and their relationship with chemical changes in moss tissues within the bryosphere were examined for two moss species in the Arctic (Racomitrium lanuginosum and Hylocomium splendens) and one moss species in Antarctica (Bryum pseudotriquetrum) (Table 2.2). Moss blocks were collected and divided into 4 to 6 layers according to the color and texture (Fig. 2.2) to analyze fungal assemblages, fungal succession, and hyphal length in relation to chemical changes in decomposing moss tissues.

Fungal Assemblages A total of 19, 18, and 14 fungal species were isolated from moss tissues of R. lanuginosum, H. splendens, and B. pseudotriquetrum, respectively (Table 2.2). Major species included Penicillium sp.1, Mortierella minutissima, Umbelopsis ramanniana, Pseudogymnoascus pannorum, and Absidia cylindrospora in R. lanuginosum, Penicillium sp.1, M. minutissima, and Cylindrocarpon sp. in H. splendens, and Phoma herbarum and P. pannorum in B. pseudotriquetrum

18 Penicillium sp. 1, Mortierela minutissima, Cylindrocarpon sp.

19 Penicillium sp.1, Mortierella minutissima, Umbelopisis rammaniana, Pseudogymnoascus pannorum, Absidia cylindrospora Turnover (Fig. 2.3a) 4446 1859 [41%] 658 [13%] Increase first and then decrease thereafter Selective decomposition of holocellulose Decrease from 60.3 to 27.5

1164 349 [30%] 107 [5%] Increase

Selective decomposition of holocellulose Decrease from 150.6 to 56.5

Turnover

Arctic Hylocomium splendens 12–18 cm (6 layers)

Arctic Racomitrium lanuginosum 14–17 cm (5 layers)

Decrease from 28.9 to 17.5

No significant difference

3224 643 [17%] No data No significant change

No turnover (Fig. 2.3b)

14 Phoma herbarum, Pseudogymnoascus pannorum

Antarctica Bryum pseudotriquetrum 6–10 cm (4 layers)

Proportions relative to the total hyphal length are given in brackets. Fungi were isolated from moss tissues using a washing method. Arctic data from Osono et al. (2012); Antarctic data from Hirose et al. (2017).

Pattern of fungal succession Hyphal Lengths Total hyphal length (mean, m/g) Darkly pigmented hyphal length (mean, m/g) Clamp-bearing hyphal length (mean, m/g) Pattern of changes in total hyphal length during decomposition Decomposition Processes Pattern of decomposition of organic chemical components Changes in C/N ratio of moss tissues during decomposition

Study Area Moss species Thickness of moss blocks (number of layers that were distinguished) Fungal Assemblages and Succession Number of species Major species

Table 2.2.  Fungi and decomposition in Arctic and Antarctic moss profiles.

20  Fungi in Polar Regions

Diversity and Ecology of Fungi in Polar Region 21 (a)

(b)

Fig. 2.2.  Photographs of moss profiles. (a) Hylocomium splendens in the Arctic, (b) Bryum pseudotriquetrum in Antarctica.

Color version at the end of the book

(Table 2.2). Pseudogymnoascus pannorum was found in both the Arctic and Antarctica, and was considered to be a cosmopolitan. Phoma herbarum also has a worldwide distribution not only in Antarctica, but also in temperate regions of the Southern and Northern hemispheres, the Tropics, and the Arctic (Domsch et al. 2007), and appears to be widely distributed in continental Antarctica (Hirose et al. 2016).

Fungal Succession Turnover of fungal species was found within the two arctic moss profiles, indicating the successional changes in fungal assemblages during decomposition of moss tissues (Table 2.2). In R. lanuginosum, Cladosporium herbarum was a major species at the uppermost green parts of moss, but the frequency of this species decreased downward (Fig. 2.3). Penicillium sp.1 and U. ramanniana increased at the middle layers, which consisted of senescent and relatively recently dead moss tissues, and then decreased at deeper layers. Finally, P. pannorum and A. cylindrospora increased at the deeper layers where moss tissues were heavily decomposed and became blackened and fragile. A similar pattern of fungal succession was found in H. splendens, where the major fungal species shifted from Penicillium sp.1 at the upper layers, to M. minutissima at the middle layers, and to Cylindrocarpon sp. at the lower layers (Osono et al. 2012). In contrast to these arctic profiles, the

22  Fungi in Polar Regions

Fig. 2.3.  Patterns of fungal succession in Arctic and Antarctic moss profiles. The profiles were divided into five and four layers in the Arctic and Antarctica, respectively, from the upper surface layer to the lower bottom layer. (a) Racomitrium lanuginosum in the Arctic. Black squares and solid line, Cladosporium herbarum; open circles and dotted line, Penicillium sp.1; open triangles and dotted line, Umbelopsis ramanniana; gray triangles and gray line, Pseudogymnoascus pannorum; gray diamonds and gray line, Absidia cylindrospora. (b) Bryum pseudotriquetrum in Antarctica. Black squares and solid line, Phoma herbarum; black circles and solid line, Pseudogymnoascus pannorum.

vertical pattern of fungal distribution differed in the Antarctic moss profile. In B. pseudotriquetrum, the frequencies of occurrence of P. herbarum and P. pannorum increased downward without turnover of the major fungal species (Fig. 2.3).

Hyphal Length Hyphal lengths in moss tissues were measured by direct observation using an agar film method. Mean values of total hyphal lengths were 1,164 m/g in R. lanuginosum, 4,446 m/g in H. splendens, and 3,224 m/g in B. pseudotriquetrum (Table 2.2). These values were within the range previously reported for tundra soils (Osono et al. 2012), but were lower than those reported previously for litter and soil in warmer regions (Osono 2015). Total hyphal length increased from the uppermost green layers toward the lower layers within both of the arctic moss profiles; it then decreased at the lowermost layer of H. splendens. In contrast, no significant changes were found for total hyphal length among vertical layers of Antarctic B. pseudotriquetrum. The lengths of darkly pigmented (melanized) hyphae, which are associated with those of dematiaceous fungi, accounted for 30, 41, and 17% of the total hyphal length in R. lanuginosum, H. splendens, and B. pseudotriquetrum, respectively (Table 2.2). The lengths of clamp-bearing hyphae, which belonged to Basidiomycota, accounted for 5% and 13% of the total hyphal length in R. lanuginosum and H. splendens, respectively (Table 2.2). No measurement of clamp-bearing hyphal length was performed for B. pseudotriquetrum.

Diversity and Ecology of Fungi in Polar Region 23

Decomposition processes The contents of organic chemical components and nitrogen were assessed within the arctic and Antarctic moss profiles (Table 2.2). The amount of holocellulose relative to recalcitrant compounds classified as acid unhydrolyzable residues (AUR, include lignin, tannin, and cutin) decreased downward from the upper to the lower layers of two arctic moss profiles, which was indicative of selective decomposition of holocellulose. This contrasted to the profile of Antarctic moss B. pseudotriquetrum, in which no significant change was found for the relative amount of holocellulose and AUR during decomposition. The carbon to nitrogen (C/N) ratio of moss tissues decreased during decomposition of all three moss profiles, leading to relative accumulation of nitrogen at the lower layers (Table 2.2).

Fungi Associated with Willow Tissues Willow in Polar Region Willows (Salix spp.) are dominant dwarf shrubs in arctic tundra of the northern hemisphere. Salix arctica (the arctic willow) is quite abundant in the polar oasis of the glacier foreland in the Oobloyah Bay area (Fig. 2.4a), establishing a wide range of microhabitats, including xeric moraines and mesic hummocks (Mori et al. 2006, 2008, 2017, Osono et al. 2006, 2016). The frequent occurrence of S. arctica in habitats ranging from recently deglaciated, non-vegetated young moraines to older, well-vegetated ones provided a unique opportunity to test the hypothesis that fungal assemblages associated with the dead remains of S. arctica change during primary succession following deglaciation (i.e., that primary seral succession of fungi occurs). Moreover, surveying leaves and stems (approximately 3 mm in diameter) of S. arctica in different stages of decomposition enabled the study of substrate succession of fungi to clarify the roles of fungi in the decomposition (Osono et al. 2014). In contrast, no indigenous vascular plants, including Salix spp., (a)

(b)

Fig. 2.4.  Photographs of dead willow materials. (a) Salix arctica in the Arctic, (b) Location where Salix spp. were planted in Antarctica.

Color version at the end of the book

24  Fungi in Polar Regions were encountered in continental Antarctica, including in the Lützow-Holm Bay area. In 1967, however, saplings of dwarf deciduous shrubs S. pauciflora and S. reinii originating from Hokkaido, Northern Japan, were transplanted at an experimental site near Syowa station (Fig. 2.4b). These saplings died within a few years because of the adverse environment of Antarctica, with their stems (approximately 3 cm in height, 1–3 mm in basal diameter, and bared of leaves) left withering and standing for 40 years at the experimental site. Isolating fungi from these woody stems of exotic willows with ground contact provided a unique opportunity to investigate the diversity and ecology of fungi associated with willow remains on continental Antarctica (Hirose et al. 2013) and to compare them with those of the Arctic.

Fungal Assemblages A total of 15, 14, and 18 fungal species were isolated from dead leaves and stems of arctic willow and dead stems of exotic willows in Antarctica, respectively (Table 2.3), which is similar to the numbers of species in arctic and Antarctic moss tissues (Table 2.2). Major species in arctic willow included Comoclathris sp., Rhizoctonia sp., an unidentified taxon of Dothideomycete, and Phialophora sp. (Table 2.3). These four major species accounted for more than two-thirds of the number of all fungal isolates obtained from dead leaves and stems. In contrast to the findings in the Arctic, Cladophora luteo-olivacea and Phialocephala sp. were major species in exotic willows in Antarctica (Table 2.3). No fungal taxa were found common to dead remains of the Arctic and Antarctic willows. Minor genera in the arctic willow included Alternaria, Botrytis, and Cladosporium, which are common components of fungal assemblages on dead plant remains in arctic and temperate regions of the Northern hemisphere (Osono et al. 2014). The fungal taxa detected in dead remains from exotic willows in Antarctica included saprobic and root-associated fungi, of which C. luteo-olivacea has also been reported from other Antarctic regions and is likely indigenous (Hirose et al. 2013).

Fungal Succession Collecting dead leaves and stems of arctic willow from moraines in different stages of ecosystem development after glacier retreat enabled the study of seral succession of fungal assemblages. Moreover, when dead leaves and stems were collected, those in different stages of decomposition were included, allowing simultaneous elucidation of substrate fungal succession. The result was that turnover of fungal species was not found during either primary succession or decomposition of either leaves or stems (Table 2.3). Only minor changes in the frequencies of occurrence were detected for major fungal taxa between leaves at different stages of decomposition. No such stage-dependent differences were found for the occurrence of major fungal species in decomposing stems (Osono et al. 2014). No such data about fungal succession were available for dead stems of exotic willows in Antarctica.

Diversity and Ecology of Fungi in Polar Region 25 Table 2.3.  Fungi and decomposition in Arctic and Antarctic dead willow tissues. Study Area

Arctic

Arctic

Antarctica

Willow species

Salix arctica

Salix arctica

Salix pauciflora, S. reinii

Tissue

Leaf

Woody stem

Woody stem 18

Fungal Assemblages and Succession Number of species

15

14

Major species

Comoclathris sp., Rhizoctonia sp., Dothideomycete sp.

Comoclathris sp., Cladophora Rhizoctonia sp., luteo-olivacea, Phialophora sp. Phialocephala sp.

Pattern of fungal succession

No turnover

No turnover

No data

No data

No data

Potential capability of major fungal Selective species to decompose organic decomposition of chemical components holocellulose Hyphal Lengths Total hyphal length (mean, m/g)

4068

1970

No data

Darkly pigmented hyphal length (mean, m/g)

1063 [30%]

369 [17%]

No data

Pattern of decomposition of organic Selective chemical components decomposition of holocellulose

No significant change

No data

Changes in relative nitrogen content in willow tissues during decomposition

No significant change

No data

Decomposition Processes

Increase

Proportions relative to the total hyphal length are given in brackets. Fungi were isolated from moss tissues with a surface disinfection method. Arctic data from Osono et al. (2014); Antarctic data from Hirose et al. (2013).

Hyphal Length Hyphal lengths in leaves and stems of arctic willow were measured by direct observation using an agar film method, the same method as used for moss tissues. Mean values of total hyphal lengths were 4,068 and 1,970 m/g in leaves and stems of arctic willow (Table 2.3), similar levels to those in arctic and Antarctic moss tissues (Table 2.2). Total hyphal length increased with the decomposition of leaves and stems, whereas it was not significantly different between moraines in different stages of ecosystem development (Osono et al. 2014). Lengths of darkly pigmented hyphae accounted for 30 and 17% of the total hyphal length in leaves and stems of arctic willow, respectively (Table 2.3), similar levels to those in arctic and Antarctic moss tissues (Table 2.2). No such data about hyphal length were available for dead stems of exotic willows in Antarctica.

26  Fungi in Polar Regions

Decomposition Processes Contents of organic chemical components and nitrogen were assessed in leaves and stems of arctic willow in different stages of decomposition (Table 2.3). The amount of holocellulose relative to recalcitrant compounds (as AUR) decreased with the decomposition of leaves, indicative of selective decomposition of holocellulose. This contrasted to the decomposition of stems, in which no significant change was found for the relative amounts of holocellulose and AUR during decomposition. Similar patterns were found for changes in the relative content of nitrogen in dead tissues of arctic willow; the relative content of nitrogen increased with decomposition in leaves, whereas it did not change in stems (Table 2.3). Pure culture tests indicated that the major fungal species (Comoclathris sp., Rhizoctonia sp., and an unidentified Dothideomycete) were capable of decomposing holocellulose selectively in sterilized leaves of arctic willow (Table 2.3), consistent with the pattern of changes in organic chemical components in decomposing leaves. In contrast to the chemical changes in the leaves, in dead stems there were no obvious chemical changes, no significant increase in hyphal length, and no turnover of fungi, which suggest that there was low biological activity of decomposition processes in the dead stems.

Discussion The number of fungal species and hyphal length were generally low in dead tissues of moss and willow in the Arctic and Antarctica, compared to those in lower latitude ecosystems. This suggests that the extremely adverse environment limits the growth and coexistence of multiple fungal species and may account for the dominance and ubiquity of a limited number of fungal species. The vertical patterns of abundance and richness of fungi and chemical changes in the Arctic and Antarctic moss profiles exhibited interesting similarities and contrasts (Table 2.2). The hyphal length and the fungal richness were generally similar, while marked differences were found for fungal succession and lignocellulose decomposition between these moss profiles. Species composition of fungi changed successively downward within the Arctic moss profiles, concomitantly with the decrease of species richness and the relative decrease of holocellulose, indicating competition for carbohydrates and competitive exclusion between fungal species. In contrast, a cumulative increase of fungi with no significant changes in LCI characterized the succession in Antarctic moss profiles. These two patterns of fungal succession and resource utilization are analogous to the models of primary succession of plants in polar deserts proposed by Svoboda and Henry (1987). Svoboda and Henry (1987) summarized plant succession in marginal arctic environments and presented three models of succession: a directional-replacement model in low resistance environments, a directionalnonreplacement model in high resistance environments, and a nondirectionalnonreplacement model in extremely resistant environments. The richness and

Diversity and Ecology of Fungi in Polar Region 27

occurrence of fungi increased in the Antarctic moss profiles, but there was little evidence of turnover or replacement in the chronosequence, indicating directionalnonreplacement succession and high environmental resistance, which represent the sum of the adverse factors hindering the success of species establishment. In contrast, the fungal succession in Arctic moss profiles is consistent with the directional-replacement model, in which species replacement takes place due to competition. These contrasting patterns of fungal succession suggest that more hostile environmental conditions in continental Antarctica than in the Arctic lead to the characteristic pattern of succession in decomposing moss tissues in Antarctica, and limit the roles of fungi in the decomposition processes there. The observations of fungal succession and decomposition of arctic willow suggest that the growth and selective decomposition of holocellulose by major fungal species led to the accumulation of nitrogen in dead leaves (Table 2.3), a similar pattern of decomposition to that in arctic moss tissues. The pattern of fungal succession in leaves also fits the directional-nonreplacement model of Svoboda and Henry (1987), in which the occurrence of major fungal species increased during decomposition, as in the case of the Antarctic moss profile. The leaves of arctic willow exhibited an interesting contrast to their stems: in their stems, no turnover of fungi was found, and there were no significant changes in chemical composition during decomposition (Table 2.3). This indicates a nondirectional-nonreplacement succession in extremely resistant environments to fungi due to not only the direct exposure to environmental fluctuation, but also to the recalcitrant nature of woody tissues. The pattern of seral succession in leaves and stems of arctic willow at the glacier foreland also fits the nondirectional-nonreplacement model. In summary, the patterns of fungal succession and decomposition in Arctic and Antarctic plant remains can be integrated into the framework of successional models of Svoboda and Henry (1987) originally proposed for plant succession in marginal arctic environments. Introduction of exotic substrata in terrestrial environments of Antarctica has significant effects on indigenous soil fungi, because the supply of dead organic remains is quite low due to low ecosystem productivity in recently deglaciated, ice-free regions (Arenz et al. 2011). Recent studies of fungal populations in historically introduced exotic materials demonstrated a significant overlap of fungi with those from pristine locations in Antarctica (Farrell et al. 2011), consistent with the findings of present study detecting a presumably indigenous fungal species, C. luteo-olivacea, in exotic stems of willows (Table 2.3). These fungi have a variety of physiological traits that enable them to tolerate the harsh environment and survive under cold and dry conditions in the Arctic and Antarctica (Robinson 2001). To the knowledge of the authors, the present study is the first to provide an integrative framework and insights that contribute to understanding the diversity and ecology of fungi in the polar regions of the Arctic and Antarctica.

28  Fungi in Polar Regions

Acknowledgments We thank Dr. H. Kanda, Dr. A.S. Mori, Dr. T. Ueno, Dr. S. Iwasaki, Mr. R.W. Howe, Dr. Y. Tanabe, Dr. S. Kudoh, Dr. K. Kato, Dr. S. Imura, and members of JARE-51 for their assistance in fieldwork; and Dr. E. Nakajima for critical reading of the manuscript. This study was supported by the National Institute of Polar Research through General Collaboration Projects no. 29-31 to T.O., Japan Society for the Promotion of Science KAKENHI Grant (No. 18K05731 to T.O.), and the ArCS Project.

References Arenz, B.E., B.W. Held, J.A. Jurgens and R.A. Blanchette. 2011. Fungal colonization of exotic substrates in Antarctica. Fun. Div. 49: 13–22. Arenz, B.E., R.A. Blanchette and R.L. Farrell. 2014. Fungal diversity in Antarctic soils. pp. 35–53. In: D.A. Cowan (ed.). Antarctic Terrestrial Microbiology. Springer-Verlag, Berlin Heiderberg, Germany. Atmospheric Environment Service. 1982. Canadian Climatic Normals 1951–1980. Temperature and precipitation, the North, Y.T. and N.W.T. Ottawa, Environment Canada, 55 p. Domsch, K.H., W. Gams and T.H. Anderson. 2007. Compendium of Soil Fungi, second edition. IHWVerlag, Eching. Farrell, R.L., B.E. Arenz, S.M. Duncan, B.W. Held, J.A. Jurgens and R.A. Blanchette. 2011. Introduced and indigenous fungi of the Ross Island historic huts and pristine areas of Antarctica. Polar Biol. 34: 1669–1677. Hirose, D., Y. Tanabe, M. Uchida, S. Kudoh and T. Osono. 2013. Microfungi associated with withering willow wood in ground contact near Syowa Station, East Antarctica for 40 years. Polar Biol. 36: 919–924. Hirose, D., S. Hobara, S. Matsuoka, K. Kato, Y. Tanabe, M. Uchida, S. Kudoh and T. Osono. 2016. Diversity and community assembly of moss-associated fungi in ice-free coastal outcrops of continental Antarctica. Fungal Ecol. 24: 94–101. Hirose, D., S. Hobara, Y. Tanabe, M. Uchida, S. Kudoh and T. Osono. 2017. Abundance, richness, and succession of microfungi in relation to chemical changes in Antarctic moss profiles. Polar Biol. 40: 2457–2468. Hudson, H.J. 1968. The ecology of fungi on plant remains above the soil. New Phytol. 67: 837–874. Kanda, H. and M. Inoue. 1994. Ecological monitoring of moss and lichen vegetation in the Syowa station area, Antarctica. Proc. NIPR Symp. Polar Biol. 7: 221–231. Kudoh, S., Y. Tanabe, M. Uchida, T. Osono and S. Imura. 2015. Meteorological features observed in Yukidori Zawa, Langhovde and Kizahashi Hama, Skarvsnes on the Sôya Coast, East Antarctica, with comparison of those observed at Syowa Station. Antarctic Record 59: 163–178 (in Japanese with English summary). Lindo, Z. and A. Gonzalez. 2010. The Bryosphere: an integral and influential component of the Earth’s biosphere. Ecosystems 13: 612–627. Mori, A., T. Osono, S. Iwasaki, M. Uchida and H. Kanda. 2006. Initial recruitment and establishment of vascular plants in relation to topographical variation in microsite conditions on a recentlydeglaciated moraine in Ellesmere Island, high arctic Canada. Polar Biosci. 19: 85–95. Mori, A., T. Osono, M. Uchida and H. Kanda. 2008. Changes in the structure and heterogeneity of vegetation and microsite environments with the chronosequence of primary succession on a glacier foreland in Ellesmere Island, high arctic Canada. Ecol. Res. 23: 363–370. Mori, A.S., T. Osono, H. Cornelissen, J. Craine and M. Uchida. 2017. Biodiversity-ecosystem function relationships change through primary succession. Oikos 126: 1637–1649. Ochyra, R., R.I. Lewis-Smith and H. Bednarek-Ochyra. 2008. The Illustrated Moss Flora of Antarctica. Cambridge, Cambridge University Press.

Diversity and Ecology of Fungi in Polar Region 29 Osono, T., A. Mori, M. Uchida and H. Kanda. 2006. Chemical property of live and dead leaves of tundra plant species in Oobloyah Valley, Ellesmere Island, high arctic Canada. Mem. Natl. Inst. Polar Res. Spec. Issue 59: 144–155. Osono, T. 2007. Ecology of ligninolytic fungi associated with leaf litter decomposition. Ecol. Res. 22: 955–974. Osono, T. and J.A. Trofymow. 2012. Microfungal diversity associated with Kindbergia oregana in successional forests of British Columbia. Ecol. Res. 27: 35–41. Osono, T., T. Ueno, M. Uchida and H. Kanda. 2012. Abundance and diversity of fungi in relation to chemical changes in arctic moss profiles. Polar Sci. 6: 121–131. Osono, T., S. Matsuoka, D. Hirose, M. Uchida and H. Kanda. 2014. Fungal colonization and decomposition of leaves and stems of Salix arctica on deglaciated moraines in high-Arctic Canada. Polar Sci. 8: 207–216. Osono, T. 2015. Hyphal length in the forest floor and soil of subtropical, temperate, and subalpine forests. J. For. Res. 20: 69–76. Osono, T., A.S. Mori, M. Uchida and H. Kanda. 2016. Accumulation of carbon and nitrogen in vegetation and soils of deglaciated area in Ellesmere Island, high-Arctic Canada. Polar Sci. 10: 288–296. Robinson, C.H. 2001. Cold adaptation in Arctic and Antarctic fungi. New Phytol. 151: 341–353. Svoboda, J. and G.H.R. Henry. 1987. Succession in marginal arctic environments. Arc. Alp. Res. 19: 373–384. Tanabe, Y., M. Uchida, T. Osono and S. Kudoh. 2012a. Limnological parameters in Skarvsnes lakes between the 49th and 50th Japanese Antarctic Research Expeditions in 2008–2009. Long-term monitoring study. JARE Data Reports 322: 1–49. Tanabe, Y., M. Uchida, T. Osono and S. Kudoh. 2012b. Limnological parameters in Skarvsnes lakes between the 50th and 51th Japanese Antarctic Research Expeditions in 2009–2010. Long-term monitoring study. JARE Data Reports 323: 1–53. Tanabe, Y., S. Yasui, T. Osono, M. Uchida, S. Kudoh and M. Yamamuro. 2017. Abundant deposits of nutrients inside lakebeds of Antarctic oligotrophic lakes. Polar Biol. 40: 603–613. Ueno, T., T. Osono and H. Kanda. 2009. Inter- and intraspecific variations of the chemical properties of high-Arctic mosses along water-regime gradients. Polar Sci. 3: 134–138. Van der Wal, A., T.D. Geydan, T.W. Kuyper and W. de Boer. 2013. A thready affair: linking fungal diversity and community dynamics to terrestrial decomposition processes. FEMS Microbiol. Rev. 37: 477–494.

3 Snow Molds and Their Antagonistic Microbes in Polar Regions Tamotsu Hoshino,1,2,3,* Hisahiro Morita,4 Yuka Yajima,5 Masaharu Tsuji,6 Motoaki Tojo7 and Oleg B. Tkacehnko8

Introduction Approximately 80% of the biosphere is constantly or seasonally cold and has temperatures below 5ºC (e.g., Gounot 1999). The biosphere, with an exception of the deep sea, is almost identical to the cryosphere. The term cryosphere was proposed by the Polish scientist, A.B. Dobrowolski and collectively describes the portions of the Earth’s surface where water exists as the frozen state—snow cover, glaciers, ice sheets and shelves, freshwater ice, sea ice, icebergs, permafrost, and ground ice (Barry and Gan 2011). The cryosphere creates a seasonally extreme environment Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1, Tsukisamu-Higashi, Toyohira-ku, Sapporo, 062-0902, Japan. 2 Graduate School of Advanced Sciences and Matter, Hiroshima University, 1-3-1, Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8530, Japan. 3 Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1, Ten-noudai, Tsukuba, Ibaraki, 305-8572, Japan. Email: [email protected] 4 Tsuno Food Industrial Co. Ltd., 94 Sinden, Katsuragi-cho, Ito-gun, Wakayama, 649-7194, Japan. 5 College of Environmental Technology, Muroran Institute of Technology, 27-1, Mizomoto-cho, Muroran, Hokkaido, 050-8585, Japan. 6 National Institute of Polar Research, 10-3, Midori-cho, Tachikawa, Tokyo 190-8518, Japan. 7 Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1, Gakuen-cho, Nakaku, Sakai, Osaka, 599-8531, Japan. 8 Main Botanical Garden Named after N.V. Tsitsin, Russian Academy of Sciences, 4 Botanicheskaya ul., Moscow, 127276, Russia. * Corresponding author 1

Snow Molds and Their Antagonistic Microbes in Polar Regions 31

in temperate and frigid zones and often disappears in summer. Fungi associated with seasonal snow, ice and frozen, ground, so far termed psychrophiles (Morita 1975), should adapt to a wide temperature range from sub-zero temperatures to over 20°C, i.e., their maximal growth temperature. Fungal species were less frequently recorded from the cryosphere than those of temperate zone despite the fact that major fungal taxa have already been found in the cryosphere (Hoshino and Matsumoto 2012, Hoshino et al. 2013a, 2013b). These records suggest a possibility that various fungi are active under diverse cold environments. Fungi normally have different cells in their life cycle; fungal thermal dependence varies according to their life cycle stages and is completely different from that of bacteria. Examples are illustrated to show that the concept of psychrophile in bacteria by Morita (1975) does not apply to fungi, and we proposed a new term “cryophilic fungi” for those that spend a certain life stage or whole life cycle (sexual and/or asexual reproduction) in the cryosphere (Hoshino and Matsumoto 2012). According to sexual reproduction, the true fungi were divided into 4 phyla (Chytridiomycota, traditional Zygomycota, Ascomycota, and Basidiomycota). Though recent phylogenetic analysis revealed that fungi are composed of 7 phyla and uncertain 4 subphyla, we will use the former 4 phyla in this review. Cryophilic fungi include not only saprophytes but also parasites, as well as symbionts (Hoshino and Matsumoto 2012, Hoshino et al. 2013a, 2013b). Some groups of cryophilic fungi attack overwintering plants and other living organisms, including fungi on/in/under snow. Snow molds are incited by many fungi belonging to various taxa (oomycetes, ascomycetes, and basidiomycetes) that attack dormant plants, such as forage crops, winter cereals and conifer seedlings under snow cover (Matsumoto and Hsiang 2016). Many studies have been carried out on snow molds from the aspect of plant protection. New findings regarding their biodiversity, their distribution, and ecophysiological strategies adapted in polar regions have been reported during the past decade by a few researchers, including the author’s group. In this chapter, we show the biodiversity, their distribution, and ecological roles of snow molds in polar regions.

Biodiversity of Phytopathogenic Fungi Associated with Snow in Polar Regions Oomycetes Pythium snow rot occurs in Japan, Iran, the United States, and Kola Peninsula in the European part of Russia in the Arctic (Petrov 1983). The pathogens, i.e., Pythium iwayamai, P. paddicum, and P. okanoganense have not been found so far in polar regions, however a heterothallic species, Pythium polare was isolated from moribund tissues of polar mosses in both poles (Tojo et al. 2012). Symptoms on mosses caused by P. polare were the same (Bridge et al. 2008, Tojo and Newsham 2012) and isolates from both poles were sexually compatible. Similar symptoms

32  Fungi in Polar Regions were also found on mosses in the Arctic, such as Finnmark (northern Norway: Hoshino et al. 2000), Svalbard (Hoshino et al. 1999, 2001a) and Greenland (Hoshino et al. 2006b) and Antarctica (Hoshino et al. 2001b, Bridge et al. 2008). Pythium sp., morphologically similar to P. ultimum var. ultimum and other Pythium spp., were isolated from moribund tissues of mosses on Svalbard (Hoshino et al. 1999). Chilling resistances in moss pathogenic fungus, P. ultimum var. ultimum, from Svalbard and in the other strains from Japan, were different. The strains from Svalbard could grow and survive at 0–5°C. In addition, chilling treatment induced irregular mycelial morphology in the Arctic strains. On the other hand, the strains from Japan did not grow at temperatures below 5°C and were destroyed after chilling stress (0°C for 3 days or at 4°C for 1 week) (Hoshino et al. 2002). Pythium species were reported to be less tolerant to chilling and freezing temperatures than other snow mold taxa (Takamatsu 1989, Hoshino et al. 2002). We investigated the frost resistance of Pythium species from Japan and Subantarctic Regions. Free mycelia and hyphal swellings, structures for survival of P. iwayamai and P. paddicum lost viability within freeze–thaw 3 cycles; however, mycelia in host plants survived the treatment (Murakami et al. 2015). Fungi in permafrost are characterized both by the presence of natural cryoprotectants in these ecotopes and by the ability to utilize their inherent mechanisms of protection (Ozerskaya et al. 2009). It is conceivable that plant substrates or derivatives thereof are natural cryoprotectants, enabling them to provide advantageous conditions to microorganisms under freezing conditions.

Chytridiomycetes The cryoseston is defined as the community of organisms living on snow (Nedbalová et al. 2008). Algae which grow on snow are named “snow algae”. Snow algae are major organisms in the cryoseston, and fungi that associate with snow algae are referred to as snow fungi. Kol (1968, 1974) listed 82 species of snow fungi, including 3 fungal pathogens of snow algae, i.e., the chytridiomycetes Chytridium chlamydococci f. cryophila (Nom. Inval.), Rhizophydium sphaerocarpum subsp. Cryophilum, and the ascomycete Oospora nivalis (Kol 1939, 1968). C. chlamydococci f. cryophila was recovered in the cells of algae, Chlamidomonas bolyaiana in South America, C. nivalis in several areas in Europe, C. sangunea in Romania and Ancylonema nordenskoiöldii in Greenland (Kol 1968). R. sphaerocarpum subsp. cryophilum was found in the cells of A. nordenskoiöldii in Alaska (Kol 1942, 1968).

Zygomycetes Zygomycetous snow molds are not found in the Northern Hemisphere, but Rhizopus sp. was pathogen on the moss Bryum anatrcticum at Cape Bird on Ross

Snow Molds and Their Antagonistic Microbes in Polar Regions 33

Island, Antarctica (Greenfield 1983). Unfortunately, the specimens and the strain are not available (L. Greenfield personal communication).

Ascomycetes Pink snow mold, Microdochium nivale is the most widespread snow mold fungus. This fungus is distributed not only in the Northern Hemisphere but also in the Southern Hemisphere (Matsumoto and Hsiang 2016). This species has a few reports in the Arctic, such as Finnmark (Årsvoll 1973), Alaska (McBeath 2002) and Greenland (T. Hoshino unpublished results). Serious damage occurs on winter cereals under snow cover lasting for 2 months or more (Årsvoll 1973). Sclerotinia borealis (syn.: Myriosclerotinia borealis) causes snow mold of winter cereals, forage crops, and conifer seedlings, and has been found in cold regions in the Northern Hemisphere. This fungus has also been found from the Arctic, such as Finnmark (Årsvoll 1973), Lapland (northern Finland and Sweden: Jamalainen 1949, Ekstrand 1955), Kola Peninsula in the European part of Russia (Petrov 1983, Tkachenko 2013), Svalbard (Hoshino et al. 2003b), Greenland (Hoshino et al. 2006a), Alaska and the Yukon (Lebeau and Longston 1958, McBeath 2002). Sclerotinia nivalis also causes snow mold of herbaceous dicots (Saito 1997). This fungus was distributed in snowy temperate zone and only found from the Russian Arctic (Tkachenko 2013). S. borealis and S. nivalis were not found from Antarctica. However, Sclerotinia antarctica on hair grass Deschampsia antarctica was found in Cierva Point, Danco Coast, Antarctic Peninsula. The fungus is morphologically similar to S. borealis (Gamundi and Spinedi 1987) and needs comparisons to S. borealis. The closelyrelated strain M. nivale from phylogenic analysis was isolated from the lake water on King George Island, maritime Antarctica (Gonçalves et al. 2012). Herpotrichia juniperi causes brown felt blight of conifers in the Northern Hemisphere (Schneider et al. 2009). This fungus was also isolated from not only Svalbard in the Arctic (Ali et al. 2013), but also from maritime Antarctica (King George Island) (Rosa et al. 2010), and its DNA was found from continental Antarctica (active layer of permafrost near Station Novolazarevskaya) (Kochkina et al. 2014). The phytopathogenic activity and ecological role of these fungi in polar regions is not reported. Nissinen (1996) showed a strong positive association between the incidence of S. borealis and depth of soil frost in November in Lapland, northern Finland. In years when the average depth of frozen soil was 21 cm or more by the middle of November, the damage caused by S. borealis was severe. Conversely, when the soil was frozen to a depth of less than 5 cm, basidiomycete, Typhula spp. caused more damage. Typhula spp. predominated when soil freezing was delayed by early establishment of a thick snow cover. Röed (1960) also reported that a thin snow cover and deep soil freezing promoted plant damage caused by S. borealis, and that a thick snow cover and unfrozen or slightly frozen soil favored the development of

34  Fungi in Polar Regions Typhula spp. and M. nivale. Thus, freezing is critical to the incidence of S. borealis. Tomiyama (1955) cultured S. borealis and T. incarnata on frozen and unfrozen potato dextrose agar (PDA) plates that were kept outside in Sapporo, Hokkaido, northern Japan, during winter. In his study, mycelial growth of T. incarnata was inhibited on frozen PDA, but S. borealis grew faster on frozen PDA than on unfrozen PDA. However, his experiments were not carried out under controlled conditions, and his results have not been reproduced by others. We confirmed his results: S. borealis grew on frozen PDA under controlled condition (Hoshino et al. 2009b, 2010a). S. borealis showed normal mycelial growth under the frozen condition, and mycelial growth rate on frozen PDA at −1°C was faster than that on unfrozen PDA at the optimal growth temperature range from 4 to 10°C. Our results support the findings of Tomiyama and previous studies by others (Röed 1960, Nissinen 1996) showing that this fungus adapts to harsh winters with soil freezing. S. borealis and basidiomycetous snow mold, Typhula ishikariensis, was found in Barentsburg, Svalbard in 1999 and 2000 (Hoshino et al. 2003b). S. borealis was also collected from Longyearbyen and Ny-Ålesund in 2007 (Hoshino et al. 2010b). Recently, climate change is probably suitable for the magnification of this fungal distribution in Svalbard. In lower latitude snowy region, Hokkaido, the recent tendency towards global warming has certainly changed snow mold microflora. S. borealis is no longer a problem due to the early onset of snow cover and warm winter. T. ishikariensis replaced S. borealis (Matsumoto and Hoshino 2013). Some ascomycetous cryophilic phytopathogens use hard substrate of conifer needles in Arctic Scandinavia. A fungus, believed to be Phacidium infestans extends web-like mycelia over the surface of snowbank during snowmelt (Hoham and Duval 2001). Cells of snow algae, Chloromonas cryophila (Hoham and Mullet 1977) and Chloromonas pinchichae (Hoham 1975) are frequently found adhered to the surface of the fungal mucelia, but there is no evidence of symbiosis as is found in lichens. Possibly, the fungus and algae may exchange metabolites extracellularly. Microscopic characterization is, unfortunately, not available on the web-like fungus. Other ascomycetous species are also recognized as snow molds in both poles. Though usually regarded as a saprobe in temperate zone, an ascomycete Trichoderma polysporum (teleomorph: Hypocrea pachybasioides) caused asympomatic infections on the moss Sanionia uncinata in Svalbard (Yamazaki et al. 2011). Several ascomycetes Thyronectria antarctica var. hyperantarctica, Coleroa turfosorum, Bryosphaeria megaspora, Epibryon chorisodontii, and an unidentified Plectomycete were recorded from ring infection and macroscopic section of mosses (Hawksworth 1973, Longton 1973, Fenton 1983). Cryophilic fungi are less in the number of species than that of fungi in temperate regions (Hoshino et al. 2013a), and flora in polar regions are different from other regions of cryosphere in temperate and frigid zones. Therefore, there must be new niches for cryophilic fungal phytopathogens in polar regions, and novel cryophilic fungi are likely to have evolved as phytopathogens to adapt in polar climate and vegetation (Hoshino et al. 2013b).

Snow Molds and Their Antagonistic Microbes in Polar Regions 35

Basidiomycetes Cottony snow mold caused by low-temperature basidiomycetes (LTB = Coprinus psychromorbidus) attacks numerous garden perennials and wild species (Smith et al. 1989). The fruit rot low-temperature basidiomycete (FRLTB) causes storage rot of apples and pears in fruit-growing areas of Oregon and British Columbia (Gaudet 2001). Sclerotial strains of LTB (SLTB) have been found in the crowns of perennial grasses and winter cereals (Gaudet 2001). The disease is first seen on snow melt in spring as bleached patches of host plants. Abundant grayish-white mycelia are often present on the edges of patches. There are no or few sclerotia as has only been found with C. psychromorbidus in regions of North America including Alaska (McBeath 2002). The genus Typhula includes about 100 species, none of which occurs in tropical regions. Most species are saprophytic and low-temperature tolerant, and only six species, namely, T. incarnata, T. ishikariensis complex (including T. borealis, T. hyperborea, and T. idahoensis), T. japonica, T. phacorrhiza, T. trifolii, and T. variabilis, are known to cause disease of grasses and forage crops as well as winter cereals (Ikeda et al. 2015). The gray snow mold T. incarnata is widely distributed in snowy regions in the Northern Hemisphere including in the Arctic, such as Finnmark (Årsvoll 1973), Lapland (Ekstrand 1955, Jamalainen 1957), Russia Arctic (Khokhryakova 1983, Shiryaev 2006, Tkachenko 2013), Svalbard (Shiryaev and Mukhin 2010), Iceland (Kristinsson and Guðleifsson 1976, Hoshino et al. 2004a), Greenland (Hoshino et al. 2006a), and Aleutian Islands (Adak Island, T. Hoshino unpublished results). T. ishikariensis complex are also distributed in snowy temperate zone to the Arctic in Finnmark (Årsvoll 1973), Lapland (Ekstrand 1955, Jamalainen 1957), Kola Peninsula in the European part of Russia (Khokhryakova 1983, Tkachenko 2013), Svalbard (Hoshino et al. 2003b, Shiryaev and Mukhin 2010), Iceland (Kristinsson and Guðleifsson 1976, Hoshino et al. 2004a), Greenland (Hoshino et al. 2006a), Alaska and the Yukon (Lebeau and Longston 1958, Anchorage; T. Hoshino unpublished results). T. variabilis and T. phacorrhiza were also found in the Arctic in Russian Arctic (Shiryaev 2006), Svalbard (Shiryaev and Mukhin 2010), and Iceland (Kristinsson and Guðleifsson 1976). These species were not found in continental climates and polar desert in the Arctic (Tkachenko 2013, T. Hoshino unpublished results). We collected tiny sclerotia (ca. 1 mm) on moribund leaves of Sea pea (Lathyrus japonicus var. aleuticus) along the Great Whale River where there is continental climate in Kuujjuarapik/Whapmagoostui, Quebec, Canada (Fig. 3.1). DNA sequences of ITS region from these sclerotia had high homology with T. japonica and T. laschii which had two-spored basidium. We should elucidate ecophysiological characteristics of this fungus in the coming future. Microbotryum bistortarum, the smut fungus, was also shown to have deleterious effects on the growth and survival of Alpine Bistort (Polygonum vivparum) under snow cover also on Svalbard (Tojo and Nishitani 2005). This fungus has a similar life cycle to scleroderris canker disease fungus Gremmeniella abietina (Yokota et al. 1974). Mycelia which had clamp connection,

36  Fungi in Polar Regions

A.

B.

C.

Fig. 3.1.  Typhula sp. in continental climate. Partial view of Kuujjuarapik/Whapmagoostui and the Great Whale River in Quebec, Canada (A). A river bank and communities of Sea pea (B). Sclerotia of Typhula sp. died leaves of Sea pea (C; bar = 1 cm).

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infected in moss carpet in Jan Mayen in the Greenland Sea (Wilson 1951). However, there is no taxonomical information of this fungus until this age. Fig. 1. Hoshino et al. Snow blight of moss, Polytrichum juniperinum, was found on King George Island, maritime Antarctica. Host died in a circle of ca. 10 to 30 cm after snow melts. Clamp connected hyphae and no sclerotia were observed on tip of host leaves. DNA sequence of ITS region from moss symptoms were perfectly matched with fruit bodies of Typhula sp. on Macquarie Island in the maritime Antarctica and high homology with Typhula cf. subvariabilis from Iran (Yajima et al. 2017). Therefore, we suggested that T. cf. subvariabilis caused snow blight on moss in Antarctica, and this is the first record of Typhula snow blight in the Southern Hemisphere. The results also suggested that fungi in same genera gained similar ecological niche in both polar regions. DNA sequences of D1/D2 region which had high similarity (ca. 98.9%) to Typhula micans were found from the soil on East Ongul Island, Lützow-holm bay, East Antarctica (M. Tsuji unpublished results). T. cf. subvariabilis and T. micans often formed fruiting body without sclerotia, and probably these fungi had higher maximal growth temperature than those of T. ishikariensis and T. incarnata. These observations suggested that eurythermal ecophysiological characteristics were one of the important factors for the succession of fungi in Antarctica. Basidiomycetous snow molds produce extracellular antifreeze proteins (AFPs), which attach to the surface of ice crystals to inhibit their growth (Hoshino et al. 2009b). Extracellular antifreeze activity is the mechanism unique to basidiomycetous snow molds, but not in other snow molds belonging to oomycetes or ascomycetes (Hoshino et al. 2003a, 2009b). We purified and cloned AFPs from basidiomycetous snow molds C. psychromorbidus and T. ishikariensis (Hoshino et al. 2003a, 2009b). Cloned genes of AFPs from T. ishikariensis did not have any similarity with known proteins when we found these genes. After our publications, isogenes were found in bacteria, algae, and animals. Therefore, these fungal AFPs were considered to be a new class of AFPs. AFP of T. ishikariensis (TisAFP) exhibited bursting ice

Snow Molds and Their Antagonistic Microbes in Polar Regions 37

growth normal to the c-axis of the ice crystal and high thermal hysteresis activity (approximately 2°C), as in the case of insect hyperactive AFPs (Xiao et al. 2010). These findings suggest that fungal AFP homologs are widely distributed in different kingdoms, implying the possibility of horizontal gene transfer between eukaryotic microbes and prokaryotes (Raymond et al. 2007, Sorhannus 2011, Raymond and Kim 2012). Diffusion of AFPs secreted in the extracellular environment does not support mycelial growth under sub-zero temperatures. AFPs and thermal hysteresis activity at concentrations are found in the extracellular polysaccharide, which surround Arctic diatoms (Krembs et al. 2002). T. incarnata and T. ishikariensis also produce extracellular polysaccharides (Hoshino et al. 2009b), and they probably bind the AFP molecules they secrete. M. nivale does not produce AFPs, but produces extracellular polysaccharides, such as cellulose (Schweiger-Hufnagel et al. 2000 ) and fructan (Cairns et al. 1995). These extracellular polysaccharides of M. nivale are considered to bind plant antifungal peptides and reduce activities of these peptides (Hoshino et al. 2009b).

Antagonistic Microbes Against Snow Molds in Polar Regions In snowy temperate and frigid zones, snow molds, several fungi were parasitic Typhula spp. Ascomycete Episclerotium sclerotipus (Saito et al. 2017) and basidiomycete Cylindrobasidium parasitic (Woodbridge and Coley-Smith 1988) were parasitic sclerotia of T. phacorrhiza and T. incarnata, respectively. Matsumoto (1989) isolated 74 fungal species from sclerotia of T. ishikariensis from Japan. An Arctic strain of Trichoderma polysporum produced growth inhibitors of P. iwayamai (Kamo et al. 2016). Four compounds from culture medium of T. polysporum showed a concentration-dependent growth-inhibitory effect against P. iwayamai. None of these compounds inhibited the growth of pathogen at 33 µg/m during 15 days incubation at 20°C. Studies of the biological control of snow molds have been carried out using non-pathogenic psychrophilic fungi, such as T. phacorrhiza (Hsiang et al. 1999) and psychrotolerant bacteria, such as Pseudomonas spp. (Ohshiman 2000, Amein et al. 2008, Andersson et al. 2012). We also sought biocontrol agents from cold environmental soils including both polar region (Hoshino et al. 2004b). Our collected bacteria were divided into 3 types of fungal growth inhibitions with co-cultures in agar plates (Fig. 3.2). First, the extension of fungal colony was prohibited by co-cultured bacteria, however, mycelial colony morphology did not change (Fig. 3.2A). Second was the formation of brown line between fungal pathogen and bacterial colonies (Fig. 3.2B). This mycelia inhibition pattern was caused by Pseudomonas spp. isolated from soil collected from Adak Island and Fairbanks in Alaska, Komi, Yakutsk, and Magadan in Russia. The third pattern was fungal colony that changed to red color by the co-cultured bacterium (Fig. 3.2C). This phenomenon is not found from previous reports in bio-control agents against snow molds, and bacterial strains probably produced volatile

38  Fungi in Polar Regions

Fig. 3.2.  Mycelial growth inhibition of snow mold, Typhula ishikariensis co-culture with various bacteria from cold enviroments. Bacillus simplex from Yakutsk, Sakha republic in Russia (A), Pseudomonas sp. from Fairbanks, Alaska, USA (B), Pseudomonas sp. from Svalbard (C) and Paenibacillus macquariensis subsp. defensor from Oblast Magadan, Russia.

Color version at the end of the book

compounds to induce reddish mycelia of T. ishikariensis. This mycelia inhibition pattern was also caused by Pseudomonas spp. isolated from soil collected from Anchorage in Alaska, Ny-Ålesund on Svalbard, Komi, Yakutsk, and Magadan in Russia. Major strains that had activities to inhibit fungal growth belonged in Pseudomonas, and we also obtained a few strains of Bacillus simplex from Yakutsk, Sporosarcina psychrophile and Paenibacillus macquariensis subsp. defensor in Magadan (Hoshino et al. 2009a). B. simplex and S. psychrophile were first records of bio-control activities against fungal pathogens. Strains of P. macquariensis subsp. defensor inhibited the mycelial growth and sclerotium production of T. ishikariensis (Fig. 3.2D). As far as is known, the inhibition of sclerotium production by a bacterium has never been demonstrated in previous studies; however, the characteristic disappeared during repeated subcultures.

Ecological Role of Snow Molds in Polar Regions Moss vegetation plays an important role in primary succession in polar regions and are a key part of the terrestrial ecosystems in polar regions (Longton 1997). Previously, several fungi, including snow molds, have been reported to actively attach mosses growing in the Arctic, such as Finnmark (Hoshino et al. 2000), Svalbard (Ridley et al. 1979, Hoshino et al. 1999, 2001a), Jan May (Wilson 1951), Greenland (Hoshino et al. 2006b), Ellesmere Island (Longton 1973), and in Antarctica, such as the South Sandwich Islands, the South Orkney Islands (Longton 1973), the South Shetland Islands (Hoshino et al. 2001b, Tojo and Newsham 2012, Tojo et al. 2012) and Cape Bird, Victoria Land (Greenfield 1983), Terra Nova Bay, Ross Sea and Skarvsnes, the Sôya Coast (Hoshino et al. unpublished results, Fig. 3.3). Several researches reported a large number of concentric bands in dead moss in the Arctic (Wilson 1951, Longton 1973, Ridley et al. 1979, Hoshino et al. 2001b, Hoshino et al. 2006b, Tojo and Newsham 2012). Pathogenic fungi invaded a moss carpet and formed patches after several years. Host moss shoots were destroyed

Snow Molds and Their Antagonistic Microbes in Polar Regions 39

in the central parts by fungal infections. We often found some higher plants in the Arctic, other mosses in both polar regions, or algae in maritime Antarctica (Hoshino et al. 2001a, 2001b), in those moribund moss patches (Fig. 3.4). Therefore, the invasion of phytopathogenic fungi in a moss carpet was thought to lead to the formation of “open spaces” where other plants easily colonized. The formation of pathogenic patches might be the first step in changes in the pattern in plant communities in polar regions. Previous theories of the succession and colonization of polar plants have focused on plant-environmental or plant-plant interactions. Our observations offer a new concept of the colonization of plants. B. B.

D. D..

Fig. 3.3.  Fungal symptoms of mosses in continental Antarctica. Terra Nova Bay, Ross Sea (A and B) and Skarvsnes, the Sôya Coast (C and D).

Color version at the end of the book (A)

(B)

(C)

Fig. 3.4.  The hypothesis of ecological impact of snow molds in polar regions. The invasion of snow molds in stable moss colony (A). Partial regrowth of moss and formation of concentric bands in dead moss area (B). The invasion of other plants (C).

40  Fungi in Polar Regions

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42  Fungi in Polar Regions Kol, E. 1942. The snow and ice algae of Alaska. Smithsonian Misc. Collect. 101: 1−36. Kol, E. 1968. Kryobiologie. Biologie und Limnologie des Schnees und Elises. I. Kryovegetation. E. Schweizerbart’sche Velagsbuchhandlung, Stuttgart, Germary. Kol, E. 1974. Trachiscia (Chlorophyta) red snow from Swedish Lapland. Ann. Hist-Nat. Mus. Nat. Hung. 66: 59−63. Krembs, C., H. Eicken, K. Junge and J.W. Deming. 2002. High concentrations of exopolymeric substances in Arctic winter sea ice: implications for the polar ocean carbon cycle and cryoprotection of diatoms. Deep Sea Res. I. 49: 2163−2181. Kristinsson, H. and B.H. Guðleifsson. 1976. The activity of low temperature fungi under the snow cover in Iceland. Acta Bot. Isl. 4: 44−57. Lebeau, J.B. and C.E. Longston. 1958. Snow mold of forage crops in Alaska and Yukon. Phytopathology 48: 148–150. Longton, R.E. 1973. The occurrence of radial infection patterns in colonies of polar bryophytes. Br. Antarc. Surv. Bull. 32: 41−49. Longton, R.E. 1997. The role of bryophytes and lichens in polar ecosystems. pp. 69−96. In: S.J. Woodin and M. Marquiss (eds.). Ecology of Arctic Environments, Blackwell, Oxford, UK. Matsumoto, N. 1989. Autecology of pathogenic species of Typhula. Res. Bull. Hokkaido Natl. Agric. Exp. Stn. 152: 91−162. Matsumoto, N. and T. Hoshino. 2013. Change in snow mold flora in eastern Hokkaido and its impact on agriculture. pp. 255−261. In: R. Imai, M. Yoshid and N. Matsumoto (eds.). Plant and Microbe Adaptations to Cold in a Changing World: Proceedings of the Plant and Microbe Adaptation to Cold Conference, 2012, Springer, New York, USA. Matsumoto, N. and T. Hsiang. 2016. Snow mold. The battle under snow between fungal pathogens and their plant hosts. Springer. McBeath, J.H. 2002. Snow mold-plant-antagonist interactions: Survival of the fittest under the snow. The Plant Health Instructor. Morita, R.Y. 1975. Psychrophilic bacteria. Bacteriol. Rev. 39: 144−167. Murakami, R., Y. Yajima, K. Kida, K. Tokura, M. Tojo and T. Hoshino. 2015. Surviving freezing in plant tissues by oomyceteous snow molds. Cryobiology 70: 208−210. Nedbalová, L., M. Kociánová and J. Lukavský. 2008. Ecology of snow algae in the Giant Mts. Opera Corcontica 45: 59−68. Nissinen, O. 1996. Analyses of climatic factors affecting snow mould injury in first-year timothy (Phleum pratense L.) with special reference to Sclerotinia borealis. Acta Univ. Oulensis A 289: 1–115. Ohshiman, K. 2000. Sodium algnate as an adjuvant of an antagonistic bacterium, Pseudomonas fluorescence strain A11RN, to enhance biocontrol of turfgrass snow mold caused by Typhula ishikariensis. J. Gen. Plant Pathol. 66: 258–263. Ozerskaya, S., G. Kochkina, N. Ivanushkina and D.A. Gilichinsky. 2009. Fungi in permafrost. pp. 85–95. In: R. Margesin (ed.). Permafrost Soils, Soil Biology, vol. 16, Springer-Verlag, Berlin, Germany. Petrov, V.F. 1983. Pathogenic microflora of root growth in perennial grasses in Khibiny (in Russian, with English abstract). Bull. Appl. Bot. Genet. Plant Breed. 82: 38–45. Raymond, J.A. and H.J. Kim. 2012. Possible role of horizontal gene transfer in the colonization of sea ice by algae. PLoS ONE 5: e35968. Raymond, J.A., C. Fritsen and K. Shen. 2007. An ice-binding protein from an Antarctic sea ice bacterium. FEMS Microbiol. Ecol. 61: 214–221. Ridley, M., C. Gillow, D. Perkins and J. Ogilvie. 1979. Oxford University expeditions to Svalbard 1978. Bull. Oxf. Univ. Explor. Club. New Series, Commem. Vol. 29–79. Röed, H. 1960. Sclerotinia borealis Bub. & Vleg., a cause of winter injuries to winter cereals and grasses in Norway. Acta Agric. Scand. 10: 74–82. Rosa, L.H., M.L.A. Vieira, I.F. Santiago and C.A. Rosa. 2010. Endophytic fungi community associated with the dicotyledonous plant Colobanthus quitensis (Kunth) Bartl. (Caryophyllaceae) in Antarctica. FEMS Microbiol. Ecol. 73: 178–189. Saito, I. 1997. Sclerotinia nivalis, sp. nov., the pathogen of snow mold of herbaceous dicots in northern Japan. Mycoscience 38: 227–236.

Snow Molds and Their Antagonistic Microbes in Polar Regions 43 Saito, I., T. Hoshino and I. Yumoto. 2007. Cultural features of Episclerotium sclerotipus parasitizing sclerotia of Typhula phacorrgiza. Jpn. Phytopathol. 73: 77–78. Schneider, M., C.R. Grunig, O. Holdenrieder and T.N. Sieber. 2009. Cryptic speciation and community structure of Herpotrichia juniperi, the causal agent of brown felt blight of conifers. Mycol. Res. 113: 887–896. Schweiger-Hufnagel, U., T. One, K. Izumi, P. Hufnagel, N. Maritia, H. Kaga, M. Maorita, T. Hoshino, I. Yumoto, N. Matsumoto, M. Yoshida, M.T. Sawada and H. Okuyama. 2000. Identification of the extracellular polysaccharide produced by snow mold fungus Microdochium nivale. Biotechnol. Lett. 22: 183–187. Shiryaev, A.G. 2006. Clavarioid fungi in Urals III. Arctic Zone (in Russian with English summary). Mycol. Phytopathol. 40: 294–304. Shiryaev, A.G. and V.A. Mukhin. 2010. Clavarioid-type fungi from Svalbard: Their spatial distribution in the European High Arctic., North Am. Fungi 5: 67–84. Smith, J.D., N. Jackson and A.R. Woolhouse. 1989. Fungal diseases of amenity turf grasses, 3rd edn. E. & F.N. Spon, London, UK. Sorhannus, U. 2011. Evolution of antifreeze protein genes in the diatom genus Fragilaripsis: Evidence for horizontal gene transfer, gene duplication and episodic diversity selection. Evol. Bioinformatics 7: 279–289. Takamatsu, S. 1989. Snow molds in winter wheat: studies on occurrence of Pythium snow rot (in Japanese with English summary). Spec. Bull. Fukui Agric. Exp. Stn. 9: 1–135. Tkachenko, O.B. 2013. Snow mold fungi in Russia. pp. 293–303. In: R. Imai, M. Yoshida and N. Matsumoto (eds.). Plant and Microbes Adaptations to Cold in a Changing World, Springer, New York, USA. Tomiyama, K. 1955. Studies of the snow mold blight disease of winter cereals (in Japanese with English summary). Hokkaido Natl. Agric. Exp. Stn. Rep. 47: 224–234. Tojo, M. and S. Nishitani. 2005. The effects of the smut fungus Microbotryum bistotarum on survival and growth of Polygonum vivparum in Svalbard. Can. J. Bot. 83: 1513−1517. Tojo, M. and K.K. Newsham. 2012. Snow moulds in polar environments. Fugal Ecol. 5: 395−402. Tojo, M., P. van West, T. Hoshino, K. Kida, H. Fujii, C. Hakoda, Y. Kawaguchi, H.A. Mühlhauser, A.H. van den Berg, F.C. Küpper, M.L. Herrero, S.S. Klemsdal, A.M. Tronsmo and H. Kanda. 2012. Pythium polare, a new heterothallic Oomycete causing brown discoloration of Sanionia uncinata in the Arctic and Antarctic. Fugal Biol. 116: 756−768. Xiao, N., K. Suzuki, Y. Nishimiya, H. Kondo, A. Miura, S. Tsuda and T. Hoshino. 2010. Comparison of functional properties of two fungal antifreeze proteins from Antarctomyces psychrotrophicus and Typhula ishikariensis. FEBS Lett. 277: 394–403. Yajima, Y., M. Tojo, B. Chen and T. Hoshino. 2017. Typhula cf. subvariabilis, new snow mould in Antarctica. Mycology 8: 147–152. Yamazaki, Y., M. Tojo, T. Hoshino, K. Kida, T. Sakamoto, H. Ihara, I. Yumoto, A.M. Tronsmo and H. Kanda. 2011. Characterization of Trichoderma polysporum from Spitsbergen, Svalbard archipelago, Norway, on species identity, infectivity to moss, and polygalacturonase activity. Fungal Ecol. 4: 15−21. Yokota, S., T. Uozumi and S. Matsuzaki. 1974. Scleroderris canker of Todo-fir in Hokkaido, northern Japan II. Physiological and pathological characteristics of the causal fungus. Eur. J. For. Path. 4: 155−166. Wilson, J.W. 1951. Observations on concentric ‘fairy rings’ in Arctic moss mat. J. Ecol. 39: 407–416. Woodbridge, B. and J.R. Coley-Smith. 1988. A new species of Cylindrobasidium parasitic on sclerotia of Typhula incarnata. Trans. Br. Mycol. 91: 166−169.

4 Pathogenic Fungi on Vascular Plants in the Arctic Diversity, Adaptation, Effect on Host and Ecosystem, and Response to Climate Change Shota Masumoto

Introduction Fungi are one of the most species-rich groups of organisms in the Arctic (Meltofte 2013). The known number of fungal species in the Arctic regions is presently about 4,350, and the total fungal-species richness in the Arctic may exceed 13,000 (Dahlberg and Bültmann 2013). Fungi constitute a large portion of arctic biodiversity and are essential to the functioning of the Arctic terrestrial ecosystems, because their various life strategies to access nutrients and energy are mutualistic, endophytic, saprotrophic, or parasitic. In nutrients and energy flow in ecosystems, plant parasitic fungi play the role of a consumer using primary production and are also often considered to be reducers of ecosystem productivity, as they cause disease on host plants. Therefore, their ecological features can directly control the host population dynamics, and indirectly affect community structure or functioning of an ecosystem through host population modification (Gilbert 2002). The most surveyed parasitic fungi in the Arctic is lichenicolous fungi, the parasite on lichen (Zhurbenko 2010), and the species richness corresponds to > 20% of the known total Graduate School of Environment and Information Sciences, Yokohama National University, 79-7 Tokiwadai, Hodogaya, Yokohama, 240-8501, Japan. Email: [email protected]

Pathogenic Fungi on Vascular Plants in the Arctic 45

number globally. In contrast, there is currently little knowledge of plant parasitic fungi in the Arctic (Dahlberg and Bültmann 2013). In this chapter, I present the diversity and ecology of plant pathogenic fungi in the Arctic. Although plant pathogenic fungi are defined as parasitic fungi able to cause disease in host plants (Kirk et al. 2008), note that I call true fungi and fungal-like organisms (e.g., oomycetes) having pathogenicity “pathogenic fungi”. The most well-studied plant pathogens in the Arctic are snow-mold fungi (Hoshino et al. 2009, Tojo and Newsham 2012, Tkachenko 2013). “Snow-mold fungi” is a general term for pathogenic fungi that cause blight, root rot, or damping-off of their host plant in cool temperature environments, thus they include various taxonomic groups of fungal or fungal-like micro-organisms, such as the genus Typhula, Sclerotinia, Racodium, Microdochium, and Pythium, and mainly infect moss or graminoid as host plant species (Hoshino et al. 2009, Tojo and Newsham 2012). Various studies on the taxonomy and ecology of snow-mold fungi and applied studies on their antifreeze proteins have been promoted (Hoshino et al. 2003, Kondo et al. 2012, Tkachenko 2013). In contrast, studies on other pathogenic fungi on vascular plants, as focused on in this chapter, are very limited. These pathogens have been reported since the late nineteenth century by Karsten (1872), which is the first overview of arctic fungi (Dahlberg and Bültmann 2013). Arctic fungal research has been primarily exploratory, focusing on the identification, description and recording of fungal taxa (Dahlberg and Bültmann 2013). There are some species of plant pathogens included in the catalogs and checklists for fungi in the various Arctic regions (Gulden and Torkelsen 1996, Elvebakk et al. 1996, Hallgrímsson and Eyjólfsdóttir 2004, Borgen et al. 2006). However, with the exception of a brief description in such catalogs as part of an exhaustive survey, detailed reports on the taxonomical characteristics or the effect on host plants is limited to a few species in certain local places (Tojo and Nishitani 2005, Smith et al. 2004, Masumoto et al. 2014). Moreover, there are only two studies that include surveys on the effect of the fungi on an ecosystem level (Olofsson et al. 2011, Masumoto et al. 2018a). The slow progression of the study for arctic pathogen would be caused by a logistical difficulty for a survey on arctic pathogens. In addition to the difficulty of access to the Arctic regions, we need to carry out a long-term survey there to clarify the pathogenic ecology because it is impossible to cultivate most pathogens in vitro. Further, another reason for the slow progression would be that ecologists are not as interested in arctic pathogens compared to organisms that symbolize adaptation to harsh environments (e.g., lichen) or are iconized in the Arctic (e.g., mammals such as the polar bear). These factors that have delayed investigations of such pathogens do not disappear easily, however, recent climate change in the Arctic regions has pushed us to study the potential importance of arctic plant pathogens. “Climate change” today is one of the most attractive topics connecting “the Arctic” with “plant pathogen”. Recently, arctic ecosystems have experienced increasingly warm temperatures, and they are known to be more sensitive to human-induced global warming than other terrestrial ecosystems due to strong positive feedback among air temperature, ice, and snow cover (ACIA 2005). The

46  Fungi in Polar Regions Arctic warming exceeds the global average warming, and precipitation in this region is generally predicted to increase in both summer and winter (IPCC 2013). Ongoing climate change has very likely had substantial impacts on the composition, abundance, and distribution of organisms in the Arctic regions (Meltofte 2013) and changed disease dynamics to various scales (Garrett et al. 2006). Climate change can influence plant disease by altering biological processes of host plants, and the pathogens or disease-spreading organisms (vectors). Direct effects on pathogens are likely to be strongest, although different pathogen life stages may vary in their climatic susceptibilities (Harvell et al. 2002). For example, many pathogens are limited by climate requirements for their overwintering or oversummering, and winter in particular is a major period of pathogen mortality, potentially killing more than 99% of the pathogen population annually (Burdon and Elmquist 1996). Several plant diseases are more severe after mild winters, which suggests that directional climate warming will alter plant disease severity (Coakley et al. 1999). Pfender and Vollmer (1999) showed that higher winter temperatures increase survivorship of overwintering rust fungi and therefore subsequent disease on host plants. Increasing temperatures and precipitation in North America could move the distribution of needle blight to further north (Woods et al. 2005). These studies have shown that climate change can alter the ecosystem through various ecological features of pathogens, such as the pathogenic impact on host survival, population of the pathogen, life stage of the host attacked by the pathogen (Eviner and Likens 2010). Therefore, basal knowledge about pathogens, such as life cycle, occurrence dynamics, and pathogenicity for the host, will become essential to understand the response of plant pathogens to climate change and assess the modification of arctic ecosystems by the combination of pathogens and climate change. To discuss the potential impact of plant pathogenic fungi on arctic ecosystems, in this chapter I present the diversity, adaptation, effect on host and ecosystem, and response to climate change of pathogenic fungi on vascular plants. In particular, I will focus on one of the pathogenic fungi, the genus Rhytisma, which causes tar spot disease on Salix spp.

Diversity and Specificity In the Arctic region, surveys of plant pathogenic fungi, except for snow-mold fungi, are mainly conducted for their taxonomy and distribution. While arctic plant pathogenic fungi have been studied for over a hundred years, it is currently unclear how many species of pathogenic fungi are within the total 4,350 fungal species in the Arctic. For a suggestion of the species diversity of arctic pathogenic fungi, I estimated species richness of pathogenic fungi in Svalbard, Norway using some fungal catalogs. There is relatively abundant data about fungal diversity in this region (Gulden and Torkelsen 1996, Elvebakk et al. 1996), thus it is optimal for a model case study compared to other Arctic regions. The total number of fungi or fungal-like microbes including lichen in the region is 1,227 species (lichen: 593, lichenicolous: 75, and other: 560). In terms of the other fungi not identified

Pathogenic Fungi on Vascular Plants in the Arctic 47

as either lichen or lichenicolous fungi, Tojo et al. (2013) showed that 176 species were found from plants, but these species include fungi from moss or those that are simply associated with plants, such as endophytes. I re-checked his list and chose the pathogenic fungi that met the following expectations, (1) notified as “parasite” or “from living leaf” in the original articles, (2) a known pathogen in other regions. Moreover, I included some wood pathogens collected from wooden material in my list. Although their main hosts do not exist in the region, I judge them as potential pathogens because they are known to have a broad host range and the dwarf wood plant does exist there. In contrast, the fungi known to be parasites without symptoms, such as Lophodermium spp., were removed. As a result, the island has 70 pathogenic fungi on at least 57 vascular plants (Table 4.1). The species richness ratio of pathogen to vascular plant is 70:173 and to total fungal richness is 70:1227 in the region. As a general relation between number of species of fungi to vascular plants to be 5–8:1 (Hawksworth 2001, Schmit and Mueller 2007), the total fungi-vascular plant ratio in the region (1227:173) seems to be appropriate. On the other hand, the richness of pathogens in this region is lower than other regions, while the richness has been estimated to be 1–6.3 per plant species (Dianese et al. 1997) or constitute 33% of existing fungi worldwide (Shivas and Hyde 1997). Although the gap between this data firstly may be caused by the insufficient number of surveys on pathogenic fungi, the trophic differences among fungi having different life strategies also might lead to such diversity differences between the pathogens and other fungi. Hillebrand (2004) showed that the trophic levels affected species richness with the gradient of latitude and parasites differed strongly between regional and local scales. Pathogenic fungi depend partially or completely on living host plants for nutrient requirements, and the dependence on their host as a life strategy might make survival difficult and restrict their diversity in the harsh environment of the Arctic. The endemism of arctic fungi is generally considered to not be higher than other regions (Dahlberg and Bültmann 2013). Some genera and species are predominately Arctic-alpine circumpolar in their distribution, while the remaining species can also exist in boreal and temperate habitats (Gulden and Torkelsen 1996, Knudsen 2006). DNA sequence analyses for arctic ectomycorrhizal fungi imply that many of them may be distributed globally and are found also in boreal or temperate biomes (Geml et al. 2012, Timling et al. 2012). In contrast, some detailed reports on the morphological and molecular characteristics of arctic plant pathogens have shown their taxonomical specificity (Smith et al. 2004, Masumoto et al. 2014). For example, Rhytisma polare (Masumoto et al. 2014), which causes tar spot disease on Salix polaris (Fig. 4.1A) on Spitsbergen Island was identified as a new species from the morphological feature of a different spore shape from other Rhytisma spp. (Fig. 4.1C). DNA analysis to identify the molecular feature also supported the difference of the species from other related species (Masumoto et al. 2014). Melampsora epitea, which causes rust on Salix arctica reported in the Canadian Arctic (Fig. 4.1D, E) also showed morphological and genetic specificity of the pathogen compared to other Melampsora epitea in North America (Smith

Basidomycota

Phytom Ascomycota

Pathogen Atopospora betulina Blumeria graminis Ciboria aschersoniana Ciborinia ciborium Diplocarpon polygoni Duplicaria empetri Gibbera barriae Graphyllium pentamerum Isothea rhytismoides Kalmusia coniothyrium Leptotrochila cerastiorum Mycosphaerella densa Mycosphaerella maculiformis Mycosphaerella pachyasca Mycosphaerella pedicularidis Mycosphaerella recutita Phaeosphaeria herpotrichoides Phaeosphaeria microscopica Pseudopeziza drabae Pseudopeziza svalbardensis Pseudorhytisma bistortae Rhytisma salicinum Rhytisma polare Sphaerotheca erigeronis-canadensis Taphrina carnea Venturia potentillae Antrodia serialis Columnocystis abietina Stereum sanguinolentum Dacrymyces stillatus Ditiola radicata Anthraconidea altera Anthraconidea elynae Anthraconidea lindebergiae Anthraconidea misandrae Entyloma dactylidis Melampsora epitea Puccinia arenariae Puccinia bistortae Puccinia curciferarum Host Plant Betula nana Poa species and Phippsia algida Carex subspathacea Sclerotinia tuberosa and Eriophorum species Bistorta vivipara Empetrum species Cassiope hypnoides several graminoid species, Dryas Dryas leaves Salix polaris Cerastium sp. herbaceous host species Dryas and many dicotyledons Eutrema edwardsii Pedicularis hirsuta and P. lanata ssp. dasyatha various monocotyledons graminoids wide range of grasses Draba species Saxifraga hirculus Bistorta vivipara Salix polaris and S. polaris × herbacea Salix polaris Taraxacum arcticum Betula nana Potentilla pulchella Old wooden material Old wooden material Old wooden material Old wooden material Old wooden material Carex misandra Carex nardina Kobresia simpliciuscula Carex misandra Dupontia and Poa species Saxifraga cespitosa, S. oppositifolia and Salix polaris Cerastium alpinum (probably C. arcticum) Bistorta vivipara Cardamine bellidifolia

Table 4.1.  Plant pathogenic fungi (including fungal-like microbes) on vascular plants in Svalbard.

Disease Name Leaf spot Powdery mildew Popcorn disease Popcorn disease Leaf spot Leaf spot Leaf spot Leaf spot Leaf spot Leaf spot Leaf spot Leaf spot Leaf spot Leaf spot Leaf spot Leaf spot Stem spot Leaf spot Leaf spot Unknown Tar spot Tar spot Tar spot Powdery mildew Leaf blister Leaf spot Heart rot Heart rot Heart rot Brown rot Brown rot Smut Smut Smut Smut Smut Rust Rust Rust Rust

48  Fungi in Polar Regions

Chytridiomycota

Oomycota

Deuteromycota

Basidomycota

Mycosphaerella pachyasca Mycosphaerella pedicularidis Mycosphaerella recutita Phaeosphaeria herpotrichoides Phaeosphaeria microscopica Pseudopeziza drabae Pseudopeziza svalbardensis Pseudorhytisma bistortae Rhytisma salicinum Rhytisma polare Sphaerotheca erigeronis-canadensis Taphrina carnea Venturia potentillae Antrodia serialis Columnocystis abietina Stereum sanguinolentum Dacrymyces stillatus Ditiola radicata Anthraconidea altera Anthraconidea elynae Anthraconidea lindebergiae Anthraconidea misandrae Entyloma dactylidis Melampsora epitea Puccinia arenariae Puccinia bistortae Puccinia curciferarum Puccinia drabae Puccinia eutremae Puccinia gibberulosa Puccinia heucherae Puccinia hieracii Puccinia oxyriae Puccinia pazschkei Schizonella melanogramma Tolyposporium junci Typhula isikariensis Microbotryum bistortarum Ustilago hyperborea Ustilago nivalis Ustilago picacea Ustilago striiformis Ustilago vinosa Ustilago violacea Ascochyta arctica Ascochyta dianthi Ascochyta arctica Dendryphion fumosum Mastigosporium album Phoma carici Phoma complanata Ramularia alborosella Septoria lychnidis Peronospora alsinearum Peronospora parasitica Olpidium brassicae

Eutrema edwardsii Pedicularis hirsuta and P. lanata ssp. dasyatha various monocotyledons graminoids wide range of grasses Draba species Saxifraga hirculus Bistorta vivipara Salix polaris and S. polaris × herbacea Salix polaris Taraxacum arcticum Betula nana Potentilla pulchella Old wooden material Old wooden material Old wooden material Old wooden material Old wooden material Carex misandra Carex nardina Kobresia simpliciuscula Carex misandra Dupontia and Poa species Saxifraga cespitosa, S. oppositifolia and Salix polaris Cerastium alpinum (probably C. arcticum) Bistorta vivipara Cardamine bellidifolia Four Draba spp. Cochlearia groenlandica and Eutrema edwardsii Ranunculus sp. six Saxifraga species Taraxacum cymbifolium Oxyria digyna Saxifraga aizoides Carex rupestris Juncus biglumis Poa hartzii Bistorta vivipara Luzula arctica ssp. confusa Sagina nivalis Koenigia islandica Poa arctica Oxyria digyna Silene acaulis and Stellaria longipes Poa and Festuca rubra Stellaria humifusa Alopecurus borealis, Festuca and Poa arctica Eutrema edwardsii Poa species Carex glareosa and C. saxatilis Pedicularis hirsuta Cerastium species Silene uralensis Cerastium alpinum, and Caryophylaceous plants Cochlearia groenlandica, and Brassicaceous plants Saxifraga sp. Leaf spot Leaf spot Leaf spot Stem spot Leaf spot Leaf spot Unknown Tar spot Tar spot Tar spot Powdery mildew Leaf blister Leaf spot Heart rot Heart rot Heart rot Brown rot Brown rot Smut Smut Smut Smut Smut Rust Rust Rust Rust Rust Rust Rust Rust Rust Rust Rust Smut Smut Snow mold Smut Smut Smut Smut Leaf smut Table 4.1 contd.… Smut Smut Leaf spot Leaf spot Leaf spot Stooty mold Leaf spot Rust Leaf spot and root canker Leaf spot Leaf spot Downy mildew Downy mildew Seedling blight

Pathogenic Fungi on Vascular Plants in the Arctic 49

Basidomycota

Chytridiomycota

Oomycota

Deuteromycota

Phytom Ascomycota

Four Draba spp. Cochlearia groenlandica and Eutrema edwardsii Ranunculus sp. six Saxifraga species Taraxacum cymbifolium Oxyria digyna Saxifraga aizoides Carex rupestris Juncus biglumis Poa hartzii Bistorta vivipara Luzula arctica ssp. confusa Sagina nivalis Koenigia Host Plantislandica Poa arctica Betula nana Oxyria digyna Poa species and Phippsia algida Silene acaulis and Stellaria longipes Carex subspathacea Poa and Festuca rubra Sclerotinia tuberosa and Eriophorum species Stellariavivipara humifusa Bistorta Alopecurusspecies borealis, Festuca and Poa arctica Empetrum Eutrema edwardsii Cassiope hypnoides Poa species several graminoid species, Dryas Carex glareosa Dryas leaves and C. saxatilis Pedicularis Salix polarishirsuta Cerastium sp. species Cerastium Silene uralensis herbaceous host species Cerastium alpinum, and Caryophylaceous plants Dryas and many dicotyledons Cochlearia groenlandica, and Brassicaceous plants Eutrema edwardsii Saxifraga sp.hirsuta and P. lanata ssp. dasyatha Pedicularis Potentilla sp. various monocotyledons graminoids wide range of grasses Draba species Saxifraga hirculus Bistorta vivipara Salix polaris and S. polaris × herbacea Salix polaris Taraxacum arcticum Betula nana Potentilla pulchella Old wooden material Old wooden material Old wooden material Old wooden material Old wooden material Carex misandra Carex nardina Kobresia simpliciuscula Carex misandra Dupontia and Poa species Saxifraga cespitosa, S. oppositifolia and Salix polaris Cerastium alpinum (probably C. arcticum) Bistorta vivipara Cardamine bellidifolia Four Draba spp. Cochlearia groenlandica and Eutrema edwardsii

Puccinia drabae Puccinia eutremae Puccinia gibberulosa Puccinia heucherae Puccinia hieracii Puccinia oxyriae Puccinia pazschkei Schizonella melanogramma Tolyposporium junci Typhula isikariensis Microbotryum bistortarum Ustilago hyperborea Ustilago nivalis Ustilago picacea Pathogen Ustilago striiformis Atopospora betulina Ustilago vinosa Blumeria graminis Ustilagoaschersoniana violacea Ciboria Ascochyta ciborium arctica Ciborinia Ascochyta dianthi Diplocarpon polygoni Ascochyta arctica Duplicaria empetri Dendryphion fumosum Gibbera barriae Mastigosporium album Graphyllium pentamerum Phoma carici Isothea rhytismoides Phoma complanata Kalmusia coniothyrium Ramularia alborosella Leptotrochila cerastiorum Septoria lychnidis Mycosphaerella densa Peronospora alsinearum Mycosphaerella maculiformis Peronospora parasitica Mycosphaerella pachyasca Olpidium brassicae pedicularidis Mycosphaerella Synchytrium potentiallae Mycosphaerella recutita Phaeosphaeria herpotrichoides Phaeosphaeria microscopica Pseudopeziza drabae Pseudopeziza svalbardensis Pseudorhytisma bistortae Rhytisma salicinum Rhytisma polare Sphaerotheca erigeronis-canadensis Taphrina carnea Venturia potentillae Antrodia serialis Columnocystis abietina Stereum sanguinolentum Dacrymyces stillatus Ditiola radicata Anthraconidea altera Anthraconidea elynae Anthraconidea lindebergiae Anthraconidea misandrae Entyloma dactylidis Melampsora epitea Puccinia arenariae Puccinia bistortae Puccinia curciferarum Puccinia drabae Puccinia eutremae

Stem spot Leaf spot Leaf spot Unknown Tar spot Tar spot Tar spot Powdery mildew Leaf blister Leaf spot Heart rot Heart rot Heart rot Brown rot Brown rot Smut Smut Smut Smut Smut Rust Rust Rust Rust Rust Rust

Rust Rust Rust Rust Rust Rust Rust Smut Smut Snow mold Smut Smut Smut Smut Name Disease Leaf spot smut Leaf Smut Powdery mildew Smut Popcorn disease Leaf spotdisease Popcorn Leaf spot spot Leaf Leaf spot spot Leaf Stooty mold Leaf spot Leaf spot spot Leaf Rust spot Leaf Leaf spot spot and root canker Leaf Leaf spot Leaf spot Leaf spot spot Leaf Downy Leaf spotmildew Downy Leaf spotmildew Seedling Leaf spotblight Leaf spot blister Leaf

50  Fungi in Polar Regions

Pathogenic Fungi on Vascular Plants in the Arctic 51

Fig. 4.1.  Tar spot disease caused by R. polare on S. polaris on Spitsbergen Island (A; bar = 2 mm) and the ascus (C; bar = 10 μm) and ascospore (D; bar = 5 μm). Rust disease caused by Melampsora epitea on S. arctica on Ellesmere Island (D) and the urediniospore (E; bar = 10 μm). The pictures A, C, and D are adapted from Masumoto et al. (2014) and B and E from Smith et al. (2004).

Color version at the end of the book

et al. 2004). In general, specificity of parasites can be shaped by their ecological adaptation to host and environmental specificities (Adamson and Caira 1993). First, the pathogenic fungi are generally known to have higher specificity against the host species due to the host-pathogen coevolution process. In the Arctic, there are 106 endemic plant species and 22 species are distributed on Svalbard (Daniëls et al. 2013). Fourteen pathogenic fungi in Table 4.1 can possibly infect these endemic plants in the Arctic, thus these pathogens might develop their specificities under the mechanisms of plant-pathogen co-evolution. Second, the specific environment of the Arctic, low temperatures, dry conditions, short summers (long winters) could select and evolve some specific features of arctic pathogens compared to other regional communities, as observed in plants and animals. In the studies shown in the previous section, the hosts of the pathogens Salix polaris and S. arctica is not an arctic endemic species, however, the pathogens were characterized by differences in morphological and molecular features from other related species. Although the specificity of arctic pathogenic fungi is unclear due to insufficient survey efforts, the environment or host specificity of the Arctic could lead the pathogens to also be specific.

52  Fungi in Polar Regions In the Arctic Biodiversity Assessment which aimed to synthesize and assess the status and trends of biological diversity in the Arctic (Meltofte 2013), while parasitic fungi were categorized as one of the main fungal groups classed by life strategies, only a few pathogens, rust, smut, and snow-mold were cited as examples. However, from summarizing the catalog of Svalbard fungi, we can find various other pathogens, such as tar spot, powdery mildew, leaf blister (see Table 4.1). Moreover, most host plants of these pathogens are distributed widely and are dominant in the circum-Arctic (Walker et al. 2005), and some pathogens were also reported to be distributed in other Arctic regions (Hallgrímsson and Eyjólfsdóttir 2004, Borgen et al. 2006). We need to maintain a field survey over the Arctic regions, in particular, with detailed descriptions of these pathogens’ morphological and genetic characteristics to clarify the diversity and specificity of arctic plant pathogens.

Carbon Cycling Fungi are pivotal for the cycling of carbon and nutrients in terrestrial ecosystems of the Arctic (Ludley and Robinson 2008, Newsham et al. 2009). Their different life strategies (mutualistic, endophytic, saprotrophic, or parasitic) play different roles in ecosystem carbon cycling. For example, lichen, which is one of the most common mutualistic fungi, plays an important role in primary production in arctic ecosystems (Webber 1974, Longton 1988). In contrast, saprotrophic fungi act as a decomposer and promote carbon release from the soil to the atmosphere. Plant pathogenic fungi have two effects on ecosystem carbon cycling: (1) a negative effect on primary production by reducing a plant’s carbon accumulation, (2) changing the ecosystem carbon flow by absorbing and consuming organic matter from plants. Only two studies have estimated the impact of pathogenic fungi on carbon cycling in arctic ecosystems (Olofsson et al. 2011, Masumoto et al. 2018a). Using long-term monitoring data, Olofsson et al. (2011) showed that Arwidssonia empetri on Empetrum hermaphroditum caused severe host community extinction under an experiment increasing snow depth and significantly decreased net ecosystem production. In this study, the pathogen heavily blighted their host, and the ecosystem lost a dominant primary producer which decreased its primary production. In contrast, Masumoto et al. (2018a) showed that a plant pathogen can change carbon cycling at the ecosystem level without decreasing annual productivity. They investigated the multiple effects of tar spot disease, Rhytisma polare, on Salix polaris and estimated that the effects can change multiple routes of tundra ecosystem carbon cycling. I will introduce this study briefly in the next paragraph. Note that the foliar pathogens have a simple life cycle as follows; they develop the ascostroma (which is the symptom part of the pathogen like a drop of tar as shown Fig. 4.1A) on the living host leaf and overwinter on the dropped leaf, then, the released spores from the matured ascostroma infect new host leaves in the next spring. To clarify the multiple effects of the pathogen on carbon dynamics of different scales, such as host plant organs, individual, population, and ecosystem, Masumoto

Pathogenic Fungi on Vascular Plants in the Arctic 53

et al. (2018a) initially evaluated the net photosynthetic production of the host plant and the multiple effects of the pathogen at the leaf level. They divided the pathogenic effect on the leaf carbon balance into three parts; an inhibition effect for productivity (the pathogenic damage reduces leaf photosynthetic productivity), and two carbon consumption effects (the pathogen absorb host organic matter and use the carbon for their ascostroma development and respiration). Based on the measurement of photosynthetic activity, gas-exchange, host leaf and pathogenic symptom area, field temperature and field radiation, the annual production per host leaf (Ph; mgC  leaf −1 h −1), the inhibition effect on photosynthesis per symptom (P e; mgC  ascostroma−1 h−1), respiration rate per symptom (Pe; mgC  ascostroma−1 h−1), and carbon content per symptom (Cp; mgC  ascostroma−1 h−1) were simulated. Figure 4.2 shows the daily dynamics of host leaf production and of the pathogenic effects. Finally, total Ph was 5.08 (mgC leaf−1), total Pe was 0.49 (mgC ascostroma−1), total Rp was 1.81 (mgC ascostroma−1), and total Cp was 3.70 (mgC ascostroma−1). The result showed a greater effect of fungal carbon consumptions (Rp and Cp) than the inhibition of host carbon productivity (Pe). The inhibition effect on host photosynthetic productivity has generally been evaluated as the main pathogenic effect on the host (Berger et al. 2007), because a decrease in host productivity is a critical problem in agricultural systems. In wild plant-pathogen interactions, however, hosts should evolve toward increased levels of resistance, and pathogens should evolve toward optimum levels of virulence to maximize their own fitness (Lenski and May 1994, Ewald 1995). From the viewpoint of the parasitic strategy to maximize their own fitness, the inhibition effect and the carbon consumption effect have different consequences for the carbon balance of a host plant and a pathogen, as shown in Fig. 4.3. The inhibition effect only decreases host productivity and does

Fig. 4.2.  Daily carbon dynamics of host leaf production and the pathogenic effects throughout the leaf period. The carbon gain per healthy leaf with leaf age (Ph: white bar). The carbon loss per infected leaf by the pathogen’s inhibition of host photosynthetic productivity (Pe: black bar), the pathogen’s carbon consumption for their respiration (Rp: hashed bar), and ascostromal growth (Cp: grey bar). (Reproduced from Masumoto et al. 2018a.)

54  Fungi in Polar Regions

Fig. 4.3.  Consequences of inhibition and consumption effect for carbon balance of host plant and its pathogen.

not change the pathogen’s carbon gain. In contrast, while the consumption effect also negatively affects the host carbon balance, it also enables the pathogenic fungi to survive because they can use the absorbed carbon for their growth or reproduction. Therefore, the pathogenic fungi that show these two effects seem to be able to maximize their fitness by minimizing the inhibition effect and maximizing the consumption effect. These results suggested that we may crucially underestimate the realistic influence of pathogens on their host plants without considering pathogens’ carbon consumption effects. For estimation of the above multiple effects of the tar spot pathogen on a tundra ecosystem, the authors integrated these effects into a carbon flow compartment model obtained from a previous study (Nakatsubo et al. 2005). Figures 4.4A and B show the carbon flow compartment models of healthy and infected communities (infection rate: 30%) in front of Austre Brøggerbreen in Spitsbergen Island. The effect of host photosynthesis inhibition was expressed by comparing the arrows (1) in Figs. 4.4A and B. From the two figures, the inhibition effect reduced the net primary production of vascular plants (34 gC m−1 y−1) by 3%, compared to that of the healthy community (35 gC m−1 y−1). As the net ecosystem production (NEP), which is obtained from total carbon input and output from atmosphere to ecosystem,

Fig. 4.4.  Compartment models showing carbon pools and flows of the tundra ecosystem in the study site (flow: gCm−2 year−1, box: gCm−2). A: Non-infected community model modified from Nakatsubo et al. (2005). The NEP was 20 gCm−2 year−1. Carbon flow from the belowground parts to the soil carbon pool (DB) was not quantified. B: Infected community model with 30% infection rate. New carbon pools and flows by the pathogen are shown in black. The NEP was 16 gCm−2 year−1. (Reproduced from Masumoto et al. 2018a.)

Pathogenic Fungi on Vascular Plants in the Arctic 55

was 20 (35 + 4 – 8 – 11) gC m−1 y−1 in the healthy community, thus the inhibition effect (1 gC m–1 y–1) reduced the NEP by 5%. In contrast, the effect of increasing carbon release by parasitic respiration (3 gC m−1 y−1) reduced the NEP by 15%. In addition, the transported carbon from the plant to the pathogen finally falls to the ground with the host leaves in the autumn. Thus, most part of the ascostroma except for released spores in the next spring play a carbon input role in the ground as a litter. Owing to ascostromata falling with host leaves, the carbon flow from aboveground biomass to the soil carbon pool in the infected community (5.5 + 7.5 = 13 gC m−1 y−1) was more than double that of the healthy community (6 gC m−1 y−1). The increasing carbon input by the pathogen suggested that such a secondary effect by the pathogen on the soil ecosystem may be potentially important. These results suggested that considering the effect of parasitic carbon consumption is also essential to understand the effect of pathogens on ecosystem carbon dynamics, because such effects can change the carbon cycling at the ecosystem level without a large reduction in annual ecosystem productivity. Most studies on the effect of wild parasitic fungi on wild host plants focus on severe symptoms, such as a strong reduction in host productivity or heavy damage on host survival (Jarosz and Davelos 1995, Gilbert 2002). Indeed these studies mainly focus on the role of pathogens as diseases causers in ecosystems. This may be because the studies of pathogens originally advanced in studies of the plant pathology of agricultural systems and researchers intend to control pathogens by investigating their ecological features or effects on host plants. Even in the natural ecosystem, many studies have also shown the impact on the host or ecosystem by pathogens that cause severe symptoms. However, most pathogenic species in nature have a mild virulence and it is rare that pathogens critically destroy their host population. The estrangement between those selected case studies and realistic observed phenomena may lead to a judgement that mild or symptomless pathogens are everywhere, but that they are not so important for the general ecosystem. However, such pathogens may show larger effects than previously understood, if we consider the pathogens’ effects that studies may have missed, such as carbon consumption. Even if at a glance the effect of a pathogen as a disease causer is small, the effect of a pathogen as a parasite may not be small and the behind-thescenes effects of seemingly mild or non-symptom pathogenic fungi might impact the ecosystem. Parasitism is the most common consumer strategy among organisms (Lafferty et al. 2008), and the biomass is shown to be comparable to fish and birds in an ecosystem (Kuris et al. 2008). When we study the effect of pathogens in natural ecosystems including the Arctic, it is important to not only focus on the disease causing aspect of the pathogens, but also the consumer role of the pathogens as parasitic organisms.

Adaptation and Occurrence Studies on the adaptation of pathogenic fungi to arctic environments have mostly focused on snow-mold fungi (Hoshino et al. 1998, 2003, 2009, Tojo and Newsham

56  Fungi in Polar Regions 2012). These studies focus on the cold tolerance of snow-mold fungi because these pathogens are considered to infect host plants under snow in the winter or early spring (Hoshino et al. 2001, 2009). The ecological features of the pathogen infecting the host leaves strongly depend on the host’s life strategy, and particularly their phenology. Foliar parasites, including common arctic pathogens, rusts, smut, and tar spot need to complete their infection and reproduction within the host’s foliage period. If the host species is deciduous, the period for completion of the pathogen’s life cycle seem to be limited because the Arctic summer is shorter than other temperate regions. Some reports indicate that the short growing season of the Arctic has pushed the pathogens, such as rust and smut to have simplified life cycles (Lind 1927, Savile 1982). Therefore, to discuss the adaptation of such foliar parasitic fungi, we need to study how their ecological features have adapted to the short and dry summers of the Arctic, in contrast to snow-mold fungi that has adapted to the long and low temperature winter. Figure 4.5 shows the growth rate of Rhytisma polare ascostroma on Salix polaris at Ny-Ålesund Spitsbergen Island (Masumoto et al. 2014) and R. filamentum ascostroma on S. integra in Nagano, Japan (Masumoto et al. 2015). As the Arctic host’s foliage period is shorter by about 3 months than that of the Japanese host, the arctic pathogen needs to grow its ascostroma within a shorter period. We can see two different points between the life cycles of the two fungi, the period from host leaf expansion to symptoms emerging and ascostroma growth rate on the host leaf. First, the quick occurrence of the arctic pathogen results from the fact that the pathogen can mature and release its ascospores immediately after snowmelt. We confirmed that the asci in ascostroma are already being formed before snowmelt, after which the pathogen can develop completely and release ascospores within about 5 days

Fig. 4.5.  Growth rate of Rhytisma polare ascostroma on Salix polaris at Ny-Ålesund Spitsbergen Island (closed circle) and R. filamentum ascostroma on S. integra in Nagano, Japan (open circle). (Reproduced from Masumoto et al. 2018b.)

Pathogenic Fungi on Vascular Plants in the Arctic 57

(Masumoto et al. 2018b). In the Japanese pathogen, in contrast, such formation of the asci immediately after snowmelt was not observed (Masumoto unpublished). Second, the Arctic species showed a rapid growth rate on the host leaf despite the lower summer temperature compared to the Japanese one. Although the metabolic mechanisms are still unclear, the rapid growth of the arctic pathogen is suggested to be a result of the pathogen’s adaptation to a host’s shorter foliage period. The dry conditions of the arctic tundra ecosystem can also restrict the survival of parasitic fungi. For foliar fungi, temperature and water availability generally interact to determine fungal infection and sporulation, because both of them often require close to 100% relative humidity (Harvell et al. 2002). Rhytisma spp. are also known to require free water for ascostroma maturation and spore discharge (Müller 1912, Jones 1925). Masumoto et al. (2018b) showed that R. polare can progress their ascostroma maturation only under water-saturated conditions in vitro and precipitation promoted spore dispersal (Fig. 4.6). Although R. polare can complete ascostroma development and release ascospores about 5 days after snowmelt in water-available conditions, precipitation in the arctic tundra often does not support any water availability during the period of ascostroma maturation and spore dispersal. As shown in Fig. 4.7, erratic precipitation in the study site during early summer seems to be insufficient for ascostroma maturation and spore dispersal. In such situations, the pathogen would use thawed-snow water which is consistently available (Masumoto et al. 2018b). Therefore, the location where free-water is consistently supplied is essential for the survival of such pathogens. In addition, the distance of spore dispersal of the pathogen showed us a meaningful suggestion for the mechanism of the pathogen’s adaptation to the Arctic dry environment. Spores of the pathogen can be observed in the plot just a few meters from the infection

Fig. 4.6.  Precipitation (white bars) and number of observed ascospores over 2 days in R. polareinfected plots (closed circles) from June 24 to July 28, 2012. (Adapted from Masumoto et al. 2018b.)

58  Fungi in Polar Regions

Fig. 4.7.  Total precipitation (white bars) and number of rainy days (grey bar) during the periods of ascostroma maturation and spore discharge; from June 15 to July 15, 2004–2013. (Adapted from Masumoto et al. 2018b.)

Fig. 4.8.  (A) Relationship between distance to plot from infected fallen leaves (that is the infected leaf in the previous year and the infection sources in the current year) and number of trapped ascospores (Log 10 fold-change). (B) Relationship between number of observed ascospores and infection rate in the current year. Both A and B are adapted from Masumoto et al. (2018b).

sources (Fig. 4.8A), and there are significant correlations between observed spores and infection rate (Fig. 4.8B). The dispersal distance of the pathogen is shorter than that of related Rhytisma species (Leith and Fowler 1988, Masumoto et al. 2018b). This was supported by the morphological features of R. polare, which is morphologically characterized by a short and fat shaped spore (Masumoto et al. 2014). In other words, a bigger volume/surface area of the spore could make the dispersal distance shorter. We can thus conclude that R. polare has two ecological features that enable it to survive in the dry conditions of the high-Arctic: (1) a dependence on free water for ascostromal maturation and ascospore dispersal, and (2) a short ascospore dispersal range. The combination of these features suggests that R. polare can only occur in locations with available water and survives in the

Pathogenic Fungi on Vascular Plants in the Arctic 59

Arctic semi-desert by restricting itself to locations where water is consistently supplied rather than having an enhanced ability to reproduce under dry conditions compared to other Rhytisma. Our long-term monitoring supported the importance of location for their occurrence (Tojo and Masumoto unpublished). In the data obtained by measuring the incidence of tar spot disease in the same study site in Ny-Ålesund during 2008‒2013, the incidence changed not only with the year but also the particular plot location, despite the plots being established within the local area. The incidence dynamics with years would be influenced by the variations in precipitation, in parallel, the plots having a higher initial incidence maintained a relatively higher incidence in the later years compared to other plots. This suggested that different water availabilities depending on the microenvironment can influence the disease incidence through affecting the pathogen’s life cycle.

Response to Climate Change Above, I showed the adaptation of R. polare to the short and dry summer of the Arctic and the occurrence pattern depending on their ecological features. The host plant, Salix polaris, is one of the most dominant plants in the study site (Muraoka et al. 2002, Jung et al. 2014). The host plant cover the ground highly and uniformly because they grow horizontally unlike low latitude trees that grow vertically, in addition, arctic plant populations are generally considered to be mainly maintained by asexual reproduction resulting in genetic homogeneity (Callaghan et al. 1997). Such homogenous host populations and genetic diversity seem to be favorable for pathogenic infections. However, because members of the genus Rhytisma require water-saturated conditions for ascostromal maturation and ascospore dispersal, these fungi are most prevalent during spring and early summer when precipitation is abundant (Müller 1912, Jones 1925, Cannon and Minter 1986). Therefore, the results shown by Masumoto et al. (2018b) suggested that the dry conditions of the Arctic would strongly restrict the pathogenic expansion and the population would be maintained within locations with available water even though other favorable situations for a pathogenic outbreak may exist. This means that water condition dynamics with climate change could change the pathogen’s occurrence pattern. Recent metrological surveys have indicated that annual precipitation in the Arctic region is increasing (IPCC 2013, Brintanja and Selten 2014). Although changes in the occurrence of pathogens depend on each pathogenic and host species’ response to an increase in precipitation, it generally could result in a more favorable environment for plant pathogenic fungi (Harvell et al. 2002, Garrett et al. 2009). For example, winter rain is critical to the persistence of Phytophthora ramorum, which causes an increase in oak blight because increases in precipitation probably create optimal conditions for the pathogen, resulting in an increase in infection rates (Venette and Cohen 2006, Sturrock et al. 2011). Increases in precipitation also increase the risk of outbreak of white pine blister rust caused by Cronartium ribicola because the pathogen’s basidiospore germination and pine needle infection require 48 hours with conditions of 100% relative humidity (Kinloch 2003). These results suggest

60  Fungi in Polar Regions that climate change can alter disease dynamics due to changes in water availability that affect a pathogen’s life cycle. Therefore, timing, frequency, or intensity of precipitation events are important to predict the population dynamics of pathogens even if a change in annual precipitation is not observed. Temperature increases with climate change is also a main factor that can affect pathogens’ ecological features and modify their occurrence dynamics. In many cases, temperature increases lead to the geographic expansion of pathogen and vector distributions. For example, Phytophthora cinnamomi in Europe was predicted to expand in response to increased temperatures as the winter warming allows for overwintering of this oomycete in new areas (Bergot et al. 2004). Increased temperature brings pathogens into contact with more potential hosts (Baker et al. 2000, Olwoch et al. 2003) and provides new opportunities for pathogen hybridization (Brasier et al. 1999, Brasier 2001). Although recent technological advances in meteorology can quantify the changes in temperature, humidity, and precipitation patterns, simulations of the impact of climate change on pathogens remain just an indicator of potential impact. Real evidence for the impact of climate change on plant disease could come from the verification of the accuracy of these projections, and this would require long-term records of disease intensity for the regions where impacts are projected and for control regions (Garrett et al. 2009). Long-term monitoring of pathogens is necessary in general to understand their ecology, and to develop predictions of their impact on plant pathology (Harvell et al. 2002). However, the lack of availability of long-term data about disease dynamics limits the analysis of climate change effects on plant pathogens (Scherm 2004, Jeger and Pautasso 2008). Although a long-term survey is difficult logistically in the Arctic, we should make further effort to understand the response of plant pathogens to climate change using the research sites that are already well-equipped.

Conclusion and Recommendation The presence and significance of pathogens on vascular plants have often been overlooked and poorly appreciated in the Arctic, despite their wide distribution, abundancy, and pivotal role in carbon and nutrient cycling. The ecological knowledge of pathogens particularly is sparser, in contrast, studies focusing on the identification, description, or recording of pathogenic fungi are better. Most of the pathogenic species shown in Table 4.1 are circumpolar and also distributed outside the Arctic. From the diversity and distribution descriptions of the pathogens, we could predict the future dynamics of arctic pathogenic fungi. The unavoidable greening of the Arctic will steadily and significantly affect the distribution and abundance of fungi, as habitat conditions gradually transform the distribution and abundance of host plants (Dahlberg and Bültmann 2013). Although studies of arctic soil fungal communities imply that the response as yet is relatively slow (Timling and Taylor 2012), subsequent progress of pathogenic fungi with transformed vegetation would be more immediate than soil fungi because pathogens have strong interaction with host plants in general. On the other hand, I have

Pathogenic Fungi on Vascular Plants in the Arctic 61

shown the specificity of arctic pathogens (see the previous section, “Diversity and specificity”). This is likely the result of adaptation to the Arctic environments and co-evolution with the arctic host plants. The specificity of pathogens to their hosts or environments can be a factor to drive severe and unpredictable disease outbreaks, if climate change alters host or pathogen geographic ranges (Davis and Shaw 2001). The famous cases that have shown the potential for such outbreaks are the imports of Asian chestnut and European white elm to the United States which led to their pathogens’ rapid expansion through their new hosts (Anagnostakis 1987, Holmes 1980). Conversely, some crops introduced to new regions have often suffered attacks from native pathogens (Carefoot 1967, Valent 1990). While pathogenic specificity is often unclear by characterizing only traits expression because parasite infectivity and host susceptibility depend on their genotype, recent advancements in molecular approaches will significantly help to increase knowledge about the specificity of arctic pathogens by the detection of these genetic characterizations. Estimation for the effect of plant pathogens at the ecosystem level is one of the most important factors to understand and manage arctic ecosystems. Plant pathogens can have large impacts on host physiology, population dynamics, and community composition (Burdon 1991, Gilbert 2002). Moreover, they can cause problems not only for their immediate hosts but also for their associated fauna and ecological communities (Havell et al. 2002). Many studies have demonstrated that epidemics affecting dominant plant species can alter a wide range of ecosystem functions, including net primary productivity, hydrology, decomposition (Waring et al. 1987, Kranz 1990, Cromack et al. 1991, Hobara et al. 2001). Even when there are no obvious visible signs of pathogen-induced damage or mortality, pathogens can substantially alter ecosystem processes (Agrios 2005, Mitchell 2003). However, in general the dynamics of pathogenic effects are difficult to study on a large scale (Garret et al. 2006). Ultimately, such large scale studies will be facilitated by remote sensing of host plant and pathogen populations. Although remote sensing technologies have advanced rapidly, field data still isn’t sufficient to identify particular host and pathogen responses to climate change and to distinguish between different types of plant stress caused by the pathogen (West et al. 2003). However, in arctic ecosystems, it would be relatively easy to demonstrate the pathogenic effect on a large scale, such as at the ecosystem level because the ecosystem has a simple diversity of pathogens and vegetation structure compared to low latitude ecosystems. As Charles Sutherland Elton once demonstrated in a study on food webs using the simple Arctic ecosystem as a model case (Elton 1927), the simple diversity of the Arctic would be suitable to clarify the role of pathogens in the ecosystem. Although, in this chapter, I have mainly focused the direct of climate change on the ecological features of the pathogen and their effect on the host, it should be noted that the indirect effect of climate change on pathogens through the response of host plants is also essential. The stress on host plants by (i) environmental factors, and (ii) disturbance by biotic factors such as insects or pathogens, also tends to increase infection by pathogens. Some pathogens can sometimes infect a healthy host and remain latent until the host is stressed. While the ability of these pathogens

62  Fungi in Polar Regions to sporulate, spread, and infect new hosts is affected by temperature and moisture, factors that stress their hosts are often critical to their successful invasion of host tissues (Sturrock et al. 2011). For example, an increased incidence of summer drought will increase the probability that trees will be infected by pathogens whose activity is facilitated by host stresses, such as root pathogens, wound colonizers, and latent colonizers of sapwood (Brasier and Scott 1994, Broadmeadow et al. 2005, Desprez-Loustau et al. 2006). In the northern high latitudes, an increase in plant growth has been observed (Myneni et al. 1997), however, such vegetation development would affect the response of pathogens both positively and negatively. In general, increased plant density will tend to make increased infection by foliar pathogens more likely (Huber and Gillespie 1992), but host plant resistance against the pathogen might be stronger due to an increase in net primary production. For a realistic prediction of the disease dynamics that accompany climate change in the Arctic, we should clarify the climate effects on both the pathogen and the host, and moreover, on the interaction between the two species. Owing to rapid climate warming in the Arctic, it is important to understand its link to the impact of pathogens to manage arctic ecosystems effectively. Although it is still currently hard to predict the impacts of climate change on disease, Harvell et al. (2002) identified four priorities for research to improve our ability to predict future impact: (1) Collect baseline data on diseases of wild populations; (2) Separate the effects of multiple climate variables (e.g., temperature, precipitation) on disease; (3) Forecast epidemics; and (4) Evaluate the role of evolution. Priorities (3) and (4) are more intricate and applied technically, but we can apply the systems developed in other regions to arctic ecosystems. In contrast, for priorities (1) and (2), we should accumulate basal data about arctic pathogens, such as their diversity, distribution, life cycle, host range, the effect on host plants, and response to environmental change. As I have emphasised in this chapter, knowledge of the pathogens in the Arctic is seriously lacking and the priority for arctic plant pathogen study is basal data accumulation.

Acknowledgments This work was also supported by the Arctic Challenge for Sustainability Project (ArCS) Grant provided by the Ministry of Education, Culture, Sports, Science, and Technology, Japan. I thank Dr. Motoaki Tojo at Osaka Prefecture University, for providing unpublished data to this chapter.

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5 DNA Metabarcoding for Fungal Diversity Investigation in Polar Regions Shunsuke Matsuoka,* Yoriko Sugiyama and Hideyuki Doi

Introduction Understanding spatial and temporal patterns of biodiversity is one of the most fundamental aims of ecological studies, and it requires comprehensive listing of the species present at various sites and times. This type of scientific exploration is important for predicting the potential consequences of future environmental changes on ecosystem properties (Botkin et al. 2007). Fungi are known to perform unique functions in nature, as they contribute to the material cycle by decomposing organic matter, and affect the growth and survival of other organisms by symbiosis or parasitism. However, despite the importance of fungi, the diversity of fungi has been poorly investigated compared to that of animals and plants. Especially in polar regions, the studies dealing with fungal diversity have been very limited, though the species there are suspected to be highly vulnerable to global warming, and thus understanding the diversity patterns there is urgently required. Traditionally, fungal diversity in various fields has been described based on the observation of fungal tissues of the sporocarp or mycorrhiza (a symbiotic organ of plants and fungi), or mycelia or spores isolated from substrates, such as litter or soil. However, since fungal tissues are poor in morphological information, identification based on these tissues is often difficult and can result in misidentification (e.g., cryptic species). Such difficulty in identification using morphological methods has been a limitation for the investigation of diversity in fungi (e.g., Bridge and Spooner 2001, Horton and Bruns 2001). Graduate School of Simulation Studies, University of Hyogo, Kobe, 650-0047, Japan. * Corresponding author: [email protected]

68  Fungi in Polar Regions In view of this situation, a new method using DNA sequence information (i.e., DNA barcoding) was applied to fungal diversity investigation in the 1990s. In this molecular method, the genetic diversity of fungi is evaluated without relying on the morphology or the success of isolation of tissues. Therefore, the molecular method spread rapidly as a tool for fungal diversity investigation. Especially, community analysis by DNA metabarcoding using high-throughput sequencing (HTS, sometimes also called next-generation sequencing), which appeared in the 2000s, requires much lower costs in terms of time and money compared to the previous molecular methods, and has been accepted as a breakthrough in fungal diversity investigations. In this chapter, we aim to introduce the workflow for fungal biodiversity investigation, especially focusing on DNA metabarcoding, which is the predominant method used for recent studies. First, we explain what the molecular method and DNA metabarcoding are, and briefly review the studies investigating fungal diversity in polar regions using DNA metabarcoding. Next, we survey the workflow of experiments and analyses of fungal diversity using DNA metabarcoding. Finally, we discuss the prospects for diversity studies using DNA metabarcoding. The method introduced here should be quite useful and would provide us with invaluable knowledge about fungal diversity in polar regions. Note that we limit the content to a brief explanation of the workflow, and more details about the workflow or the background of the molecular methods can be found in other publications (Lindahl et al. 2013, Bálint et al. 2014, Osono 2014).

Molecular Methods of Fungal Diversity Investigation DNA Barcoding In DNA barcoding, the taxonomic identity of an organism is inferred based on its DNA sequence information. First, base sequences of barcoding regions (i.e., molecular/genetic markers) are determined by Sanger sequencing, and then the taxonomic classification of an organism is performed by comparing the sequences obtained with those in databases. In this method, even when databases do not contain sequences identical to the obtained sequences, and the taxonomy that could be inferred from the obtained sequences was not known a priori, the taxonomic position can be inferred from the sequences using phylogenetic analysis. Such a DNA barcoding method has been applied to studies of fungi diversity in polar regions, by targeting sporocarp samples (e.g., Geml et al. 2012) or mycelia isolated from plant tissues (e.g., Hirose et al. 2016). However, this method using Sanger sequencer has a major limitation, namely, that it is only applicable in cases in which DNA from a single fungal species has been isolated in a sample, and not in cases in which several species are included in a sample. This is because when several species were included, the sequencing signals of the different species overlap and cannot be distinguished from each other. In substrates such as soils or plant tissues, several fungi usually coexist, and thus

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the above method using Sanger sequencing cannot be used directly to determine the fungal diversity in these substrates. When one aims to use Sanger sequencing to investigate fungal diversity associated with soils or plant tissues, one must first do isolation of species or clone DNA of each individual species from the mixture of DNAs with different sequences in the sample.

Environmental DNA and DNA Metabarcoding The above situation was largely changed in the late 2000s by the development of HTS, which enabled the analysis of several species’ DNA in parallel without isolation of the individual DNAs. The mixture of DNAs from several species present in a certain substrate or environment is called environmental DNA. Here, “substrate” can include matter such as soil, live/dead plant tissue or river water, and does not have any limitation in size or materials. By obtaining and analyzing the DNA sequences of several species in this environmental DNA in parallel, we can evaluate the genetic diversity of communities in that environment. This method is called DNA metabarcoding (Taberlet et al. 2012a,b). With the method of DNA metabarcoding, we can directly investigate the fungal diversity in any environment, without relying on the observation of sporocarps or isolation of individual fungi and their DNA. In DNA metabarcoding, only the information of base sequences can be obtained, and thus the fungal diversity is usually evaluated in terms of operational taxonomic units (OTUs) instead of species. OTUs are clusters of related sequences, which are defined on the basis of sequence similarity (described in detail below). HTS is suitable for DNA metabarcoding because it can produce 0.45–50 billion base pairs of DNA sequence from numerous species in one run (Metzker 2010, Shendure and Ji 2008) by parallelizing the sequencing reaction, without any complicated procedures, such as isolation or cloning of individual DNAs. Thus, we can efficiently acquire fungal DNA sequences from various environments. The use of DNA metabarcoding using HTS spread explosively among researchers as a breakthrough in fungal diversity investigation, and has helped reveal the high diversity of fungi in many substrates and regions (e.g., Buée et al. 2009, Jumpponen and Jones 2009, Jumpponen et al. 2010). Among the studies employing DNA metabarcoding of fungi, we will introduce some examples conducted in polar regions in the next section.

Fungal Diversity Studies with DNA Metabarcoding in Polar Regions So far, several DNA metabarcoding studies have been conducted in polar regions to investigate fungal diversity (Table 5.1). In polar regions, materials such as dead/ alive plant tissue, soil, ice cores, water, and feces of animals can be candidates for DNA metabarcoding samples. Indeed, most of these materials have served at least once as samples for fungal DNA metabarcoding studies, and have been proven to harbor diverse fungal DNA. For example, Zhang and Yao (2015) conducted DNA metabarcoding of endophytic fungi in the Ny-Alesund region of Svalbard. They

70  Fungi in Polar Regions Table 5.1.  Examples of fungal diversity studies in polar regions using DNA meta-barcoding. Publication

Target Groups

Main Aim

Semenova et al. 2015 Soil

Substrate

Ascomycota

Effect of long-term soil warming on fungal community

Semenova et al. 2016 Soil

Soil fungi

Effect of snow depth on fungal community

Cox et al. 2016

Soil

Soil fungi

Relative importance of environmental filtering and dispersal limitation

Grau et al. 2017

Soil

Soil fungi

Difference in fungal community among habitat

Coleine et al. 2018

Sandstone

Cryptoendolithic fungi

Description of cryptoendolithic fungal community

Hassett et al. 2017

Ice core/water All fungi

Description of fungal community in ice and water

Zhang et al. 2015

Leaf and stem Endophyte of plants

Description of endophytic fungal community

Eusemann et al. 2016 Pine needle

All fungi

Description of endophytic fungal community

Blaalid et al. 2014

Plant root

Root associated fungi

Effects of environmental differences on fungal community

Mundra et al. 2015

Plant root

Root associated fungi

Effects of environmental differences on fungal community

Botnen et al. 2014

Plant root

Root associated fungi

Difference in fungal community among host plants

collected 12 samples from four plant species and obtained 184,242 reads (of which 76,691 remained after quality filtering and chimera removal), which finally resulted in 250 fungal OTUs. Also, Botnen et al. (2014) investigated the root-associated fungal community in Blomsterdalen, Svalbard. They collected 60 root system samples from three plant species and obtained 157,181 reads (of which 117,837 remained after quality filtering and chimera removal), which resulted in 354 fungal OTUs. Hassett et al. (2017) investigated 15 water and 25 ice-core samples in Arctic and sub-Arctic regions across the western Arctic, and obtained 7,222,652 reads (of which 4,055,401 remained after quality filtering and chimera removal) which clustered into 450 fungal OTUs. Moreover, the effects of environmental change on fungal diversity have been examined with DNA metabarcoding. In these studies, responses of fungal communities to natural environmental gradients (e.g., vegetative succession) and/ or artificially manipulated environmental differences (e.g., temperate and snow depth) were measured. These studies demonstrated that differences in edaphic condition, vegetation, and climate can change fungal community diversity and composition (e.g., Blaalid et al. 2014, Semenova et al. 2015, 2016). For example, Semenova et al. (2015) addressed the effects of 18 years of experimental warming on the soil ascomycetes community in Alaska. They showed that the proportion

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of lichenized and moss-associated fungi decreased with warming, while the proportion of pathogens and saprotrophic species was higher with the warming treatment. These studies demonstrated that DNA metabarcoding is useful for the detection of DNAs from diverse fungi from various substrates and their responses to environmental changes.

Workflow of DNA Metabarcoding Using High-Throughput Sequencing From here we will describe the concrete details of the method for DNA metabarcoding using HTS. DNA metabarcoding is achieved by performing roughly three steps: sampling, molecular analysis, and bioinformatics (Fig. 5.1). The full procedure varies in accordance with the type of sequencer used for the molecular analysis, and in this chapter, we will present an example in which MiSeq (the most widely used sequencing system for fungal DNA metabarcoding studies as of 2018) is used. Most operating procedures and cautionary notes for MiSeq are common to those for other types of sequencers and would even be useful if one used other sequencers.

Fig. 5.1.  Workflow of DNA metabarcoding using high-throughput sequencer.

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Sampling and Sample Storage The sampling design and strategy are important points in testing one’s hypotheses about ecological systems. For example, sampling methods, sampling effort, and sequencing depth (how many reads one obtains per sample) have been reported to affect the results of diversity investigations and community analyses. Regarding this point, previous studies showed that in metabarcoding of fungi in forest soil, tens of thousands of sequence reads are required for each sample (Smith and Peay 2014, Oono 2017). In diversity investigations, the spatial structure of the fungal community is another point that should be considered when planning the sample collection. For example, as the ectomycorrhizal fungal community in boreal forests is known to show spatial autocorrelation within about three meters (Lilleskov et al. 2004), samples should generally be taken at least three meters apart from each other. Also, taking subsamples from several points within a sampling site and pooling them into a single sample per site for analysis would enhance the rate of detection of fungi (Song et al. 2015). When doing this, pooling subsamples after sequencing yields more diverse fungal sequences compared to pooling them before DNA extraction (Song et al. 2015). Hence, in order to obtain sufficient samples or sequence depth to test one’s hypothesis, a priori examination of the sampling design should be done, taking into consideration the subsequent flow of analysis, as explained in this section. After sampling, the collected samples must be preserved with caution to prevent DNA degradation and changes in the fungal community composition. Basically, freezing or drying samples immediately after sampling is recommended, but beware that thermal drying can sometimes cause degradation of the DNA and should be avoided. If you cannot immediately freeze or dry your samples, you can preserve your samples by soaking in some appropriate reagents. For example, samples can be preserved by soaking in cetyltrimethylammonium bromide (CTAB) solution when one is planning to do DNA extraction from plant tissue samples using the CTAB method (described below). Also, 70% or higher concentrations of ethanol can delay the DNA degradation, but ethanol has to be removed before DNA extraction. There are other specific reagents that are useful for the preservation of nucleic acids, e.g., RNAlater (QIAGEN), but in any case, it is recommended to store samples in a cool, dark place and immediately proceed to DNA extraction after sampling.

DNA Extraction DNA extraction is a key procedure that can affect both further experimental procedures and analytical results. To avoid biases during sample preparation, the same DNA extraction protocol should ideally be used for all samples (Tedersoo et al. 2010). The appropriate protocol can vary depending on the kind and condition of substrates, and one should select the appropriate protocol in accordance with their sample.

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For soil samples, using the PowerSoil DNA Isolation Kit (QIAGEN) may be one of the most accepted ways for DNA isolation. Usually, soil samples contain large amounts of humic substances, which strongly inhibit polymerase chain reaction (PCR) amplification, and the PowerSoil DNA Isolation Kit is useful for removing these PCR-inhibiting substances. In DNA extraction, the amount of sample used can be an important point since DNA of rare species may be missed in samples with low amount of DNA. Song et al. (2015) tested the effects of differences in the extraction procedures, such as the amount of soil sample (0.25, 1.0 or 10 g wet weight) and the inclusion of additional steps before extraction (freeze/thaw cycles, sonication, or hot water bath incubation), on the results of diversity analysis, and showed that, as long as the PowerSoil DNA Isolation Kit was used, the analytical results did not significantly differ. Nevertheless, when other extraction methods are used, these points should be carefully examined. When dealing with plant tissue samples, one should first consider how to wash and lyse the sample according to their aim. For example, when one aims to investigate the fungal community inhabiting plant roots, one must first wash out the soil particles attached to the root surface, because these soils contain DNAs of soil-inhabiting fungi, and can be a source of contamination. When only fungi existing inside plant tissues are targeted, cleaning samples by ultrasonication would be appropriate (e.g., Ushio et al. 2016). After the cleaning procedure, samples must be lysed to disperse the fungal mycelia from tightly attached plant cells and to break fungal cell walls. Some machines, such as Tissue Lyser II (QIAGEN), can be used for this lysing procedure. Then one can proceed to the extraction procedure. Among the available DNA extraction kits for use with plant tissues, the DNeasy Plant Mini Kit (QIAGEN) is widely used. Protocols for these kits include steps for removing PCR-inhibiting substances, such as polysaccharides derived from plant tissues. Also, the CTAB method (Murray and Thompson 1980) is often used for DNA extraction from plant tissues (e.g., Mundra et al. 2015). The CTAB method is inexpensive, but PCR-inhibiting substances cannot be fully removed with this method. Thus, if PCR inhibition is observed, the DNA extraction products should be further purified before the PCR. For such purification, purification kits with spin-columns are widely used.

PCR Molecular Marker In the PCR step, the selection of a barcoding region (i.e., a region of DNA sequenced and used for taxonomic identification) and PCR primers are among the most important points. The major sequencers used today can read no more than approximately 500 bp of sequences, so ideally, the selected barcoding region should be able to detect and identify all the fungal species—but here we do not intend to discuss what the species are—within this length. However, no such ideal regions have been found so far, and thus you have to choose the best regions and primer

74  Fungi in Polar Regions pairs in accordance with your targeted taxa or functional groups, and your purpose (for DNA barcoding, you can also refer to Seifert 2009, Lindahl et al. 2013, Osono et al. 2014, Vu et al. 2019). When one aims to investigate fungal diversity with targeting of all fungi, the internal transcribed spacer (ITS) region of rDNA (Fig. 5.2) is the prime candidate for the barcoding region. This region is widely accepted as a barcoding region for fungi, as it has a sufficiently high evolutionary rate to enable accumulation of enough variations for distinguishing species, and has a large number of reference sequences recorded in databases compared to other regions (Schoch et al. 2012). Also, optimized primer pairs for this region are being developed to lessen the amplification bias among taxa and to make them more fungus-specific (e.g., Toju et al. 2012). Fungi have two ITS regions—ITS1 and ITS2, and because of the limitation of sequenceable length of fragments, one can target only one of these two regions in metabarcoding. Regarding the choice between these two regions, previous studies showed that the use of either of the regions yielded similar numbers and composition of OTUs, and the results of ecological analyses were also the same between them (Bazzicalupo et al. 2013, Blaalid et al. 2013). On the other hand, the ITS region is not recommended for some taxa and purposes. For example, for more ancestral groups (e.g., those traditionally classified as Chytridiomycota or Zygomycota), large subunit (LSU) or small subunit (SSU) genes of rDNA (Fig. 5.2) are often adopted as barcoding regions. Indeed, in an investigation of the diversity of Glomeromycota (known as arbuscular mycorrhizal fungi), SSU shows stronger detection power and good correlation with morphological species, and thus is thought to be more suited than ITS or LSU for barcoding (Thiéry et al. 2016). Also, as the rate of evolution is large in the ITS region, it sometimes becomes very difficult to infer the taxonomic position of ITS sequences that have no related ones in databases. In such cases, more conserved regions, such as the D1/D2 regions of LSU, can be used to infer the approximate taxonomic position relative to all fungi by doing phylogenetic analysis (Mueller et al. 2016). Finally, though the amount of sequence data in databases is rapidly increasing in recent years, there are still cases in which ITS or LSU is insufficient for species identification (Vu et al. 2019). One should carefully consider the limitations of the primer sets they choose and what can be determined by using them.

Fig. 5.2.  A schematic diagram of region in fungal rDNA around ITS region.

Fusion Primers and Multiplexing In DNA metabarcoding using HTS, sequencing adapters need to be added on both ends of the PCR-amplified fragments. Also, at the same time, sample-specific tag

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sequences can be added to the fragments. By adding sample-specific tag sequences, fragments from several samples can be mixed and sequenced in a single run, and then the retrieved sequences are assigned to the respective samples based on the sequence tags. There are several ways to add these tag sequences (Lindahl et al. 2013), but usually fusion primers, that is, primers with both sequencing adapter and tag sequence adapter, are used. For more detailed procedures for adding tag and adapter sequences, one can refer to other publications (e.g., Lindahl et al. 2013, Bálint et al. 2014). PCR The key point in doing PCR is to minimize the generation of PCR chimeras and PCR errors. Sequences containing these errors can be regarded as artifactual ones, generated during the experimental procedures, but are hard to distinguish from authentic sequences of fungus. Therefore, the existence of these sequences with errors can strongly affect the results. In order to lessen the production of these errors during PCR, several measures have been proposed: using high-fidelity PCR enzymes, such as KAPA HiFi DNA Polymerase (Kapa Biosystems); reducing the number of PCR cycles; or raising the annealing temperature. Also, employing slower ramp speed (the speed of temperature change) in PCR has been reported to reduce the generation of PCR chimeras (Stevens et al. 2013). However, as the appropriate PCR conditions vary depending on the primer pairs and targeted taxa, ideally the PCR conditions should be examined for each sample. After PCR amplification, the products will be purified to remove unreacted primers and primer dimers. Residual unreacted primers and primer dimers can affect the sequencing results, so they should be removed before sequencing. To purify the PCR products, several methods have been developed, but the method using AMPure XP (Beckman Coulter) is especially convenient because multiple samples can be processed in parallel by using 96-well plates. In the purification with AMPure XP, primer dimers shorter than 150 bp can be removed by adding a 0.8-fold volume of AMPure XP to the PCR products.

Library Mixing and Sequencing When sequencing several samples with different tags in the same run, one mixes these samples in advance. When performing such mixing, by making the DNA concentration (or molality) the same for all samples, one can obtain similar numbers of reads among samples. Then, the mixed libraries are checked for the size distribution of DNA fragments with TapeStation (Agilient Technologies) or other machines with a similar function. If one finds any primer-dimers or fragments with non-specific amplification at this stage, one should use E-Gel SizeSelect (Thermo Fisher Scientific) in order to collect only the fragments corresponding to the targeted barcoding regions. Then the DNA concentration is measured with a suitable device such as Qubit (Thermo Fisher Scientific) and is adjusted. For MiSeq, the final DNA

76  Fungi in Polar Regions concentration should be adjusted to 4 nM. For the sequencing procedure, please check the manual provided by the manufacturer for each sequencer.

Bioinformatics In bioinformatics analysis, a FASTQ file generated by sequencing is used. In a FASTQ file, the base sequence and its quality (i.e., the sequence read accuracy) are recorded. Raw sequence data contain both unreliable sequences generated by PCR and sequencing errors, and reliable ones, which in general correspond to many related sequences that originate from a single species or individual. In bioinformatics, we first eliminate the unreliable sequences and group the reliable sequences that are related to each other into clusters. For this sequencing data processing, several platforms are now available (Bálint et al. 2014, Anslan et al. 2018), and by using these platforms, we can do sequence processing, clustering, and molecular identification collectively. However, as the results of these analyses can vary among platforms, careful selection of a platform is required. For example, Anslan et al. (2018) compared the analytical results from five platforms using the same data set, and showed that, though no platform seemed to have processed and clustered the sequence data completely correctly, the platforms that used VSEARCH (Rognes et al. 2016) for the quality-filtering and assembly procedures were relatively accurate. Most platforms work on LINUX-based operating systems, but the required performance of devices varies among platforms. Also, there are some web-based platforms (e.g., Protax-fungi, Abarenkov et al. 2018), so bioinformatic analysis can be done even if an appropriate analysis environment cannot be arranged with one’s own computer. For detailed commands and settings for the operation of these platforms, please refer to their manuals. Here, as an example, we will give an explanation of the procedure for using Claident (Tanabe 2018), a platform that allows one to complete all steps from sequence processing to molecular identification. This platform uses VSEARCH for the quality-filtering and assembly procedures. If tag sequence information is inputted before sequencing, MiSeq divides the obtained sequences into samples according to the tag sequences (demultiplexing process), and outputs sequences with the same tag into one FASTQ file. In Claident, one can also do this demultiplexing process by oneself, by calling the demultiplexing program provided by Illumina. If sequences were read from both the 5’ and 3’ ends of the barcoding region (i.e., paired-end read), two FASTQ files, read from either the 5’ or the 3’ end, would be outputted per sample or tag. In this case, these two sequences should first be concatenated into one sequence, with removal of low quality reads. Then, noisy and chimeric sequences assumed to be generated by PCR and/or sequencing errors are removed. After that, the resultant sequences are grouped into species or individuals based on their sequence similarity. This process is called clustering, and the groups of sequences generated by the clustering process are referred to as OTUs. In this

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clustering process, two main points are considered: the similarity threshold and the clustering method. So far, in general, the same similarity threshold value is adopted for all the obtained sequence data, and for the ITS region, this value is usually 97% (Osono 2014). This value is based on analyses that found that the mean rate of intraspecific variation of recorded ITS sequences is about 3% (Nilsson et al. 2008). Also, the ITS region is a multicopy region and has variations even within a genome, but the rate of this intra-genome variation is also reported to be about 3–5% (Lindner et al. 2013). However, the rate of variation can vary among species, and there are, of course, species with a variation rate larger or smaller than 3% (e.g., Smith et al. 2007, Nilsson et al. 2008). Therefore, though adapting the uniform similarity threshold is good in the sense that the diversity of a targeted region can be evaluated objectively, it should always be noted that the OTUs obtained do not necessarily correspond to known, morphologically defined species. Also, when using a uniform similarity threshold, a prior survey is indispensable, as the appropriate threshold value varies depending on the targeted barcoding region and taxa. Indeed, in arbuscular mycorrhizal fungi, one study showed that the average intraspecific variation rates were 99, 96, and 94% for SSU, LSU, and ITS, respectively (Thiéry et al. 2016). For the clustering step, several methods have been proposed, and the selection of a method must be based on the purpose of the study. For example, when making OTUs with 97% similarity threshold, one can either make OTUs within the obtained sequences (commonly referred to as de novo clustering, see also Lindahl et al. 2013), or map the obtained sequences to known fungal sequences in databases with 97% similarity. When the former method is employed, sequences that have no related sequences in databases can be clustered into OTUs, though it is uncertain whether these unknown OTUs are really from the fungus or are artifactually generated ones. On the other hand, when the latter method is employed, only sequences of known species or lineages (i.e., related sequences that are already registered in databases) can be clustered into OTUs. By analyzing the same data set, Cline et al. (2017) reported that more OTUs can be obtained by de novo clustering than by the mapping method, but the results of community analysis do not differ significantly between these two clustering methods. The obtained OTUs may possibly include PCR chimeras. In Claident, we can detect these chimeras by using the UCHIME algorithm (Edgar et al. 2011), a chimera-detection algorithm. A mock community (an artificially made community of known species) study targeting the ITS region showed that the UCHIME algorithm could detect chimeras without false negatives, and that the proportion of chimeric OTUs was about 0.2% of the total OTUs (Bjørnsgaard Aas et al. 2017). However, UCHIME sometimes seems to mis-detect non-chimeric OTUs as chimeric OTUs (i.e., produces false positives). The resultant OTUs are then usually identified based on the information of known sequences. In this identification process, BLAST search using International Nucleotide Sequence Database (INSD) is commonly used. In INSD, various sequences from all over the world are recorded, but there is the problem that the INSD sequence corpus is in part compromised by the presence

78  Fungi in Polar Regions of incorrectly annotated, chimeric, or otherwise substandard entries. Therefore, referring to other databases, such as UNITE (ITS; Kõljalg et al. 2013), SILVA (SSU and LSU; Quast et al. 2013), or MaarjAM (SSU; Glomeromycota; Öpik et al. 2009) databases depending on your target regions and taxa, along with INSD, would be useful. In addition, regarding identification based on DNA metabarcoding, there is another problem concerning the identification criteria, that is, the question of with what percentage of sequence similarity we can decide to use the reference sequence data. Concerning this problem, Claident performs molecular taxonomic identification of OTUs based on the database search algorithm of the query-centric auto-k-nearest-neighbor (QCauto) method (Tanabe and Toju 2013) and subsequent taxonomic assignment with the lowest common ancestor (LCA) algorithm (Huson et al. 2007). We do not give a detailed explanation about these algorithms here, but these algorithms enable us to infer the taxonomy of OTUs at objectively reliable taxonomic levels (Tanabe and Toju 2013). For these obtained OTUs, diversity analysis and statistical analysis can be carried out (Buttigieg and Ramette 2014).

Errors and Contamination in DNA Metabarcoding In DNA metabarcoding, errors and contamination are often problematic, because the methods and sequencers used here have very high detection power. Therefore, in order to check the occurrence of errors or contaminations, both negative-controls (samples with no fungal tissues or DNA) and positive-controls (samples that only contain known fungal DNA) should be prepared and analyzed throughout all the steps of experiments and analyses (Nguyen et al. 2014). Also, the DNA concentration of the PCR products is unusually high, and this highly concentrated DNA solution can be a source of contamination. Therefore, when doing DNA metabarcoding experiments, before-PCR samples should ideally be processed in a different room from the room where PCR products are processed.

Future Perspectives and Concluding Remarks Polar regions are highly vulnerable to global warming, and it is feared that the biodiversity there, too, is susceptible. Therefore, clarifying the response of fungal communities to environmental change, by investigating fungal diversity there, is urged. DNA metabarcoding should be suitable for investigation of this diversity, as it can efficiently detect a much wider diversity of fungi compared to traditional methods. Especially, for studies in polar regions, DNA metabarcoding provides the possibility to detect the presence of fungi whose taxonomy has not yet been extensively investigated, and to infer their diversity and functions. However, studies in polar regions are still limited compared to those in other regions, and polarregion studies conducted in settings that can be compared to those in other regions are also scarce. Thus, the characteristics of fungal communities in polar regions and their responses to environmental changes are largely unknown. Considering

DNA Metabarcoding for Fungal Diversity Investigation in Polar Regions 79

the possibility that future environmental changes will alter the fungal community composition in polar regions, more case studies are urgently needed to understand the fungal community patterns there. In this chapter, we introduced only studies which focused on the taxonomic diversity (e.g., species or OTU richness). However, the taxonomy of fungi does not necessarily reflect their function (Baldrian 2009). In order to clarify the functional responses of fungal communities to environmental change, metabarcoding of functional genes (e.g., genes encoding organic carbon degradation enzymes) and measuring of enzyme activities (e.g., Bödeker et al. 2014) would be an important future focus. Also, general availability of sequencers that can yield long-read sequences, such as PacBio (Pacific Biosciences), can be expected in the near future (Tedersoo et al. 2018). This technical improvement will enable us to accurately estimate the phylogenetic position of unknown fungi, which will further improve the investigation of the diversity of fungi in polar regions. Another focus of studies in polar regions would be the communities confined to permafrost or ice. Analyses of these communities may give us invaluable opportunities to infer the past diversity (Willerslev and Cooper 2005). For example, we can investigate the fungal diversity in the past by doing DNA metabarcoding analysis of ice cores or frozen sediments (Epp et al. 2012, Bellemain et al. 2013), and with these analyses, we may obtain long-term temporal data about the correspondence between past environmental changes and community/population dynamics (Rawlence et al. 2014). This data, which can only be obtained in polar regions, will provide us important knowledge about how organisms and communities will respond and evolve in this era of climate change.

Acknowledgments We thank Dr. E. Nakajima for critical reading of the manuscript. This study was supported by a Japan Society for the Promotion of Science KAKENHI Grant (No. 17K15199 to S.M.), the Environment Research and Technology Development Fund of Environmental Restoration and Conservation Agency (4-1602), and the ArCS Project.

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80  Fungi in Polar Regions Bazzicalupo, A.L., M. Bálint and I. Schmitt. 2013. Comparison of ITS1 and ITS2 rDNA in 454 sequencing of hyperdiverse fungal communities. Fungal Ecol. 6: 102–109. Bellemain, E., M.L. Davey, H. Kauserud, L.S. Epp, S. Boessenkool, E. Coissac et al. 2013. Metabarcoding of ancient arctic permafrost fungi. Environ. Microbiol. 15: 1176–1189. Bjørnsgaard Aas, A., M.L. Davey and H. Kauserud. 2017. ITS all right mama: investigating the formation of chimeric sequences in the ITS2 region by DNA metabarcoding analyses of fungal mock communities of different complexities. Mol. Ecol. Resour. 17: 730–741. Blaalid, R., S. Kumar, R.H. Nilsson, K. Abarenkov, P.M. Kirk and H. Kauserud. 2013. ITS1 versus ITS2 as DNA metabarcodes for fungi. Mol. Ecol. Resour. 13: 218–224. Blaalid, R., M.L. Davey, H. Kauserud, T. Carlsen, R. Halvorsen, K. Høiland et al. 2014. Arctic rootassociated fungal community composition reflects environmental filtering. Mol. Ecol. 23: 649–659. Bödeker, I.T., K.E. Clemmensen, W. de Boer, F. Martin, Å. Olson and B.D. Lindahl. 2014. Ectomycorrhizal Cortinarius species participate in enzymatic oxidation of humus in northern forest ecosystems. New Phytol. 203: 245–256. Botkin, D.B., H. Saxe, M.B. Araújo, R. Betts, R.H.W. Bradshaw, T. Cedhagen et al. 2007. Forecasting effects of global warming on biodiversity. Bioscience. 57: 227–236. Botnen, S., U. Vik, T. Carlsen, P.B. Eidesen, M. L. Davey and H. Kauserud. 2014. Low host specificity of root-associated fungi at an Arctic site. Mol. Ecol. 23: 975–985. Bridge, P. and B. Spooner. 2001. Soil fungi: diversity and detection. pp. 147–154. In: D.S. Powlson, G.L. Bateman, K.G. Davies, J.L. Gaunt and P.R. Hirsch (eds.). Interactions in the Root Environment: An Integrated Approach. Springer, Dordrecht, Netherlands. Buée, M., M. Reich, C. Murat, E. Morin, R.H. Nilsson, S. Uroz et al. 2009. 454 Pyrosequencing analyses of forest soils reveal an unexpectedly high fungal diversity: Research. New Phytol. 184: 449–456. Buttigieg, P.L. and A. Ramette. 2014. A guide to statistical analysis in microbial ecology: A communityfocused, living review of multivariate data analyses. FEMS Microb. Ecol. 90: 543–550. Cline, L.C., Z. Song, G.A. Al-Ghalith, D. Knights and P.G. Kennedy. 2017. Moving beyond de novo clustering in fungal community ecology. New Phytol. 216: 629–634. Coleine, C., J.E. Stajich, L. Zucconi, S. Onofri, N. Pombubpa, E. Egidi et al. 2018. Antarctic cryptoendolithic fungal communities are highly adapted and dominated by lecanoromycetes and dothideomycetes. Front. Microbiol. 9: 1392. Cox, F., K.K. Newsham, R. Bol, J.A.J. Dungait and C.H. Robinson. 2016. Not poles apart: Antarctic soil fungal communities show similarities to those of the distant Arctic. Ecol. Lett. 19: 528–536. Edgar, R.C., B.J. Haas, J.C. Clemente, C. Quince and R. Knight. 2011. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27: 2194–2200. Epp, L.S., S. Boessenkool, E.P. Bellemain, J. Haile, A. Esposito, T. Riaz et al. 2012. New environmental metabarcodes for analysing soil DNA: Potential for studying past and present ecosystems: Metabarcodes to analyse soil DNA. Mol. Ecol. 21: 1821–1833. Eusemann, P., M. Schnittler, R.H. Nilsson, A. Jumpponen, M. B. Dahl, D.G. Würth et al. 2016. Habitat conditions and phenological tree traits overrule the influence of tree genotype in the needle mycobiome—Picea glauca system at an arctic treeline ecotone. New Phytol. 211: 1221–1231. Geml, J., I. Timling, C.H. Robinson, N. Lennon, H.C. Nusbaum, C. Brochmann et al. 2012. An arctic community of symbiotic fungi assembled by long-distance dispersers: phylogenetic diversity of ectomycorrhizal basidiomycetes in Svalbard based on soil and sporocarp DNA: Biodiversity of arctic ectomycorrhizal fungi. J. Biogeogr. 39: 74–88. Grau, O., J. Geml, A. Pérez-Haase, J.M. Ninot, T.A. Semenova-Nelsen and J. Peñuelas. 2017. Abrupt changes in the composition and function of fungal communities along an environmental gradient in the high Arctic. Mol. Ecol. 26: 4798–4810. Hassett, B.T., A.-L.L. Ducluzeau, R.E. Collins and R. Gradinger. 2017. Spatial distribution of aquatic marine fungi across the western Arctic and sub-arctic: Arctic marine fungal communities. Environ. Microbiol. 19: 475–484. Hirose, D., S. Hobara, S. Matsuoka, K. Kato, Y. Tanabe, M. Uchida et al. 2016. Diversity and community assembly of moss-associated fungi in ice-free coastal outcrops of continental Antarctica. Fungal Ecol. 24: 94–101.

DNA Metabarcoding for Fungal Diversity Investigation in Polar Regions 81 Horton, T.R. and T.D. Bruns. 2001. The molecular revolution in ectomycorrhizal ecology: peeking into the black-box: Molecular approaches for studies of em ecology. Mol. Ecol. 10: 1855–1871. Huson, D.H., A.F. Auch, J. Qi and S.C. Schuster. 2007. MEGAN analysis of metagenomic data. Genome Res. 17: 377–386. Jumpponen, A. and K.L. Jones. 2009. Massively parallel 454 sequencing indicates hyperdiverse fungal communities in temperate Quercus macrocarpa phyllosphere. New Phytol. 184: 438–448. Jumpponen, A., K.L. Jones, J. David Mattox and C. Yaege. 2010. Massively parallel 454-sequencing of fungal communities in Quercus spp. ectomycorrhizas indicates seasonal dynamics in urban and rural sites. Mol. Ecol. 19: 41–53. Kõljalg, U., R.H. Nilsson, K. Abarenkov, L. Tedersoo, A.F.S. Taylor, M. Bahram et al. 2013. Towards a unified paradigm for sequence-based identification of fungi. Mol. Ecol. 22: 5271–5277. Lilleskov, E.A., T.D. Bruns, T.R. Horton, D. Taylor and P. Grogan. 2004. Detection of forest stand-level spatial structure in ectomycorrhizal fungal communities. FEMS Microbiol. Ecol. 49: 319–332. Lindahl, B.D., R.H. Nilsson, L. Tedersoo, K. Abarenkov, T. Carlsen, R. Kjøller et al. 2013. Fungal community analysis by high-throughput sequencing of amplified markers—a user’s guide. New Phytol. 199: 288–299. Lindner, D.L., T. Carlsen, R. Henrik Nilsson, M. Davey, T. Schumacher and H. Kauserud. 2013. Employing 454 amplicon pyrosequencing to reveal intragenomic divergence in the internal transcribed spacer rDNA region in fungi. Ecol. Evol. 3: 1751–1764. Metzker, M.L. 2010. Sequencing technologies—the next generation. Nat. Rev. Genet. 11: 31–46. Mueller, R.C., L. Gallegos-Graves, D.R. Zak and C.R. Kuske. 2016. Assembly of active bacterial and fungal communities along a natural environmental gradient. Microb. Ecol. 71: 57–67. Mundra, S., R. Halvorsen, H. Kauserud, E. Müller, U. Vik and P.B. Eidesen. 2015. Arctic fungal communities associated with roots of Bistorta vivipara do not respond to the same fine-scale edaphic gradients as the aboveground vegetation. New Phytol. 205: 1587–1597. Murray, M.G. and W.F. Thompson. 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8: 4321–4326. Nguyen, N.H., D. Smith, K. Peay and P. Kennedy. 2014. Parsing ecological signal from noise in next generation amplicon sequencing. New Phytol. 205: 1389–1393. Nilsson, R.H., E. Kristiansson, M. Ryberg, N. Hallenberg and K.H. Larsson. 2008. Intraspecific ITS variability in the kingdom Fungi as expressed in the international sequence databases and its implications for molecular species identification. Evol. Bioinform. Online 4: 193–201. Oono, R. 2017. A confidence interval analysis of sampling effort, sequencing depth, and taxonomic resolution of fungal community ecology in the era of high-throughput sequencing. PLoS ONE 12: e0189796 Öpik, M., M. Metsis, T.J. Daniell, M. Zobel and M. Moora. 2009. Large-scale parallel 454 sequencing reveals host ecological group specificity of arbuscular mycorrhizal fungi in a boreonemoral forest. New Phytol. 184: 424–437. Osono, T. 2014. Metagenomic approach yields insights into fungal diversity and functioning. pp. 1–23. In: T. Sota, H. Kagata, Y. Ando, S. Utsumi and T. Osono (eds.). Species Diversity and Community Structure. Springer, Berlin, Germany. Quast, C., E. Pruesse, P. Yilmaz, J. Gerken, T. Schweer, P. Yarza et al. 2013. The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools. Nucleic Acids Res. 4: D590–D596. Rawlence, N.J., D.J. Lowe, J.R. Wood, J.M. Young, G.J. Churchman, Y.-T. Huang et al. 2014. Using palaeoenvironmental DNA to reconstruct past environments: progress and prospects: Using palaeoenvironmental DNA to reconstruct past environments. J. Quat. Sci. 29: 610–626. Rognes, T., T. Flouri, B. Nichols, C. Quince and F. Mahé. 2016. VSEARCH: a versatile open source tool for metagenomics. PeerJ. 4: e2584. Schoch, C.L., K.A. Seifert, S. Huhndorf, V. Robert, J.L. Spouge, C.A. Levesque et al. 2012. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc. Natl. Acad. Sci. USA 109: 6241–6246. Seifert, K.A. 2009. Progress towards DNA barcoding of fungi. Mol. Ecol. Resour. 9: 83–89.

82  Fungi in Polar Regions Semenova, T.A., L.N. Morgado, J.M. Welker, M.D. Walker, E. Smets and J. Geml. 2015. Long-term experimental warming alters community composition of ascomycetes in Alaskan moist and dry arctic tundra. Mol. Ecol. 24: 424–437. Semenova, T.A., L.N. Morgado, J.M. Welker, M.D. Walker, E. Smets and J. Geml. 2016. Compositional and functional shifts in arctic fungal communities in response to experimentally increased snow depth. Soil Biol. Biochem. 100: 201–209. Shendure, J. and H. Ji. 2008. Next-generation DNA sequencing. Nature Biotechnol. 26: 1135–1145. Smith, D.P. and K.G. Peay. 2014. Sequence depth, not PCR replication, improves ecological inference from next generation DNA sequencing. PLoS ONE 9: e90234. Smith, M.E., G.W. Douhan and D.M. Rizzo. 2007. Intra-specific and intra-sporocarp ITS variation of ectomycorrhizal fungi as assessed by rDNA sequencing of sporocarps and pooled ectomycorrhizal roots from a Quercus woodland. Mycorrhiza 18: 15–22. Song, Z., D. Schlatter, P. Kennedy, L.L. Kinkel, H.C. Kistler, N. Nguyen et al. 2015. Effort versus reward: Preparing samples for fungal community characterization in high-throughput sequencing surveys of soils. PLoS ONE 10: e0127234. Stevens, J.L., R.L. Jackson and J.B. Olson. 2013. Slowing PCR ramp speed reduces chimera formation from environmental samples. J. Microbiol. Methods 93: 203–205. Taberlet, P., E. Coissac, M. Hajibabaei and L.H. Rieseberg. 2012a. Environmental DNA: Environmental DNA. Mol. Ecol. 21: 1789–1793. Taberlet, P., E. Coissac, F. Pompanon, C. Brochmann and E. Willerslev. 2012b. Towards next-generation biodiversity assessment using DNA metabarcoding: Next-generation DNA metabarcoding. Mol. Ecol. 21: 2045–2050. Tanabe, A.S. and H. Toju. 2013. Two new computational methods for universal DNA barcoding: A benchmark using barcode sequences of bacteria, archaea, animals, fungi, and land plants. PLoS ONE 8: e76910. Tanabe, A.S. 2018. Claident v0.2.2018.05.29 software distributed by the author at. https://www.claident. org/ (Accessed 15 January 2019). Tedersoo, L., R.H. Nilsson, K. Abarenkov, T. Jairus, A. Sadam, I. Saar et al. 2010. 454 Pyrosequencing and Sanger sequencing of tropical mycorrhizal fungi provide similar results but reveal substantial methodological biases. New Phytol. 188: 291–301. Tedersoo, L., A. Tooming-Klunderud and S. Anslan. 2018. PacBio metabarcoding of fungi and other eukaryotes: errors, biases and perspectives. New Phytologist 217: 1370–1385. Thiéry, O., M. Vasar, T. Jairus, J. Davison, C. Roux, P.-A. Kivistik et al. 2016. Sequence variation in nuclear ribosomal small subunit, internal transcribed spacer and large subunit regions of Rhizophagus irregularis and Gigaspora margarita is high and isolate-dependent. Mol. Ecol. 25: 2816–2832. Toju, H., A.S. Tanabe, S. Yamamoto and H. Sato. 2012. High-Coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples. PLoS ONE 7: e40863. Ushio, M., S. Aiba, Y. Takeuchi, Y. Iida, S. Matsuoka et al. 2017. Plant–soil feedbacks and the dominance of conifers in a tropical montane forest in Borneo. Ecol. Monogr. 87: 105–129. Vu, D., M. Groenewald, M. de Vries, T. Gehrmann, B. Stielow, U. Eberhardt et al. 2019. Large-scale generation and analysis of filamentous fungal DNA barcodes boosts coverage for kingdom fungi and reveals thresholds for fungal species and higher taxon delimitation. Stud. Mycol. 92: 135–154. Willerslev, E. and A. Cooper. 2005. Review paper. Ancient DNA. Prc. R. Soc. B 272: 3–16. Zhang, T. and Y.-F. Yao. 2015. Endophytic fungal communities associated with vascular plants in the high arctic zone are highly diverse and host-plant specific. PLoS ONE 10: e0130051.

6 Oomycetes in Polar Regions Motoaki Tojo

Introduction Oomycetes, synonyms Oomycota and Peronosporomycetes, are fungus-like microorganisms belonging to the eukaryotic kingdom Chromista (Cavalier-Smith 1981, Dick 2001). Oomycetes and true fungi resemble each other in their gross structure, and comprise a mass of branched hyphae. This similarity is, however, thought to be the result of the convergent evolution of Oomycetes and true fungi sharing the common nutritional resources and the living environments. On the other hand, Oomycetes are quite different from true fungi at the cellular and molecular levels. For example, Oomycetes have diploid thallus, cell walls of glucan-cellulose and have no chitin, and zoospores with two flagella (Sietsma et al. 1969, Dick 2001, Mélida et al. 2013). Oomycetes are comprised of more than 900 species (Kirk et al. 2008). They are saprobic or parasitic on both plant and animal matters in water and soil over the world (Sparrow 1960). Only a limited number of Oomycetes species, Peronospora alsinearum, Peronospora parasitica, Pythium sp., Pythium polare, Pythium ultimum var. ultimum, and Saprolegnia sp., have been recorded from polar regions (Table 6.1). Therefore, there are still a few reports on the other Oomycete genus in polar regions except for Pythium. This is in contrast with the true fungi, which show an excessive diversity in the regions (Arenz and Blanchette 2011, Elvebakk et al. 1996, Hughes et al. 2003, Tojo et al. 2013, Tsuji et al. 2013, Zhang et al. 2016). The reason behind this difference is unknown, but probably related to requirements of free water or high humidity on reproductions for many of the Oomycetes species. The severe cold stress with little availability of free water in polar environments (Hisdal 1998) may limit the number of species which can be Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuencho, Nakaku, Sakai, 599-8531, Japan. Email: [email protected]

Host or Substrate Alpine chickweed (Cerastium alpinum), and other Caryophyllaceous plants Danish scurvygrass (Cochlearia groenlandica), and other Brassicaceous plants Sanionia moss (Sanionia uncinata) Sanionia moss (Sanionia uncinata) Leafy liverwort (Cephaloziella varians) Antarctic hair grass (Deschampsia Antarctica) Soil Carrot (Daucus carota) in a greenhouse Sanionia moss (Sanionia uncinata), Fresh water

Lake water

Oomycete Species

Peronospora alsinearum

Peronospora parasitica

Pythium sp. (originaly described as Pythium ultimum var. ultimum)

Pythium sp.

Pythium sp.

Pythium sp.

Pythium sp.

Pythium ultimum var. ultimum

Pythium polare

Saprolegnia sp.

King George Island (Antarctic)

Spitsbergen Island (Arctic), Baffin Island (Arctic), Greenland (Arctic), Norway mainland (Arctic), King George Island (Antarctic)

Spitsbergen Island (Arctic)

Humble Island (Antarctic)

Signy Island (Antarctic)

Adelaide Island (Antarctic)

Gonçalves et al. (2012)

Tojo et al. (2012)

Tojo et al. (2001)

Arenz and Blanchette (2011)

Bridge et al. (2008)

Hughes et al. (2003)

Hoshino et al. (2000, 2001, 2006)

Hoshino et al. (1999)

Spitsbergen Island (Arctic) Finnmark (Arctic), Greenland (Arctic)

Hagen (1941)

Hagen (1941)

References

Spitsbergen Island (Arctic)

Spitsbergen Island (Arctic)

Distribution

Table 6.1.  Oomycetes recorded in polar regions.

84  Fungi in Polar Regions

Oomycetes in Polar Regions 85

distributed in the environments. It is also interesting that there is no record for the other major Oomycetes, such as Phytophthora. This is maybe due to limited distributions of vascular plants in the polar regions, as most of Phytophthora spp. are parasites of vascular plants (Erwin and Ribeiro 1996). This chapter, therefore, focuses on Pythium spp., which are the most common genus of Oomycetes in polar regions, although there are a few reports on Peronospora and Saprolegnia (Table 6.1).

Taxonomy and Distribution In polar regions, Pythium spp. can cause snow rot on mosses (Fig. 6.1). Pythium is the most cosmopolitan genus among Oomycetes, and holds more than 200 species through the world. Among of them, several Pythium species occur in polar regions. The first record of isolation of Pythium in the polar region was from a moribund Sanionia moss in Spitsbergen Island, Norway (Hoshino et al. 1999). This isolate was originally described as P. ultimum var. ultimum (Hoshino et al. 1999), but was re-identified as an unknown Pythium sp. afterwards with further taxonomic analysis (M.T. unpublished). Since then, several species of Pythium have been recorded from polar regions (Hoshino et al. 2000, 2001, Tojo et al. 2001, Hughes et al. 2003, Bridge et al. 2008, Arenz and Blanchette 2011, Tojo et al. 2012). Most of Pythium

Fig. 6.1.  Snow rot of Sanionia moss (Sanionia uncinata) associating Pythium spp. in Spitsbergen Island, Norwegian arctic. Brawn and circular color patches (arrow heads) appear just after the snow melts (A and B). The circular patches are ranging in size from 2 to 15 centimeters on the moss colonies. Sporangium of Pythium polare found on the tissue of the Sanionia moss causing snow rot (C).

86  Fungi in Polar Regions spp. in polar regions were still unnamed, and P. polare is the only named Pythium across the regions. Pythium polare is the most common Oomycetes in polar regions which has a bipolar distribution (Tojo et al. 2012). This species is heterothallic, which requires antheridial and oogonial isolates to produce oospores—even in between the Arctic antheridial isolate and the Antarctic oogonial isolate (Fig. 6.3). P. polare is characterized by globose sporangia with discharge tubes of various lengths that release zoospores (Fig. 6.3). The species is taxonomically closely related with P. iwayamai, P. paddicum, and P. okanoganense, which are known as snow rot Pythium in Japan (Iwayama 1933, Hirane 1955, 1960, Takamatsu 1989a,b, Takamatsu and Takenaka 2001, Masumoto et al. 2009) and USA (Lipps 1980, Lipps and Bruehl 1978, 1980) (Fig. 6.2). P. polare is distinguished from these snow rot Pythium by longer discharge tubes to release zoospores and aplerotic oospores, usually with one to five antheridia (Fig. 6.3). P. polare is also closely related with the moss parasitic P. barbulae (Ueta and Tojo 2016) (Fig. 6.2). P. polare and P. barbulae are the only Pythium which have been reported as moss parasites in natural environmental conditions. P. polare, P. iwayamai, P. paddicum, P. okanoganense,

Fig. 6.2.  Phylogenetic positions of Pythium polare from polar regions, P. barbulae from Osaka Japan, P. iwayamai from Sapporo, Japan, and other Pythium species in clade G (Lévesque and de Cock 2004). All of the three Pythium spp. can grow at 0ºC, and, therefore, have the common feature of the lowtemperature properties. The phylogenetic trees of Minimum-Evolution (ME) and Neighbor-Joining (NJ) were based on the rRNA gene-ITS regions using ME as the base topology. Pythium ultimum var. ultimum and P. splendens from clade I were used as outgroups. Numbers along nodes indicate bootstrap confidence levels (gaining more than 50% support) for ME and NJ, respectively.

Oomycetes in Polar Regions 87

Fig. 6.3.  Life cycle of Pythium polare. In or on moss tissues, P. polare produces a white and rapid growing mycelium. The mycelium gives rise to sporangium which germinates directly by producing germ tube(s). The mycelium also produces spherical oogonia and club-shaped antheridia. P. polare shows heterothallism and has the oogonial isolate and the antheridial isolate. Oospores are formed after fertilization of oogonia and antheridia, which are produced by the oogonial isolate and the antheridial isolate, respectively. Oospores have thick walls and are resistant to adverse conditions in the polar environments, such as freezing and drying. Oospores can germinate by producing a germ tube. In melt water, sporangium produces a discharge tube, at the end of which forms a balloon-like secondary sporangium called vesicle. In the vesicle, 4–20 zoospores are produced, which, when released, swarm about for a few hours towards moss tissues. After arriving on the moss tissues, zoospores round off to form a cyst, and germinate by producing a germ tube. The germ tube usually penetrates the moss tissues and starts a new infection.

and P. barbulae taxonomically belong to Pythium clade G described by Lévesque and de Cock (2004). All of the major snow rot Pythium belong to the clade G (Tojo and Newsham 2012, Ueta and Tojo 2016) (Fig. 6.2). P. polare is, therefore, one of the snow rot Pythium showing a wide distribution throughout the polar regions. Several Pythium species which belong to groups other than the clade G have been found from polar regions, and most of them are included in the clade E of the genus (M.T. unpublished). Pythium kandovanense, which belongs to the clade E, has been recently described to cause snow rot symptoms on Lolium perenne in a natural grassland in Iran (Bouket et al. 2015). These suggest that Pythium spp. of the clade E also can be inhabitants in the polar environments. The clades E and G are taxonomically closely related to each other and belong to the single genus Globisporangium of the modern Pythium taxonomy, which divided the

88  Fungi in Polar Regions former Pythium group into four new genus: Ovatisporangium, Globisporangium, Elongisporangium, and Pilasporangium, based on phylogeny and morphology (Uzuhashi et al. 2010). Therefore, most of the polar Pythium belong to Globisporangium. This taxonomical uniqueness maybe results from independent evolutionally pathways of the snow rot Pythium from other Pythium spp. Pythium occurrence in greenhouses in polar regions is exceptionally treated. P. ultimum var. ultimum has been found in a greenhouse in Spitsbergen Island (Tojo et al. 2001). Pythium spp. other than the clades E and G can spread when they are introduced inside the greenhouses in polar regions. Considering the severe cold stress and few available host plants in the natural environments of polar regions (Hisdal 1998), the temperate region Pythium can only likely survive in polar regions under greenhouse conditions.

Ecology Pythium polare is able to infect Sanionia moss and cause brown discoloration (Tojo et al. 2012) (Fig. 6.1). P. polare is also isolated from the non-symptomatic moss and fresh water (Tojo et al. 2012). Bridge et al. (2008) isolated a Pythium species from Signy Island, which is almost identical to P. polare based on ITS sequences, and demonstrated that it can be a causative agent of snow rot on Antarctic hairgrass (Deschampsia antarctica). A Pythium species, which has an identical ITS sequence to P. polare, inhabits an alpine haircap moss (Polytrichastrum alpinum) on King George Island (Yu and Hur unpubl.). These suggest that P. polare infects a broad range of plants, including mosses and monocotyledons. The main host Sanionia moss has a bipolar distribution (Smith 1996, Virtanen et al. 1997) and grow predominantly in locations with a steady supply of snow melt water (Davey 1997, Victoria et al. 2009). P. polare requires snow melt water to produce zoospores for dissemination (Fig. 6.1). Moreover, the frost resistance of P. polare was enhanced when the mycelia were present in the living Sanionia moss tissues (Murakami et al. 2015). P. polare is, therefore, highly dependent on Sanionia moss to survive in the polar regions, although this Oomycete has a greater frost resistance than the other snow rot Pythium (Murakami et al. 2015). It is also interesting that Trichoderma polysporum, which has antagonistic activity to Pythium, exists in Sanionia moss in the polar region (Kamo et al. 2016). This suggests that the polar Pythium also receives microbial stress for surviving in polar regions, as well as Pythium spp. in the temperate and tropical regions (Naseby et al. 2000). When the populations were investigated on P. polare and the other moss inhabiting Pythium in summer seasons from 2003 to 2014 at Spitsbergen Island, they were increased during 2003 to 2012, and decreased from 2012 to 2014 with different patterns of population change among the Pythium spp. (M.T. unpublished). P. polare was the dominant among the Pythium in several locations in Spitsbergen Island (M.T. unpublished). It was recently reported that P. polare is infected by a toti and toti-like virus, which is named Pythium polare RNA virus 1 (PpRV1) (Sasai et al. 2018).

Oomycetes in Polar Regions 89

Advantages and/or disadvantages of PpRV1 infection on P. polare host is unclear, and remains a topic for further study.

Acknowledgments This work was supported from the Japan Society for the Promotion of Science by the grant-in-aid for scientific research No. 15K00626.

References Arenz, B.E. and R.A. Blanchette. 2011. Distribution and abundance of soil fungi in Antarctica at sitesonthe Peninsula, Ross Sea Region and McMurdo Dry Valleys. Soil Biol. Biochem. 43: 308–315. Bouket, A.C., M. Arzanlou, M. Tojo and A. Babai-Ahari. 2015. Pythium kandovanense sp. nov., a fungus-like eukaryotic microorganism (Stramenopila, Pythiales) isolated from snow covered ryegrass leaves. Int. J. Syst. Evol. Microbiol. 65: 2500–2506. Bridge, P.D., K.K. Newsham and G.J. Denton. 2008. Snow mould caused by a Pythium sp.: a potential vascular plant pathogen in the maritime Antarctic. Plant Pathol. 57: 1066–1072. Cavalier-Smith, T. 1981. Eukaryotic kingdoms: seven or nine? BioSystems 14: 461–481. Davey, M.C. 1997. Effects of continuous and repeated dehydration on carbon fixation by bryophytes from the maritime Antarctic. Oecologia 110: 25–31. Dick, M.W. 2001. The Peronosporomycetes. pp. 39–72. In: D. McLaughlin, E. McLaughlin and P. Lemke (eds.). The Mycota, Springer-Verlag, New York, NY, USA. Elvebakk, A., H.B. Gjærum and S. Sivertsen. 1996. Part 4. Fungi II. Myxomycota, oomycota, chytridiomycota, zygomycota, ascomycota, deutromycota, basidiomycota: uredinales and ustilaginales. pp. 207–259. In: A. Elvebakk and P. Prestrud (eds.). A Catalogue of Svalbard Plants, Fungi, Algae and Cyanobacteria. Norsk Polarinstitutt, Oslo, Norway. Erwin, D.C. and O.K. Ribeiro. 1996. Phytophthora Diseases Worldwide. APS Press, St. Paul. Gonçalves, V.N., A.B.M. Vaz, C.A. Rosa and L.H. Rosa. 2012. Diversity and distribution of fungal communities in lakes of Antarctica. FEMS Microbiol. Ecol. 82: 459–471. Hagen, A. 1941. Micromycetes from Vestspitsbergen collected by dr. Emil Hadač in 1939. Medd Norges Svalb Ishavs–Unders 49: 1–11. Hirane, S. 1955. Studies on the control of Pythium snow blight of wheat and barley. Nat. Inst. Agric. Sci., Japan 60: 1–86 (in Japanese). Hirane, S. 1960. Studies on Pythium snow blight of wheat and barley, with special reference to the taxonomy of the pathogens. Trans. Mycol. Soc. Jpn. 2: 82–87. Hisdal, V. 1998. Svalbard nature and history. Norsk Polarinstitutt, Oslo, Norway. Hoshino, T., M. Tojo, G. Okada, H. Kanda, S. Ohgiya and K. Ishizaki. 1999. A filamentous fungus, Pythium ultimum Trow var. ultimum, isolated from moribund moss colonies from Svalbard, northern islands of Norway. Polar Biol. Sci. 12: 68–75. Hoshino, T., M. Tojo and A.M. Tronsmo. 2000. Pythium blight of moss colonies (Sanionia uncinata) in Finnmark. Polarflokken 24: 161–164. Hoshino, T., M. Tojo, H. Kanda and A.M. Tronsmo. 2001. Ecological role of fungal infections of moss carpet in Svalbard. Mem. Natl. Inst. Polar Res., Spec Issue 54: 507–513. Hoshino, T., M. Tojo and I. Yumoto. 2006. Blight of moss caused by Pythium sp. in Greenland. Meddelelser on Grønland, Serie: Bioscience 56: 95–98. Hughes, K.A., B. Lawley and K.K. Newsham. 2003. Solar UV-B radiation inhibits the growth of Antarctic terrestrial fungi. Appl. Environ. Microbiol. 69: 1488–1491. Iwayama, S. 1933. A new snow-rot disease of cereal plants caused by Pythium sp. Agri. Exp. St. Rep., Toyama-Ken, Japan. pp. 1–20 (in Japanese).

90  Fungi in Polar Regions Kamo, M., M. Tojo, Y. Yamazaki, T. Itabashi, H. Takeda, D. Wakana and T. Hosoe. 2016. Isolation of growth inhibitors of the snow rot pathogen Pythium iwayamai from an arctic strain of Trichoderma polysporum. J. Antibiot. 69: 451–455. Kirk, P.M., P.F. Cannon, D.W. Minter and J.A. Stalpers. 2008. Dictionary of the fungi, 10th edn. CABI Europe, Oxford, UK. Lévesque, C.A. and A.W.A.M. de Cock. 2004. Molecular phylogeny and taxonomy of the genus Pythium. Mycol. Res. 108: 1363–1383. Lipps, P.E. and G.W. Bruehl. 1978. Snow rot of winter wheat in Washington. Phytopathology 68: 1120–1127. Lipps, P.E. 1980. The influence of temperature and water potential on asexual reproduction by Pythium spp. associated with snow rot of wheat. Phytopathology 70: 794–797. Lipps, P.E. and G.W. Bruehl. 1980. Infectivity of Pythium spp. zoospores in snow rot of wheat. Phytopathology 70: 723–726. Masumoto, S., T. Shigyo and M. Tojo. 2009. Pythium snow blight of Kentucky bluegrass turf in a golf course in Hokkaido, Japan. J. Japanese Soc. Turfgrass. Sci. 38: 33–36. Mélida, H., J.V. Sandoval-Sierra, J. Diéguez-Uribeondo and V. Bulone. 2013. Analyses of extracellular carbohydrates in oomycetes unveil the existence of three different cell wall types. Eukaryotic Cell 12: 194–203. Murakami, R., Y. Yajima, K. Kida, K. Tokura, M. Tojo and T. Hoshino. 2015. Surviving freezing in plant tissues by oomycetous snow molds. Cryobiology 70: 208–210. Naseby, D.C., J.A. Pascual and J.M. Lynch. 2000. Effect of biocontrol strains of Trichoderma on plant growth, Pythium ultimum population, soil microbial communities and soil enzyme activities. J. Appl. Microbiol. 88: 161–169. Sasai, S., K. Tamura, M. Tojo, M.L. Herrero, T. Hoshino, S.T. Ohki and T. Mochizuki. 2018. A novel nonsegmented double stranded virus from an Arctic isolate of Pythium polare. Virology 522: 234–243. Sietsma, J.H. D.E. Eveleigh and R.H. Haskins. 1969. Cell wall composition and protoplast formation of some Oomycete species. Biochim. Biophys. Acta 184: 306–317. Sparrow, F.K., Jr. 1960. Aquatic Phycomycetes. The University of Michigan Press. Ann. Arbor., MI, USA. Smith, R.I.L. 1996. Terrestrial and freshwater biotic components of the western Antarctic Peninsula. pp. 15–59. In: R.M. Ross, E.E. Hofmann and L.B. Quentin (eds.). Foundations of Ecological Research West of the Antarctic Peninsula Antarctic Research Series, vol. 70. American Geophysical Union, Washington DC, USA. Takamatsu, S. 1989a. Ecological study of Pythium snow rot of wheat and barley. Spe Bull Fukui Pref. Agric. Exp. Stn. 9: 1–135 (in Japanese). Takamatsu, S. 1989b. Distribution of Pythium snow rot fungi in paddy fields and upland fields. Japan Agricultural Research Quarterly 23: 100–108. Takamatsu, S. and S. Takenaka. 2001. Snow rot caused by Pythium species. pp. 87–100. In: N. Iriki, D.A. Gaudet, A.M. Tronsmo, N. Matsumoto, M. Yoshida and A. Nishimune (eds.). Low Temperature Plant-Microbe Interactions Under Snow. Hokkaido National Agricultural Experiment Station, Sapporo, Japan. Tojo, M., T. Hoshino, M.L. Herrero, S.S. Klemsdal and A.M. Tronsmo. 2001. Occurrence of Pythium ultimum var. ultimum in a greenhouse on Spitsbergen Island, Svalbard. Eur. J. Plant Pathol. 107: 761–65. Tojo, M., P. Van West, T. Hoshino, K. Kida, H. Fujii, H. Hakoda et al. 2012. Pythium polare, a new heterothallic Oomycete causing brown discoloration of Sanionia uncinata in the Arctic and Antarctic. Fungal Biol. 116: 756–768. Tojo, M. and K.K. Newsham. 2012. Snow mould in polar environments. Fungal Ecol. 5: 395–402. Tojo, M., S. Masumoto and T. Hoshino. 2013. Phytopathogenic fungi and fungal-like microbes in Svalbard. pp. 263–284. In: R. Imai, M. Yoshida and N. Matsumoto (eds.). Plant and Microbe Adaptations to Cold in a Changing World. Springer, New York, USA. Tsuji, M., S. Fujiu, N. Xiao, Y. Hanada, S. Kudoh, H. Kondo et al. 2013. Cold adaptation of fungi obtained from soil and lake sediment in the Skarvsnes ice-free area, Antarctica. FEMS Microbiol. Lett. 346: 121–130.

Oomycetes in Polar Regions 91 Ueta, S. and M. Tojo. 2016. Pythium barbulae sp. nov. isolated from the moss, Barbula unguiculata; morphology, molecular phylogeny and pathogenicity. Mycoscience 57: 11–19. Uzuhashi, S., M. Tojo and M. Kakishima. 2010. Phylogeny of the genus Pythium and description of new genera. Mycoscience 51: 337–365. Victoria, F.C., A.B. Pereira and D.P. Costa. 2009. Composition and distribution of moss formations in the ice-free areas adjoining the Arctowski region, Admiralty Bay, King George Island, Antarctica. Iheringia, Série Botânica 64: 81–91. Virtanen, R.J., P.A. Lundberg, J. Moen and L. Oksanen. 1997. Topographic and altitudinal patterns in plant communities on European Arctic islands. Polar Biol. 17: 95–113. Zhang, T., N.F. Wang, Y.Q. Zhang, H.Y. Liu and L.Y. Yu. 2016. Diversity and distribution of aquatic fungal communities in the Ny-Ålesund region, Svalbard (high arctic). Microb. Ecol. 71: 543–554.

7 Biotechnological Potentials of Arctic Fungi Purnima Singh* and R. Kanchana

Introduction Polar habitats are exciting places on Earth where psychrophiles and psychrotolerant fungi live. These fungi, through novel biochemical adaptations, survive the extreme environmental conditions, such as low temperature, intermittent freezing and ultraviolet light. The fungal diversity existing in Arctic realm, although scanty compared to the temperate or tropical regions, are much more distinctive and chemically well-equipped. The adaptation strategies employed by the psychrophilic/ psychrotolerant fungi lead to the production of several metabolic compounds. These compounds, such as cold tolerant enzymes, antimicrobials, antioxidants, and antifreeze proteins, find immense applications in the field of pharmaceutical and biotechnological industries, and therefore provide opportunities for bioprospecting in the Arctic.

Taxonomy of Fungi Around 2.3% of the world’s fungal biota exists in the Arctic (Fig. 7.1a). Glacier habitats include one of the largest unexplored and extreme biospheres of Earth (Buzzini et al. 2012). Recently, Hoshino and Matsumoto (2012) coined a new category, ‘cryophilic’, for cold-adapted yeast and filamentous fungi, and this term has been adopted in the recent studies. Conventional and molecular techniques have been used to characterize culturable psychrophilic yeasts and filamentous fungi from the Arctic. Morphological characteristics and physiological tests were taken Parvatibai Chowgule College of Arts & Science, Margao, Goa-403602, India. Email: [email protected] * Corresponding author

Biotechnological Potentials of Arctic Fungi 93

Fig. 7.1. (a) Landscape of Svalbard Arctic showing different habitats where fungal biota exists. (b) Arctic wild mushroom Lycoperdon molle collected from Svalbard, Arctic. (c) A novel species of yeast contributed from Svalbard, Arctic. (d) A novel species of filamentous fungi contributed from Svalbard, Arctic. (e) Strong enzyme (lipase) activity of an ice core yeast collected from Svalbard, Arctic. (f) A Cryoconites yeast (Rhodoturala svalbardensis sp. nov.) showing positive AFPs activity.

Color version at the end of the book

into account for initial identification of yeasts by following standard procedures of Yarrow (1998) and Kurtzman et al. (2011). However, initial identification of filamentous fungi was achieved on the basis of morphotaxonomy with the help of standard literatures (Barnett 1960, Barron 1977, Carmichael et al. 1980, Ellis 1971, 1976, Kirk et al. 2008). Strains with similar morphological and physiological characteristics were grouped together, and representative strains of each group were

94  Fungi in Polar Regions used for sequence analysis of D1/D2 domain of the rDNA gene. BLAST search was carried out with NCBI database (http:// www. ncbi. nlm. nih. gov/BLAST/).

a) Filamentous Fungi Arctic fungi have been studied from various substrates and habitats: ice (GundeCimerman et al. 2003, Sonjak et al. 2006), superficial horizons of landscapes (Zabawski 1982, Bab’eva and Sizova 1983, Bergero et al. 1999, Kirtsidely 1999a,b, 2001, 2002, Chernov 2002, Etienne 2002, Callaghan et al. 2004, Callaghan 2005, Wallenstein et al. 2007), and plant substrates (Karatygin et al. 1999, Yamazaki et al. 2011). The mycobiota of Arctic permafrost have also been recorded (Kochkina et al. 2001, Ozerskaya et al. 2004, Panikov and Sizova 2007, Ozerskaya et al. 2009). Of the 2,500 species of fungi that have been reported from Arctic, ~ 1,750 species are known from the Russian Arctic (Karatygin et al. 1999). Aquatic fungi in Arctic show relatively high diversity, especially Chytridiales and Saprolegniales. However, Basidiomycets and Ascomycetes showed lower diversity (17 families, 30 genera, and about 100 species) in comparison to sub-Arctic (50 families, about 300 genera, and 1,200 species) (Miller and Farr 1975). Mycorrhizal symbionts are common in Arctic ecosystems (Michelsen et al. 1998). Borgen et al. (2005) estimated about 250 ectomycorrhizal fungal species in Greenland. Mycological exploration in Svalbard began with the studies of Karsten (1872), Lind (1928), Hagen (1941), Kobayashi et al. (1968), Zabawski (1976), and Tamotsu et al. (1999). The diversity of fungi in soils of Bellsund has been studied (Kurek et al. 2007), and new genera and species have been described from the region (Pang et al. 2008, 2009). Elvebakk et al. (1996), in their comprehensive account of known Svalbard fungi, listed 389 species belonging to Myxomycota, Oomycota, Chytridiomycota, Zygomycota, Ascomycota, Deuteromycota, and Basidiomycota. However, the authors imply that the mycobiota studies of the region are only fragmentary and that these 389 species represent a very small part of the actual mycobiota. A recent catalog of ‘macro- and micromycetes recorded for Norway and Svalbard’ (Aarnæs 2002) also indicates that diverse groups of fungi exist in the area. Recently, a list of soil filamentous fungi from Svalbard has been contributed (Singh et al. 2012). CFU of filamentous fungi ranged 4 × 103 to 1.2 × 104 and taxonomic studies based on morphology and sequence data showed the presence of Phialophora alba, Articulospora tetracladia, and Varicosporium sp. from the Arctic cryoconites (Singh and Singh 2011, Edwards et al. 2013). Recently, a few novel species of filamentous fungi (Fig. 7.1d) have been contributed from the Arctic (Sonjak et al. 2007, Singh et al. 2013b). Bird feather fungi from Svalbard Arctic have also been contributed (Singh et al. 2016). Studies on diversity and distribution of fungi from hydro-terrestrial, marine environments, and from lichens in Svalbard have also been carried out using 454 pyrosequencing (Zhang et al. 2015a,b, 2016a,b).

Biotechnological Potentials of Arctic Fungi 95

b) Yeasts The Arctic harbors 46 species of yeasts (Buzzini et al. 2012) which have been isolated from various habitats, such as Siberian sands, sediments, and permafrost layers (Dmitriev et al. 1997, Golubev 1998, Gilichinsky et al. 2005, Vishniac and Takashima 2010), Iceland soils (Vishniac 2002, Birgisson et al. 2003), ancient Greenland ice cores (Ma et al. 1999), Svalbard glacier associated habitats (Butinar et al. 2007, Lee et al. 2010, Pathan et al. 2010), and glaciers in Alaska (Uetake et al. 2012). Evidence of colonization and succession of Arctic glaciers by psychrophilic microbes has been reported (Skidmore et al. 2000, 2005, Mindl et al. 2007). Recently, Singh and Singh (2011) reported Cryptococcus gilvescens, Rhodotorula sp., Mrakia sp., Thelebolus sp. from cryoconites, and the colony forming unit (CFU) of yeast per gram of sediment sample was about 7 × 103 to 1.4 × 104. A few novel species of yeasts (Fig. 7.1c) have been contributed from the Arctic (Singh et al. 2014, Tsuji et al. 2018a and b). The studies on yeasts in glacial cryoconites and ice cores are unique habitats where true psychrophiles live, and is therefore highly desirable to focus on the investigations. Information on yeast diversity, distribution, and biotechnological potential from the Arctic ice-cores is scanty. Isolates obtained from Arctic ice cores have been shown to be metabolically active even under sub-zero temperature. There is very low culturable yeast diversity and only three species have been reported from Alaskan Pt. Barrow tundra (Bunnell et al. 1980). Butinar et al. (2007) studied the yeast from the ice cores of Austre Lovénbreen and Austre Brøgerbreen while Singh et al. (2013a) characterized 10 strains of psychrophilic yeasts from glacier ice cores of Midre Lovénbreen glacier Svalbard. The ice core melt water of Svalbard glacier contained about 3 × 103–1 × 104 CFUs/mL, and 18S rDNA (Deoxy Ribonucleic Acid) sequence analysis using D1/D2 domain identified five yeast species, namely Cryptococcus albidosimilis, C. adeliensis, C. saitoi, Rhodotorula mucilaginosa, and Rhodosporidium sp. (Singh et al. 2013). Recently, Tsuji et al. 2016 contributed 8 species of yeasts (Cryptococcus gilvescens, Cryptococcus victoriae, Mrakia gelida, Mrakia robertii, Mrakia psychrophila, Rhodotorula glacialis, Rhodotorula psychrophenolica and Thelebolus microsporus) from deglaciated area of Svalbard.

Biotechnological Potentials of Arctic Fungi Arctic fungi find applications in the field of biotechnology because they produce substances such as enzymes, polyunsaturated fatty acids, antifreeze proteins, and secondary metabolites. As decomposers, these fungi also form an important part of the nutrient cycle. Biochemical adaptations enable macromycete species to overcome extreme environmental hazards—including low temperatures, intermittent freezing, and relatively high exposure to ultraviolet rays. These acclimatizations might influence the production of cold tolerant enzymes and antioxidant compounds with biotechnological potentials.

Amylase – – – – ++ ++ ND +++ – – –

Name of Filamentous Fungi

Acremonium fusidioides NFCCI-2142 (Nicot) W. Gams

Acremonium roseolum NFCCI-2143 (G.Sm).W. Gams

Arthriniumphaeospermum NFCCI-2144gene GU266274.1 (97%) Articulospora tetracladia (CCP-V) Ingold Articulospora sp. Cry-FB1 Articulospora sp. Cry-FB2

Aspergillus aculeatus NFCCI-2137IizukaFJ876653.1 (100%)

Aspergillus flavus NFCCI-2139 Link ex Gray

Aspergillus niger NFCCI-2140 van Tieghem

Aspergillus niger NFCCI-2141 Gr.

Aureobasidium pullulans strain 1

Pectinase +

–

–

–

++

ND

ND

ND

–

–

–

Cellulase +++

–

–

–

+++

++

++

–

–

–

–

Esterase –

ND

ND

ND

ND

++

+

–

ND

ND

ND

Protease –

ND

ND

ND

ND

++

++

–

ND

ND

ND

–

+++

+++

–

–

ND

ND

–

–

–

–

Phosphatase

Table 7.1.  Biotechnological potentials of Arctic filamentous fungi.

+

ND

ND

ND

ND

+

+

–

ND

ND

ND

Urease



Keratinase ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

References Gawas-Sakhalkar and Singh 2011

Singh et al. 2012

Singh et al. 2012

Singh et al. 2012

Singh et al. 2012

Singh et al. 2016

Singh et al. 2016

Singh and Singh 2011

Singh et al. 2012

Singh et al. 2012

Singh et al. 2012

96  Fungi in Polar Regions

– – – – – – – ND – + – ND – – –

Aureobasidium pullulans strain 2 Aureobasidium sp. Botrytis verrucosa Cladosporium chlorocephalum

Cladosporium cladosporioides NFCCI-2145 (Fresen.) de Vries Cladosporium cladosporioides

Cladosporium tenuissimum NFCCI-2146 Cooke Cladosporium herbarum PG246 (AB916505)

Corynespora cassiicola NFCCI-2147 (Berk. & Curt.) Wei.

Emericella nidulans NFCCI-2138 (Eidam) Vuill. Fusarium oxysporum

Geomyces pannorum NFCCI-2148 (Link) Sigler et J.W. Carmich. Geomyces pannorum strain 1 Geomyces pannorum strain 2 Microdochium sp. strain 1

+

–

–

ND

+

–

–

ND

–

++

–

–

+

–

++

+++

–

–

++

+

–

–

ND

++

–

+++

–

–

+

+++

–

+

++

ND

++

ND

ND

ND

ND

+

ND

–

+

–

–

–

+

+

ND

–

ND

ND

ND

ND

–

ND

–

+

+

++

++

++

+++

ND

++

–

–

ND

–

–

–

–

+++

–

–

++

+++

+++

ND

–

ND

ND

ND

ND

–

ND

–

+++

+

+

ND

ND

ND

ND

ND

ND

ND

++

ND

ND

ND

ND

ND

ND

ND

Table 7.1 contd. …

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Singh et al. 2012

Gawas-Sakhalkar and Singh 2011

Singh et al. 2012

Singh et al. 2012

Singh et al. 2015

Singh et al. 2012

Gawas-Sakhalkar and Singh 2011

Singh et al. 2012

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Biotechnological Potentials of Arctic Fungi 97

Amylase – – – – – – – – – – – –

Name of Filamentous Fungi

Microdochium sp. strain 2

Mortierella sp. NFCCI-2151EF601628.1 98% Mortierella alpina strain 1 Mortierella alpina strain 2 Mortierella alpina strain 3 Mortierella alpina strain 4 Mortierella schmuckeri Mortierella simplex Mortierella sp.

Mucor hiemalis NFCCI-2152 Wehmer Mucor hiemalis strain 1 Mucor hiemalis strain 2

…Table 7.1 contd. Pectinase –

–

–

–

+

–

–

–

–

–

–

–

Cellulase –

–

–

+++

–

–

–

–

–

–

–

++

Esterase –

–

ND

–

–

–

–

–

–

–

ND

+

Protease –

–

ND

–

–

–

+

–

–

+

ND

–

Phosphatase ++

++

–

–

+

+

–

–

–

+++

–

++

Urease ++

++

ND

–

++

–

–

+

–

–

ND

++

Keratinase ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

References Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Singh et al. 2012

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Singh et al. 2012

Gawas-Sakhalkar and Singh 2011

98  Fungi in Polar Regions

– – – ND ++ + – – +++ – – – – –

Mucor hiemalis strain 3

Myrothecium roridum NFCCI-2153 Tode ex Steudel

Penicillium chrysogenum NFCCI-2154 Thom. Penicillium commune PG291 (AB916511) Penicillium citrinum strain 1 Penicillium citrinum strain 2 Penicillium citrinum strain 3 Penicillium citrinum strain 4 Penicillium citrinum strain 5 Penicillium citrinum strain 6 Penicillium citrinum strain 7 Penicillium citrinum strain 8 Penicillium frequentans Penicillium rugulosum +++

+++

–

+

–

+

+++

++

+

++

ND

–

–

–

+

+++

+++

+++

–

+++

+

+

+

++

ND

–

–

–

–

+

–

–

–

+

–

–

++

–

ND

ND

ND

–

+

–

–

+

–

+

+

–

+

–

ND

ND

ND

–

+++

+++

–

+++

–

+++

–

+++

+++

++

ND

–

–

+

+

–

–

–

–

–

–

–

+++

+

ND

ND

ND

++

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

++

ND

ND

ND

Table 7.1 contd. …

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Singh et al. 2015

Singh et al. 2012

Singh et al. 2012

Gawas-Sakhalkar and Singh 2011

Biotechnological Potentials of Arctic Fungi 99

Amylase – – – – – – – – +++ ND ND

Name of Filamentous Fungi

Penicillium sp.

Phialophora alba (CCP-I) J.F.H. Beyma

Phialophora fastigiata NFCCI-2155 (Lagerb. &Melin) Conant Phialophora sp. strain 1 Phialophora sp. strain 2 Phialophora sp. strain 3 Phialophora sp. strain 4 Phialophora sp. strain 5 Pithomyces chartatum

Preussia sp. NFCCI-2149 OY2307 FJ571483.1 (86%) Pyrenochaetopsis pratorum PG293 (AB916515)

…Table 7.1 contd.

Pectinase ND

ND

+

+

++

++

++

+

–

ND

–

Cellulase ND

++

++

++

+

–

+++

+++

+++

+

++

Esterase ND

ND

–

–

+

–

–

–

ND

ND

–

Protease ND

ND

–

–

–

–

–

–

ND

ND

–

Phosphatase ND

ND

++

–

+

+

+

–

–

ND

–

Urease ND

ND

+++

+++

–

–

++

+

ND

ND

–

Keratinase +++

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

References Singh et al. 2015

Singh et al. 2012

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Singh et al. 2012

Singh and Singh 2011

Gawas-Sakhalkar and Singh 2011

100  Fungi in Polar Regions

– + – – – – – – –

Xylaria sp. NFCCI-2150 FJ884195.1 (98%) NSM 1 NSM 2 NSM 3 (Zygomycete) NSM 4 NSM 5 NSM 6 (Zygomycete) NSM 7 (Zygomycete) NSM S1-J, S2-T, S3-f, S4i –

–

–

–

–

+++

–

+

–

++

–

++

+

++

–

++

++

+

+++

–

–

–

–

+

+++

–

+

ND

–

+

++

–

–

–

+

+

ND

–

–

+

–

–

–

–

–

++

–

+

+

–

+

+

–

–

+

ND

++

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

Singh et al. 2012

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Gawas-Sakhalkar and Singh 2011

Singh et al. 2012

Gawas-Sakhalkar and Singh 2011

Foot Note: NSM = Non-sporulatingmorphotype, Enzyme Activity (+++ = Good activity, ++ = Moderate activity, + = Low activity, – = No activity, ND = not detected)

–

Trichosporiella cerebriformis

Biotechnological Potentials of Arctic Fungi 101

ND

ND ND

+ + +

Cryptococcus adeliensis MLB-18 (JX192655)

Cryptococcus albidosimilis MLB-19 (JX192656)

Cryptococcus albidosimilis MLB-24 (JX192661)

ND

ND – + +

Mrakia blollopis PG256 (AB916516) Mrakia sp. CCP-III-WY Thelebolus sp. Cry-YB 240 Thelebolus sp. Cry-YB 241

ND

ND

+

ND

ND

ND

ND

+

Cellulase +

–

–

ND

ND

++

++

++

–

–

++

++

++

+

+

+

+

+

Esterase –

+

–

ND

ND

+

++

+

–

+

++

++

++

+

++

++

++

++

Protease +

++

–

ND

ND

++

++

+

–

–

++

++

++

++

++

++

++

ND

ND

ND

+

ND

ND

ND

ND

ND

–

–

ND

ND

ND

ND

ND

ND

ND

++

Urease +++

+

–

ND

ND

++

++

+

–

–

++

+

++

++

++

+

++

ND

Keratinase ND

ND

ND

+

+++

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

Singh et al. 2016

Singh et al. 2016

Singh and Singh 2011

Singh et al. 2015

Singh et al. 2015

Singh et al. 2013

Singh et al. 2013

Singh et al. 2016

Singh and Singh 2011

Singh and Singh 2011

Singh et al. 2013

Singh et al. 2013

Singh et al. 2013

Singh et al. 2013

Singh et al. 2013

Singh et al. 2013

Singh et al. 2013

Singh et al. 2013

References

Foot Note: NSM = Non-sporulatingmorphotype, Enzyme Activity (+++ = Good activity, ++ = Moderate activity, + = Low activity, – = No activity, ND = Not detected)

– ND

–

Thelebolus microsporus PG278 (AB916508)

ND

++

Rhodotorula sp. Cry-FB3

Rhodotorula mucilaginosa MLB-27 (JX192664)

Rhodosporidium lusitaniae MLB-20 (JX192657)

–

+

Rhodotorula sp. CCP-II

–

+ –

Cryptococcus albidosimilis MLB-25 (JX192662)

Cryptococcus gilvescens (CCP-III-OY) Chernov & Babeva

ND

ND

ND

+ +

ND

Cryptococcus saitoi MLB-23 (JX192660)

Cryptococcus saitoi MLB-22 (JX192659)

Pectinase

Cryptococcus saitoi MLB-26 (JX192663)

+ +

Cryptococcus adeliensis MLB-21 (JX192658)

Amylase

Name of Yeasts

Phosphatase

Table 7.2.  Biotechnological potentials of Arctic yeasts.

102  Fungi in Polar Regions

Biotechnological Potentials of Arctic Fungi 103

i) Enzyme Potentials of Arctic fungi Cold-active enzymes, such as amylases, catalase, cellulases, invertase, lactase, lipases (Fig. 7.1e) pectinases, and proteases produced by Arctic fungal strains find potential applications in the food, medicine, and detergent industries. Studies on psychrophilic yeasts from glacier habitats have been a gap area of research for the opportunities of biotechnology in the Arctic Region (Cavicchioli et al. 2002, Feller and Gerday 2003, Buzzini et al. 2012). Enzyme catalase (Fiedurek et al. 2003) and invertase (Skowronek et al. 2003) have been recorded from Spitsbergen fungi. Recently, systematic studies on the cold-active enzymes from the Arctic regions have been conducted (Gawas-Sakhalkar and Singh 2011, Singh and Singh 2011, Singh et al. 2011, Gawas-Sakhalkar et al. 2012, Singh et al. 2012a, Singh et al. 2013, Singh et al. 2016). The amylase enzymes can be used as detergent additive, in textile processing, and in the food industry. Degradation of cellulose and urea by these polar isolates at low temperatures demonstrates their immense biotechnological importance. Presence of heterotrophic organisms such as filamentous fungi and yeasts in the Arctic glacier cryoconites find paramount importance as they drive the process of degradation of organic macromolecules through the secretion of extracellular hydrolytic cold-adapted enzymes and thus assist in nutrient cycling in sub-glacial environments.

ii) Antioxidant Potentials Antioxidants play important roles in scavenging obnoxious free radicals produced during any physiological stress in the Arctic environment. Singh et al. (2012b) investigated the chemical composition, antioxidant potential (FRS, ILP TEAC activities), and antimicrobial activity of the Arctic wild mushroom Lycoperdon molle (Fig. 7.1b). Arctic mushroom species could be used in food and pharmaceutical industries as natural antioxidants. L. molle produce a number of secondary metabolites, several of which were phenolics and polysaccharides. ESI-MS analysis of the L. molle extract also showed the presence of compounds—namely phosphatidylglycerol, phosphoethanolamine, phosphoserine, phosphoionositol, and lysophosphatidylcholine. The chemical reactions that generally take place in the Arctic, viz, ROS-induced peroxidation and de-esterification of glycerolipids, caused by dehydration and rehydration cycles, are responsible for cell membrane disruptions. Compounds such as polyols, might act as osmoprotectants, and quench the ROS, thereby protecting the cellular structures in the Arctic’s harsh environmental conditions. Based on findings (Singh et al. 2012b), it can be concluded that the mushrooms available in the Arctic region can be a potential source of natural antioxidants applicable to the pharmaceuticals and therapeutics. Therefore Arctic mushroom species must essentially be explored systematically and cultivated in laboratory conditions for mass production.

104  Fungi in Polar Regions

iii) Antifreeze Potentials Antifreeze proteins (AFPs) play an important role in the survival of polar organisms, and in health care applications. So far, there are only a few publications on AFPs of Arctic fungi. Park et al. (2012) characterized the ice-binding protein from Arctic yeast Leucosporidium sp. AY30, and subsequently, Koh et al. (2015) studied the effect of the antifreeze protein of Leucosporidium sp. AY30 on cryopreservation. An AFP from a yeast strain Leucosporidium sp., isolated from an ice core of an Arctic pond in Svalbard was reported by Lee et al. (2010). Recently, AFPs have also been screened from icecores and cryoconites of Svalbard, Arctic (Singh et al. 2013a, Singh et al. 2014). Only few Cryoconites yeast have shown positive activity (Fig. 7.1f), whereas all the isolates of ice core yeasts showed negative results for extracellular and intracellular antifreeze proteins (AFPs). The AFPs probably also play an important role in the survival of other non-AFP-producing organisms coexisting in the cryoconite ecosystem. AFPs potentially may find applications in the health (cryopreservation of blood and organs) sector, agriculture, and industry.

iv) PUFA Potentials Polyunsaturated fatty acids (PUFAs) at colder temperatures regulate the membrane fluidity of organisms, thereby improving the ability of the organisms to survive at low temperatures (Robinson 2001). Enzymes, such as desaturase present in yeasts, through repetitive desaturation, are responsible for the synthesis of PUFAs. At low temperatures, the membranes of psychrophilic yeasts accumulate high concentration of unsaturated fatty acids (Pathan et al. 2010). The genera Mrakia, Leucosporidium, and Rhodotorula have indicated that at low and sub-zero temperatures, linolenic and linoleic acids predominated in fatty acid composition. An increase in temperature led to the increase in oleic acid percentage (Singh et al. 2013a). Singh et al. (2013a) have also compared percent concentrations of fatty acids in psychrophiles grown at different temperatures, and observed that while the PUFA concentration increased with decrease in temperature, the concentration of monounsaturated fatty acids (MUFA) decreased. In the fatty acid composition of psychrophilic yeasts from the puddles in the vicinity of Midre Lovénbreen glacier, Pathan et al. (2010) did not measure the percent concentration of linolenic acid (C18:3); the concentration of linoleic acid (C18:2) alone was higher than the MUFA or saturated fatty acid values obtained during the study. Singh et al. 2013 observed that linoleic acid (C18:2) values alone are higher than MUFA or saturated fatty acids (SFA) at low temperature. The analysis on the fatty acid profile further reveals that decrease in temperature increases the concentration of total unsaturated fatty acids. The major fatty acids found were linoleic acid (C18:2n6c) and linolenic acid (C18:3n3), contributing to the prevalence of PUFA. Oleic acid (C18:1n9c) was the most abundant MUFA in all the Arctic yeasts, while palmitic acid (C16:0) was the major SFA. These fatty acids possibly help the fungal strains to survive in glacial cold environment. PUFA have immence potential for healthcare applications.

Biotechnological Potentials of Arctic Fungi 105

Discussion and Conclusion The viable cell counts showed that the Arctic glacier cryoconites at higher latitude support thriving of more yeast species than filamentous fungi. At the lower altitude, filamentous fungi were more abundant than the yeast. This could be likely because the temperature at higher altitudes is lower than the downstream segment of the glacier, and yeast rather than filamentous fungi survive at lower temperatures. This is in agreement with Gostinčar et al. (2006) and Butinar et al. (2007), who described the evolution of yeast and yeast-like fungal populations in the basal Arctic ice. In the Svalbard ice cores, the number of viable yeast cells decreased with the increase in the depth of ice core (Singh et al. 2013). This indicates that the yeast cells are reasonably equipped to thrive in the porous surface layer of ice compared to the deeper layers. Butinar et al. (2007) however, observed that the abundance of yeast is less in the surface layer as compared to the basal layer. Pathan et al. (2010) studied yeast from the puddles near the Midre Lovén breen glacier, which showed presence of unsaturated branched atty acid as recorded in the present study. Fungi are the principle recyclers of nutrients in the Arctic, and are essential to the survival of the entire ecosystem (Ludley and Robinson 2008, Singh and Singh 2011, Edwards et al. 2013). Baseline data on fungal community composition and their potentials is an absolute prerequisite for being able to estimate the effects that climate change will have on the Arctic ecosystem. The rapid loss of glacier thickness represents a serious threat to fungal biota inhabiting Arctic extreme ecological niches, in addition to global warming. Due to the essential effect of fungi on the ecosystem as a whole, climate change influence on fungal diversity of such niches are extremely important for a better understanding of different habitat-microbial interaction in the climate system. Whole genome sequencing of a few Arctic microbes have revealed the presence of several genes encoding industrially important enzymes, biosynthesis of plant growth promoting hormones, and ability for resistance to heavy metals and toxic compounds (Singh et al. 2015, Singh et al. 2017, Kapse et al. 2017). Analysis of annotated genome sequence revealed immense biotechnological potentials of Arctic microbes. Thereore, there is need for extensive studies on genomics and proteomics of Arctic fungi for its immense application in the health sector, agriculture, and industry.

Acknowledgments Authors are thankful to Parvatibai Chowgule College of Arts & Science for facilities, and Science and Engineering Research Board (SERB) for financial support (PDF/2016/003707).

References Aarnæs, J-O. 2002. Catalogue over macro- and micromycetes recorded for Norway and Svalbard. Synopsis Fungorum 16. Oslo: Fungiflora.

106  Fungi in Polar Regions Bab’eva, E.N. and T.P. Sizova. 1983. Micromycetes in soils of arctotundra ecosystem. Soil Sci. (Pochvovedenie) 10: 98–101. Barnett, H.L. 1960. Illustrated genera of imperfect fungi, 2nd edn. Burgess Publishing Company, USA. Barron, G.L. 1977. The genera of hyphomycetes from soil. Robert E. Krieger Pub. Comp, INC. Huntington, New York. Bergero, R., M. Girlanda, G.C. Varese, D. Intili and A.M. Luppi. 1999. Psychrooligotrophic fungi from Arctic soils of Franz Joseph Land. Polar Biol. 21(6): 361–368. Birgisson, H., O. Delgado, L. Garcı´a Arroyo, R. Hatti-Kaul and B. Mattiasson. 2003. Cold-adapted yeasts as producers of cold-active polygalacturonases. Extremophiles 7: 185–193. Borgen, T., S.A. Elborne and H. Knudsen. 2005. A checklist of the greenland basidiomycota. Meddelser om Grönland, Bioscience 56: 37–59. Bunnell, F., O.K. Miller, P.W. Flanagan and R.E. Benoit. 1980. Tundra microflora: composition, biomass and environmental relations. pp. 255–290. In: Brown, et al. (eds). An Arctic Ecosystem: The Coastal Tundra of Northern Alaska. Dowden, Hutchinson and Ross, Inc., Straudsburg, Pa. Butinar, L., I. Spencer-Martins and N. Gunde-Cimerman. 2007. Yeasts in high Arctic glaciers: The discovery of a new habitat for eukaryotic microorganisms. Antonie Leeuwenhoek 91: 277–289. Buzzini, P., E. Branda, M. Goretti and B. Turchetti. 2012. Psychrophilic yeasts from worldwide glacial habitats: diversity, adaptation strategies and biotechnological potential, FEMS MicrobiolEcol, http://dx.doi.org/ 10.1111/j.1574-6941.2012.01348.x. Callaghan, T.V. 2005. Arctic tundra and polar desert ecosystems In: Arctic climate impact assessment -scientific report. Cambridge University Press, Cambridge, pp. 243–352. Callaghan, T.V., L.O. Björn, Y. Chernov, T. Chapin, T.R. Christensen, B. Huntley and R.A. Ims et al. 2004. Biodiversity, distributions and adaptations of Arctic species in the context of environmental change. Ambio 33: 404–417. Carmichael, J.W., W. BryceKendrick, I.L. Conners and L. Sigler. 1980. Genera of hyphomycetes. The University of Alberta Press, Canada. Cavicchioli, R., K.S. Siddiqui, D. Andrews and K.R. Sowers. 2002. Low temperature extremophiles and their applications. CurrOpinBiotechnol. 13: 253–261. Chernov, Y.I. 2002. Arctic biota: Taxonomic diversity. Zoologicheski Zhurnal 81: 1411–1431. Dmitriev, V.V., D.A. Gilichinsky, R.N. Faizutdinova, I.N. Shershunov, W.I. Golubev and V.I. Duda. 1997. Occurrence of viable yeasts in 3-million-year-old permafrost in Siberia. Mikrobiologiya 66: 655–660. Ellis, M.B. 1971. Dematiaceous hyphomycetes. CMI, Kew, England. Ellis, M.B. 1976. More dematiaceous hyphomycetes. CMI, Kew, England. Elvebakk, A., H.B. Gjaerum and S. Sivertsen. 1996. Myxomycota, oomycota, chytridiomycota, zygomycota, ascomycota, deuteromycota, basidiomycota: uredinales and ustilaginales. In: A. Elvebakk and P. Prestrud (eds.). A Catalogue of Svalbard Plants, Fungi, Algae and Cyanobacteria. Part 4. Fungi II. 207–259. Edwards, A., B. Douglas, A.M. Anesio, S.M. Rassner, T.D.L. Irvine-Fynn, B. Sattler and G.W. Griffith. 2013. A distinctive fungal community inhabiting cryoconite holes on glaciers in Svalbard. Fungal Ecol. 6(2): 168–176. Etienne, S. 2002. The role of biological weathering in periglacial areas: A study of weathering rinds in south Iceland. Geomorphology 47: 75–86. Feller, G. and C. Gerday. 2003. Psychrophilic enzymes: Hot topics in cold adaptation. Nat. Rev. Microbiol. 1: 200–208. Fiedurek, J., A. Gromada, A. Słomka, T. Korniłowicz-Kowalska, E. Kurek and J. Melke. 2003. Catalase activity in Arctic microfungi grown at different temperatures. ActaBiol. Hung. 54: 105–112. Gawas-Sakhalkar, P. and S.M. Singh. 2011. Fungal community associated with terrestrial Arctic moss, Tetraplodon minioides. Curr. Sci. 100: 1701–1705. Gawas-Sakhalkar, P., S.M. Singh, S. Naik and R. Ravindra. 2012. High temperature optima phosphatases from psychrotrophic Arctic fungus, Penicillium citrinum. Polar Res. 31: 11105, http://dx.doi. org/10.3402/polar.v31i0.11105.

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108  Fungi in Polar Regions Mindl, B., A.M. Anesio, K. Meirer, A.J. Hodson, J. Laybourn-Parry, R. Sommaruga and B. Sattler. 2007. Factors influencing bacterial dynamics along a transect from supralgacial runoff to proglacial lakes of a high Arctic glacier. FEMS Microbiol. Ecol. 59: 307–317. Michelsen, A., C. Quarmby, D. Sleep and S. Jonasso. 1998. Vascular plant 15N abundance in heath and forest tundra ecosystems is closely correlated with presence and type of mycorrhizal fungi in roots. Oecologia 115: 406–418. Miller, O.K. and D.E. Farr. 1975. Index of the common fungi of North America (synonymy and common names). Bibliotheca Mycologia 44: 206–230. Ozerskaya, S., G. Kochkina, N. Ivanushkina and D.A. Gilichinsky. 2009. Fungi in permafrost. In: Margesin, R. (ed.). Permafrost Soils. Soil Biol., Vol. 16. Springer 85–95. Ozerskaya, S.M., N.E. Ivanushkina, G.A. Kochkina, R.N. Fattakhova and D.A. Gilichinsky. 2004. Mycelial fungi in cryopegs. Int. J. Astrobiol. 3: 327–331. Pang, K.L., M.W. Chiang and L.L.P. Vrijmoed. 2008. Havisporalongyearbyenensis gen. et sp. nov.: An arctic marine fungus from Svalbard, Norway. Mycologia 100: 291–295. Pang, K.L., M.W. Chiang and L.L.P. Vrijmoed. 2009. Remisporaspitsbergenensis sp. nov., a marine lignicolousascomycete from Svalbard, Norway. Mycologia 101: 531–534. Panikov, N.S. and M.V. Sizova. 2007. Growth kinetics of microorganisms isolated from Alaskan soil and permafrost in solid media frozen down to −35°C. FEMS Microbiol. Ecol. 59: 500–512. Park, K.S., H.Do, J.H. Lee, S.I. Park, Ej. Kim, S.J. Kim, S.H. Kang and H.J. Kim. 2012. Characterization of the ice-binding protein from Arctic yeast Leucosporidium sp. AY30. Cryobiology 64: 286–96. doi: 10.1016/j.cryobiol.2012.02.014. Pathan, A.A.K., B. Bhadra, Z. Begum and S. Shivaji. 2010. Diversity of yeasts from puddles in the vicinity of Midre Lovenbreen glacier, Arctic and bioprospecting for enzymes and fatty acids. Curr. Microbiol. 60: 307–314. Robinson, C.H. 2001. Cold adaptation in Arctic and Antarctic fungi. New Phytol. 151: 341–353. Singh, P. and S.M. Singh. 2011. Characterization of yeast and filamentous fungi isolated from cryoconite holes of Svalbard, Arctic. Polar Biol. 35: 575–583. Singh, S.M., L. Yadav, S.K. Singh, P. Singh, P.N. Singh and R. Ravindra. 2011. Phosphate solubilizing ability of Arctic Aspergillus niger strains. Polar Res. 30: 7283, doi:10.3402/polar.v30i0.7283. Singh, S.M., S.K. Singh, L. Yadav, P.N. Singh and R. Ravindra. 2012a. Filamentous soil fungi from Ny-Ålesund, Spitsbergen and screening for extracellular enzymes. Arctic 65: 35–55. Singh, P., A. Singh, L.M. D`Souza, U. Roy and S.M. Singh. 2012b. Chemical constituents and antioxidant activity of arctic mushroom Lycoperdon molle Pers. Polar Res. 31: 17329, doi:10.3402/polar. v31i0.17329. Singh, P., M. Tsuji, S.M. Singh, U. Roy and T. Hoshino. 2013a. Taxonomic characterization, adaptation strategies and biotechnological potential of cryophilic yeasts from ice cores of MidreLovénbreen glacier, Svalbard, Arctic. Cryobiology 66: 167–175. Singh, S.M., L.S. Yadav, P.N. Singh, R. Hepat, R. Sharma and S.K. Singh. 2013b. Arthrinium rasikravindrii sp. nov. from Svalbard, Norway. Mycotaxon 122(1): 449–460. Singh, P., S.M. Singh, M. Tsuj, G.S. Prasad and  T. Hoshino. 2014. Rhodotorula svalbardensis sp. nov., a novel yeast species isolated from cryoconite holes of Ny-Alesund, Arctic. Cryobiology 68(1): 122–8. Singh, S.M., M. Tsuji, P. Gawas-Sakhalker, M.J.J.E. Loonen and T. Hoshino. 2016. Bird feather fungi from Svalbard Arctic. Polar Biol. 39(3): 523–532. Singh, P., M. Tsuji and U. Roy. 2016. Characterisation of yeast and filamentous fungi from Brøggerbreen glaciers, Svalbard. Polar Rec. 52(4): 442–449. Singh, P.,  N. Kapse, P. Arora, S.M. Singh and P.K. Dhakephalkar. 2015. Draft genome of Cryobacterium sp. MLB-32, an obligate psychrophile from glacier cryoconite holes of high Arctic. Mar Genomics 21: 25–26. doi:10.1016/j.margen.2015.01.006. Singh, P.,  N. Kapse, U. Roy, S.M. Singh and P.K. Dhakephalkar. 2017. Draft genome sequence of permafrost bacterium Nesterenkonia sp. strain PF2B19, revealing a cold adaptation strategy and diverse biotechnological potential. Genome Announc 5(15). pii: e00133–17. Skidmore, M.L., J.M. Foght and M.J. Sharp. 2000. Microbial life beneath a high Arctic glacier. Appl. Environ. Microbiol. 66: 3214–3220.

Biotechnological Potentials of Arctic Fungi 109 Skidmore, M.L., S.P. Anderson, M.J. Sharp, J.M. Foght and B.D. Lanoil. 2005. Comparison of microbial community composition of two subglacial environments reveals a possible role for microbes in chemical weathering processes. Appl. Environ. Microbiol. 71: 6986–6997. Skowronek, M., J. Kuszewska, J. Fiedurek and A. Gromada. 2003. Invertase activity of psychrotrophic fungi. Ann. UnivMariae Curie-Skłodowska Lublin-Polonia 58: 1–9. Sonjak, S., J.C. Frisvad and N. Gunde-Cimerman. 2006. Penicillium mycobiota in arctic subglacial ice. Microb. Ecol. 52(2): 207–16. Sonjak, S., V. Ursic, J.C. Frisvad and N. Gunde-Cimerman. 2007. Penicillium svalbardense, a new species from Arctic glacial ice. Antonie Leeuwenhoek  92(1): 43–51. Tamotsu, H., M. Tojo, G. Okada, H. Kanda, S. Ohgiya and K. Ishizaki. 1999. A filamentous fungus, Pythiumultimum Throw var. ultimum, isolated from moribund moss colonies from Svalbard, northern island of Norway. Polar Biosci. 12: 68–75. Tsuji, M., J. Uetake and Y. Tanabe. 2016. Changes in the fungal community of Austre Brøgger breen deglaciation area, Ny-Ålesund, Svalbard, high arctic. Mycoscience 57: 448e451. https://doi. org/10.1016/j.myc.2016.07.006. Tsuji, M., Y. Tanabe, W.F. Vincent and M. Uchida. 2018a. Gelidatrema psychrophila sp. nov., a novel yeast species isolated from an ice island in the Canadian High Arctic. Mycoscience 59: 67e70. Tsuji, M., Y. Yukiko Tanabe, F. Warwick, W.F. Vincent and M. Uchida. 2018b. Mrakia arctica sp. nov., a new psychrophilic yeast isolated from an ice island in the Canadian High Arctic. Mycoscience 59: 54e58. Uetake, J., Y. Yoshimura, N. Nagatsuka and H. Kanda. 2012. Isolation of oligotrophic yeasts from supra glacial environments of different altitude on the Gulkana Glacier (Alaska). FEMS Microbiol. Ecol. 82: 279–86. Vishniac, H.S. 2002. Cryptococcus tephrensis, sp. nov., and Cryptococcus heimaeyensis, sp. nov., new anamorphic basidiomycetous yeast species from Iceland. Can J. Microbiol. 48: 463–467. Vishniac, H.S. and M. Takashima. 2010. Rhodotorulaarcica sp. nov., a basidiomycetous yeast from Arctic soil. Int. J. SystEvolMicrobiol. 60: 1215–1218. Wallenstein, M.D., S. McMahon and J. Schimel. 2007. Bacterial and fungal community structure in Arctic tundra tussock and shrub soils. FEMS Microbiol. Ecol. 59: 428e435. Yamazaki, Y., M. Tojo, T. Hoshino, K. Kida, T. Sakamoto, H. Ihara, I. Yumoto, A.M. Tronsmo and H. Kanda. 2011. Characterization of Trichoderma polysporum from Spitsbergen, Svalbard archipelago, Norway, with species identity, pathogenicity to moss, and polygalacturonase activity. Fungal Ecol. 4: 1 5e2 1. Yarrow, D. 1998. Methods for the isolation, maintenance and identification of yeasts. pp. 77–100. In: C.P. Kurtzman and J.W. Fell (eds.). The Yeasts. A Taxonomic Study, Elsevier, Amsterdam. Zabawski, J. 1976. Soil fungi isolated from peat bogs in Hornsund region (West Spitsbergen). In: New recognitions of peatland and peat, Vol 2. Proceedings of the 5th International Peat Congress, 21– 25 September 1976, Poznan, Poland. Czasopism Techn. Wydawn. 158–170. Zabawski, J. 1982. Soil microfungi of peats in the Hornsundregion (West Spitsbergen). Acta Univ Wratisl. 525: 269e279. Zhang, T., N.F. Wang, Y.Q. Zhang, H.Y. Liu and L.Y. Yu. 2015a. Diversity and distribution of fungal communities in the marine sediments of Kongsfjorden, Svalbard (High Arctic).  Sci Rep. 23;5: 14524. doi:10.1038/srep14524. Zhang, T., X.L. Wei, Y.Q. Zhang, H.Y. Liu and L.Y. Yu. 2015b. Diversity and distribution of lichen-associated fungi in the Ny-Ålesund Region (Svalbard, High Arctic) as revealed by 454 pyrosequencing. Sci. Rep. doi:10.1038/srep14850. Zhang, T., N.F. Wang, Y.Q. Zhang, H.Y. Liu and L.Y. Yu. 2016a. Diversity and distribution of aquatic fungal communities in the Ny-Ålesund region, Svalbard (High Arctic). Microb. Ecol. 71(3): 543–554. Zhang, T., N.F. Wang, HY. Liu, Y.Q. Zhang and L.Y. Yu. 2016b. Soil pH is a key determinant of soil fungal composition in the Ny-Ålesund region, Svalbard (High Arctic). Front Microbiol. doi:10.3389/fmicb.2016.00227.

8 Dairy Wastewater Treatment Under Low-Temperature Condition by an Antarctic Basidiomycetous Yeast Masaharu Tsuji,1,* Sakae Kudoh1,2 and Tamotsu Hoshino3

Introduction Milk fat curdle in sewage is an intractable material for active sludge treatment under cold temperature conditions. Since dairy wastewater is discharged either untreated or only partially treated, the drainage from dairy farms and milk factories, as well as the wastewater from the cleaning milk transport pipes and tanks, may pollute rivers and groundwater. This effluent may also contain detergents, bactericides, mucus, lactose, and milk fat (Healy et al. 2007). Systems have been developed for treating dairy wastewater. For example, under low-temperature conditions, wastewater may be treated by passage through bio-filters and a reed bed system (Shah et al. 2002, Kato et al. 2010, Biddlestone et al. 1991). However, these systems are not used widely because of their high running costs to operate, and large space requirements. Therefore, an activated sludge system (AS) is currently widely used for the industrial

National Institute of Polar Research (NIPR), 10-3 Midori-cho, Tachikawa, Tokyo 190-8518, Japan. Department of Polar Science, SOKENDAI (The Graduate University for Advanced Studies), 10-3 Midori-cho, Tachikawa, Tokyo 190-8518, Japan. Email: [email protected] 3 Bio-production Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-higashi, Toyohira-ku, Sapporo, Hokkaido 062-8517, Japan. Email: [email protected] * Corresponding author: [email protected] 1 2

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treatment of dairy wastewater (Ying et al. 2010), which is easier to maintain and has lower running costs. However, this system does not work efficiently in winter due to the adverse effects of low temperatures on microbial function. The use of microorganisms isolated from polar regions to remove nitrogen and phosphorus compounds from wastewater under low-temperature conditions has been reported by Chevalier et al. (2000) and Hirayama-Katayama et al. (2003), but it has not yet been applied for milk fat. Cryophilic yeast (Hoshino and Matsumoto 2012) Mrakia spp. has been found in the Arctic, Siberia, Alaska, the Alps, the Apennines, Patagonia, and Antarctica (Singh and Singh 2012, Tsuji et al. 2013c, Fell et al. 1969, Thomas-Hall et al. 2010, Margesin and Fell 2008). Di Menna (1966) reported that the genus Mrakia accounts for about 24% of the culturable yeast in Antarctic soil. Moreover, we reported that about 35% of culturable fungi isolated from lake sediment and soil from East Antarctica is Mrakia spp. (Tsuji et al. 2013a). These reports suggest that Mrakia spp. is the dominant yeast in Antarctica and is the fungi most adaptive to the Antarctic and to other cold environments.

Screening for Fungi Having Milk Fat Decomposing Activity In an attempt to identify an appropriate agent for bio-remediation of milk fat curdle, fungi were collected from Skarvsnes ice-free area, East Antarctica. A total of 305 strains were isolated and tested for their ability to decompose milk fat on fresh cream agar at 10°C. The results revealed that only Antarctic basidiomycetous yeast of the genus Mrakia have the ability to degrade of milk fat (Shimohara et al. 2012). Based on this result, we isolated 27 new Mrakia strains from Skarvsnes ice free area and assessed their ability to decompose milk fat at temperatures ranging from 4°C to 15°C, over a 3-week period. All 27 strains demonstrated milk fat degrading ability at 4°C and 10°C, but they had varying degrees of degrading effectiveness and temperature sensitivities. Overall, results of the assessment identified Mrakia blollopis strain SK-4 as having the highest ability to degrade milk fat at both 4°C and 10°C (Tsuji et al. 2015). There are no reports regarding the ability of fungi to degrade milk fat, other than our reports. However, there have been several reports about the activities of extracellular enzyme of cold-adapted yeast Mrakia isolated during bioprospecting expeditions. We reported that Mrakia blollopis expresses extracellular enzymes, such as cellulase, β-glucosidase, catalase, amylase, and lipase under coldtemperature conditions (Tsuji et al. 2013b). De Garcia et al. (2007) reported that yeasts isolated from Patagonia, Argentina show higher lipolytic activity at 4°C than at 20°C. Moreover, De Garcia et al. (2012) reported that Mrakia spp. isolated from the Patagonian Andes, Argentina produce active esterases at 5°C. Turchetti et al. (2008) demonstrated that about 77% of Mrakia spp. isolated from alpine glaciers exhibit lipolytic activity at 4°C. Pathan et al. (2010) reported that Mrakia sp. YSAR-9, which has high sequence homology (> 99%) with M. blollopis SK-4, shows higher lipolytic reactivity at 22ºC than at 8ºC, using Tween 20 as a substrate.

112  Fungi in Polar Regions In contrast, when SK-4 is inoculated onto an agar plate containing Tween 20 as a substrate, higher reactivity is observed at 4ºC than at 10ºC or 20ºC (unpublished data). Over the past 5 years, we have focused on identifying extracellular enzymes, especially enzymes capable of decomposing milk fat, from cold-adapted yeast isolated from polar regions, such as Antarctica, and the Arctic. Over 300 strains isolated from these regions have been tested for enzymatic activity. When inoculated on agar plates containing Tween 20 or Tween 80 as a substrate, some of these strains produced a positive reaction, but no yeast strains have been identified that show a strong degradation ability of milk fat under low-temperature conditions, except for strains of the genus Mrakia. To the best of our knowledge, to date, Mrakia is the only genus known that exhibits this high ability to degrade milk fat under lowtemperature conditions. Milk fat is composed of fatty acids of various chain lengths (Mansson 2008). A lipase of M. blollopis SK-4 shows a high reactivity to substrates with various chain lengths (Tsuji et al. 2013a). Characterization of the SK-4 lipase suggests that other Mrakia spp. also have enzymes that can decompose milk fat containing fatty acids of various chain lengths. Currently, it is unknown why the ability to degrade milk fat differs by strain and temperature. Further experiments are required to elucidate the precise details and mechanisms involved in the milk-fat degradation ability of genus Mrakia, including genome-based analyses, metabolomics and microarray analyses, during the decomposition of milk fat.

Effects of Nitrogen Concentration and Culturing Temperature on Morphology of Mrakia blollopis SK-4 To determine the effect of nutrient concentration on M. blollopis SK-4 colony morphology, SK-4 has been evaluated on potato dextrose agar (PDA) and fresh cream agar at various culturing temperatures. When M. blollopis SK-4 is incubated on PDA for 4 weeks at 4ºC, its colony morphology closely resembles a yeast form. However, when this fungus is cultured on PDA at 10 or 15°C, the ratio of mycelial that forms in the colony relative to the yeast morphology gradually increases, corresponding to the increase in culturing temperature (Fig. 8.1). When the yeast was inoculated on 1/5 × concentration PDA, colony morphology is more myceliallike than yeast-like, and the extent of the mycelial form increases relative to the increase in culturing temperature. (Fig. 8.2). In contrast, the yeast morphology of SK-4 colonies is maintained on the eutrophic medium (2 × concentration PDA) regardless of the culturing temperature (Fig. 8.3). Based on the results of culturing on eutrophic and oligotrophic media, it is believed that only the nutrients directly under the colony are utilized by the growing fungi. Since SK-4 fails to retain the yeast morphology when utilizing only nutrients under each colony on an oligotrophic medium, SK-4 may undergo a change in morphology in order to obtain nutrients beyond the colony boundary through germination and the extension of mycelia. The optimal growth temperature of SK-4 is 15°C. Therefore, colony morphology of SK-4 on PDA is thought to be related to the rate of growth and nutrient concentration under the colony. Moreover, we evaluated cell morphology

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Fig. 8.1.  Colony morphology rates of Mrakia blollopis SK-4 colony on PDA. Ratios of yeast colony morphology and mycelial colony morphology of M. blollopis SK-4 at 4 weeks after inoculation.

Fig. 8.2.  Colony morphology rates of Mrakia blollopis SK-4 for 4 weeks on 1/5 × concentration PDA at different temperatures. Ratios of yeast colony morphology and mycelial colony morphology of M. blollopis SK-4 at 4 weeks after inoculation.

of SK-4 during secretion of lipase using fluorescence in situ hybridization (FISH) and optical microscopy. The cell morphology of SK-4 during lipase secretion is exclusively the yeast form (Tsuji et al. 2014). These results are in accordance with the consideration that SK-4 adopts the yeast form in aquatic environments, and that this form may secrete more lipase than the mycelial form. M. blollopis SK-4, as detailed above, is a unique microorganism with a set of characteristics making it an ideal candidate for use in the bio-remediation of milk fat under conditions of low temperature. Therefore, SK-4 was chosen for use as the inoculum for the model dairy wastewater treatment study.

114  Fungi in Polar Regions

Fig. 8.3.  Colony morphology of Mrakia blollopis SK-4 grown for 4 weeks on 2 × concentration PDA at different temperatures (a) 4°C (b) at 10°C (c) at 15°C. Solid lines indicate yeast form and dashed lines indicate mycelial form. Bars: a–c 1 cm.

Model Dairy Wastewater Treatment by Batch Process Physiological Characterizations of Mrakia blollopis SK-4 The maximum growth temperature of Mrakia blollopis SK-4 was 22ºC. Maximum growth temperatures of other related species are less than 20ºC. M. blollopis SK-4 differed from the other strains in substrate utilization as well. The strain SK-4 is also able to thrive well on media with lactose, D-arabinose, and inositol as carbon sources. Unlike other strains, this strain also grows on vitamin-free medium. A comparison of fermentabilities shows that M. blollopis SK-4 is able to ferment typical sugars, such as glucose, sucrose, galactose, maltose, lactose, raffinose, trehalose, and melibiose, while other related species are unable to strongly ferment such a variety of sugars (Table 8.1). These results suggested that strain SK-4 is a potential candidate for a biological agent to decompose under low temperature conditions the various sugars contained in milk parlor wastewater.

Assessment of Milk Fat Decomposition in Model Wastewater In an experimental environment, AS was cultivated at room temperature with aeration using cow’s milk as the substrate. After one month, the sludge was divided into two portions. One portion of AS was mixed with M. blollopis SK-4 (1.4 g/L, dry weight), and the other portion was used as a control. Separated AS was prepared with mixed liquor suspended solids (MLSS; 3000 mg/L) and cow’s milk, and incubated at 10ºC with aeration. One week later, the prepared AS was added to cow’s milk, and the biochemical oxygen demand (BOD5) of the wastetreated water was measured after 24 hours. The BOD5 assay was performed using a coulometer (Ohkura Electric, Saitama, Japan). Model wastewater containing cow’s milk as a substrate of milk fat was prepared to equivalent levels of BOD sludge loading that is typically seen in standard wastewater treatment (0.35 kg-BOD/kg-MLSS.d). The AS containing M. blollopis SK-4 had a BOD removal rate of 83.1%, higher than that of the control (63.8%).

Wastewater Treatment by Antarctic Basidiomycetous Yeast 115 Table 8.1.  Comparison of physiological characteristics of Mrakia blollopis SK-4 and other Mrakia species. Characteristic

M. blollopis SK-4

M. blollopis CBS8921T

M. psychrophhila AS2.1971T

M. frigida CBS5270T

Maximum growth temperature

22ºC

20ºC

18ºC

17ºC

Assimilation of Lactose

+

W

+

V

Inositol

+

w/+

+

V

D-arabinose

+

w/–

+

V

Ethanol

w/–

+

+

+

Growth on 50% glucose

w/–

–

+

–

Growth on vitamin-free medium

+

w

+

–

+

–

–

W

Fermentation of Galactose Lactose

+

–

–

–

Raffinose

+

–

–

W

Maltose

+

–

–

W

Main physiology test results for characteristics of M. blollopis SK-4 and related species are shown. Physiological data were taken from Fell et al. (1969), Xin and Zhou (2007), Thomas-Hall et al. (2010), and the current study. +, positive; w, weak; –, negative; v, variable; nd, no data.

When the BOD volume load was adjusted to 1.5-times that of standard wastewater treatment (0.52 kg-BOD/Kg-MLSS.d), the BOD removal rate by AS containing M. blollopis SK-4 was 80.1%, again higher than that of the control (65.2%). Regardless of the BOD volume load, the AS containing M. blollopis SK-4 had a 1.25-fold higher BOD removal rate than that of the control (Table 8.2). Table 8.2.  Performance of activated-sludge model treatment system of milking-parlor wastewater. Components* BOD Loading Rate BOD Sludge Loading Load of Finish of Removal (kg-BOD/m3•d) (kg-BOD/kg-MLS•d) BOD (mg/L) BOD (mg/L) Rate (%) AS containing M. blollopis SK-4

1.0

0.35

1210

204

83.1

AS (control)

1.0

0.35

1260

456

63.8

AS containing M. blollopis SK-4

1.5

0.52

1840

366

80.1

AS (control)

1.5

0.52

1790

623

65.2

Abbreviations for Table 8.2 are biochemical oxygen demand (BOD), activated sludge (AS), and activated sludge containing Mrakia blollopis SK-4 (ASM).

116  Fungi in Polar Regions

Model Dairy Wastewater Treatment by Continuous Condition Under Changing Temperatures Wastewater Treatment Conditions and Measurement of Biochemical Oxygen Demand A continuous model dairy wastewater treatment test was carried out in a 3.5 L wastewater treatment tank consisting of a 2.9-L aeration tank and a 0.6-L settling tank (Fig. 8.4). The test was performed in a low-temperature incubator (Model LTI-601SD, Tokyo Rikakikai, Tokyo, Japan). AS was prepared with mixed liquor suspended solids (MLSS, 4000 mg/L), and the 4000 mg/L of MLSS was maintained. The model dairy wastewater consisted of synthetic wastewater (Table 8.3) containing cow’s milk at a BOD of 900 mg/L. The flow rate of the model wastewater was maintained at 1900 mL/d at 10, 20, and 25ºC, but was changed to 850 mL/d at 3ºC. The BOD volume load was adjusted to 0.15 kg-BOD/ kg-MLSS∙d at 10, 20, and 25ºC, and adjusted to 0.07 kg-BOD/kg-MLSS∙d at 3ºC. The BOD5 of the treated wastewater was measured using a coulometer (Ohkura Electric, Saitama, Japan).

Fig. 8.4.  Experimental system. (From left) tubing tank, aeration tank and setting tank.

Continuous Model Dairy Wastewater Treatment Under Conditions of Changing Temperature The colony counts of SK-4 gradually decreased between the start of the experiment and week 23 of the experiment. After 1 week, the colony counts decreased by approximately 80%. When the water temperature was changed to 3ºC after 4 weeks from the start of the experiment, the colony counts increased slightly, but decreased when the temperature was increased to 20ºC–25ºC between 8 and 23 weeks after the onset of the experiment. The temperature was then changed to 3ºC after 23 weeks from the start of the experiment, and the colony counts gradually increased through 30 weeks to a final colony count of 6.4 × 104 CFU/mL, which provided a

Wastewater Treatment by Antarctic Basidiomycetous Yeast 117

total MLSS of 0.25%. The average BOD decrease was about 86.0% at 10ºC, 96.5% at 20ºC, 97.3% at 25ºC, and 83.5% at 3ºC. BOD removal rates greater than 80% were obtained under all temperature conditions evaluated (Fig. 8.5). The water temperature of actual dairy wastewater in Hokkaido, Japan may range from 3ºC in winter to 25ºC in summer (unpublished data). Since the maximum growth temperature of SK-4 is 22ºC, one of our aims was to assess the rate of BOD removal with the addition of SK-4 to the AS, and to determine if SK-4 is able to survive at high temperatures, such as 20ºC and 25ºC. Model dairy wastewater treatment experiments showed that the colony counts of SK-4 gradually decreased over a 23-week period, then increased through the end of the experiment, when the temperature was again changed to 3ºC. Therefore, we conclude that this yeast strain is able to survive at 20–25°C for 15 weeks and fix the activated sludge. Table 8.3.  Composition of synthetic wastewater. Compounds

Concentration (mg/L)

KH2PO4

570

Na2HPO4

770

NH4Cl

190

CaCl2∙2H2O

20

MgSO4∙7H2O

20

FeCl3

1.5

Fig. 8.5.  Continuous model wastewater treatment by activated sludge containing M. blollopis SK-4. The left-hand side of the vertical axis shows the viable cell counts (CFU/mL) and right-hand side of the vertical axis indicates the BOD removal rate (%). The horizontal axis indicates time (wk).

118  Fungi in Polar Regions These results suggest that dairy wastewater treatment with SK-4 in the AS should be efficient under low-temperature conditions. Moreover, strain SK-4 satisfies the criteria for classification as a biosafety level-l agent: the strain is harmless to animals, plants, and humans, and presents the lowest risk to the environment. Therefore, SK-4 is a promising biological agent for dairy wastewater treatment, even in cold regions of the world. These studies were applied for a patent entitled “Basidiomycetous yeast from the South Pole having milk fat decomposition ability and use of the same,” and has been registered as Patent P5867954 in Japan. Based on this patent, a private company in Japan presently sells this wastewater treatment system. This is the first time that research results from the Japanese Antarctic Research Expedition (JARE) in the field of biology have been commercially developed.

Acknowledgments This work was carried out as part of the Science Program of JARE-48. It was supported by NIPR under MEXT, Japan. This work was supported by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for C Young Scientists (A) [grant numbers 16H06211].

References Biddlestone A.J., K.R. Gray and G.D. Job. 1991. Treatment of dairy farm wastewaters in engineered reed bed systems. Process Biochem. 26: 265–268. Chevalier, P., D. Proulx, P. Lessard, W.F. Vincent and J. de la Noue. 2000. Nitrogen and phosphorus removal by high latitude mat-forming cyanobacteria for potential use in tertiary wastewater treatment. J. Appl. Phycol. 12: 105–112. De Garcia, V., S. Brizzio, D. Libkind, P. Buzzini and M. van Broock. 2007. Biodiversity of cold adapted yeasts from glacial meltwater rivers in Patagonia Argentina. FEMS Microbiol. Ecol. 59: 331–341. De Garcia, V., S. Brizzio and M.R. Broock. 2012. Yeasts from glacial ice of Patagonia Andes, Argentina. FEMS Microbiol. Ecol. 82: 540–550. Di Menna, M.E. 1966. Yeasts in Antarctic soil. Antonie van Leeuwenhoek 32: 29–38. Fell, J.W., A.C. Statzell, I.L. Hunter and H.J. Phaff. 1969. Leucosporidium gen. n., the heterobasidiomycetous stage of several yeasts of the genus Candida. Anton Leeuw. Int. J. G. 35: 433–462. Healy, M.G., M. Rodgers and J. Mulqueen. 2007. Treatment of dairy wastewater using constructed wetland and intermittent sand filter. Bioresour. Technol. 98: 2268–2281. Hirayama-Katayama K., Y. Koike, K. Kobayashi and K. Hirayama. 2003. Removal of nitrogen by Antarctic yeast cells at low temperature. Polar Biosci. 16: 43–48. Hoshino, T. and N. Matsumoto. 2012. Cryophilic fungi to denote fungi in cryosphere. Fungal Biol. Rev. 26: 102–105. Hoshino, T., Y. Yokota, M. Tsuji, I. Yumoto and S. Kudoh. 2016. Basidiomycetous yeast from the south Pole having milk fat decomposition ability and use of the same. JP Patent P5867954. Kato, K., H. Ietsugu, T. Koba, H. Sasaki, N. Miyaji, T. Yokota et al. 2010. Design and performance of hybrid reed bed systems for treating high content wastewater in the cold climate. 12th Int. Conf. Wetland Syst. Water Pollut. Contr. Mansson, H.L. 2008. Fatty acids in bovine milk fat. Food Nutr. Sci. 52: 1–3. Margesin, R. and J.W. Fell. 2008. Mrakiella cryoconiti gen. nov., sp. nov., a psychrophilic, anamorphic, basidiomycetous yeast from alpine and arctic habitats. Int. J. Syst. Evol. Microbiol. 58: 2977–2982.

Wastewater Treatment by Antarctic Basidiomycetous Yeast 119 Pathan, A.A.K., B. Bhadra, Z. Begum and S. Shivaji. 2010. Diversity of yeasts from puddles in the vicinity of Midre Lovénbreen glacier, Arctic and bioprospecting for enzymes and fatty acids. Curr. Microbiol. 60: 307–314. Shah, S.B., D.K. Bhumbla, T.J. Basden and L.D. Lawrence. 2002. Cool temperature performance of a wheat straw biofilter for treating dairy wastewater. J. Environ. Sci. Health B. 37: 493–505. Shimohara, K., S. Fujiu, M. Tsuji, S. Kudoh, T. Hoshino et al. 2012. Lipolytic activities and their thermal dependence of Mrakia species, basidiomycetous yeast from Antarctica. J. Water Waste. 54: 691–696 (Japnese). Singh, P. and S.M. Singh. 2012. Characterization of yeast and filamentous fungi isolated from cryoconite holes of Svalbard, Arctic. Polar Biol. 35: 575–583. Thomas-Hall, S.R., B. Turchetti, P. Buzzini, E. Branda, T. Boekhout, B. Threelen et al. 2010. Coldadapted yeasts from Antarctica and Italian Alps-description of three novel species: Mrakia robertii sp. nov., Mrakia blollopis sp. nov. and Mrakiella niccombsii sp. nov. Extremophiles 14: 47–59. Tsuji, M., S. Fujiu, N. Xiao, Y. Hanada, S. Kudoh, H. Kondo et al. 2013a. Cold adaptation of fungi obtained from soil and lake sediment in the Skarvsnes ice-free area, Antarctic. FEMS Microbiol. Lett. 346: 121–130. Tsuji, M., S.M. Singh., Y. Yokota., S. Kudoh. and T. Hoshino. 2013b. Influence of initial pH on ethanol production by Antarctic Basidiomycetous yeast Mrakia blollopis. Biosci. Biotech. Bioch. 77: 2483–2485. Tsuji, M., Y. Yokota., K. Shimohara, S. Kudoh and T. Hoshino. 2013c. An application of wastewater treatment in a cold environment and stable lipase production of Antarctic basidiomycetous yeast Mrakia blollopis. PLoS One 8: e59376. Tsuji, M., Y. Yokota, S. Kudoh and T. Hoshino. 2014. Effects of nitrogen concentration and culturing temperatureon lipase secretion and morphology of the Antarctic basidiomycetous yeast Mrakia blollopis. Int. J. Res. Eng. Sci. 2: 49–54. Tsuji, M., Y. Yokota, S. Kudoh and T. Hoshino. 2015. Comparative analysis of milk fat decomposition activity by Mrakia spp. isolated from Skarvsnes ice-free area, East Antarctica. Cryobiology 70: 293–299. Turchetti, B., P. Buzzini, M. Goretti, E. Branda, G. Diolaiuti, C. D’Agata et al. 2008. Psychrophilic yeasts in glacial environments of alpine glaciers. FEMS Microbiol. Ecol. 63: 73–83. Xin, M. and P. Zhou. 2007. Mrakia psychrophila sp. Nov., a new species isolated from Antarctic soil. J. Zhejiang Univ-Sc. B 8: 260–265. Ying, C., K. Umetsu, I. Ihara, Y. Sakai and T. Yamashiro. 2010. Simultaneous removal of organic matter and nitrogen form milking parlor wastewater by a magnetic activated sludge (MAS) process. Bioresour. Technol. 101: 4349–4353.

9 Ethanol Fermentation by the Basidiomycetous Yeast Mrakia blollopis Under Low Temperature Conditions Masaharu Tsuji1,* and Tamotsu Hoshino2

Introduction Cryophilic yeasts (Hoshino and Matsumoto 2012) of the genus Mrakia have been found in the cold environments worldwide, such as Arctic, Siberia, Alaska, Alps, Apennines, Patagonia, and Antarctica (Margesin et al. 2005, De Garcia et al. 2007, Pathan et al. 2010, Thomas-Hall et al. 2010, De Garcia et al. 2012, Singh et al. 2013, Tsuji et al. 2013a). Di Menna (1966) reported that the genus Mrakia accounts for approximately 24% of the culturable yeasts in Antarctic soil. Moreover, we previously reported that Mrakia spp. constitute approximately 35% of the culturable fungi isolated from lake sediments and soils of East Antarctica (Tsuji et al. 2013b). These reports suggest that Mrakia spp. are the dominant culturable fungi in Antarctica, and are well-adapted to the Antarctic environment. The information regarding the ethanolic fermentation in basidiomycetous yeasts is limited. Fermentative ability has been reported for Mrakia spp. (Jones and Slooff 1966), Xanthophyllomyces spp. (Fell et al. 2011), and Bandoniozyma spp. (Valente et al. 2012). Other species, namely, Rhodotorula mucilaginosa, National Institute of Polar Research (NIPR), 10-3 Midori-cho, Tachikawa, Tokyo 190-8518, Japan. Bio-production Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-higashi, Toyohira-ku, Sapporo, Hokkaido 062-8517, Japan. Email: [email protected] * Corresponding author: [email protected] 1 2

Ethanol Fermentation by Mrakia blollopis 121

R. minuta, R. pallida, and Cryptococcus saitoi, were originally reported as unable to ferment ethanol (Fonseca et al. 2011, Sampio 2011). However, Rao et al. (2008) reported that some strains of these species can ferment ethanol obtained from xylose. Nine Mrakia species are known—M. aquatica, M. arctica, M. blollopis, M. cryoconiti, M. frigida, M. gelida, M. niccombsii, M. psychrophila, and M. robertii (Thomass-Hall et al. 2010, Liu et al. 2015, Tsuji et al. 2018). Most of the species in this basidiomycetous yeast genus are known for their ability to ferment sugars, and all can ferment glucose and sucrose except Mrakia aquatica and M. cryoconiti (Fell and Margesin 2011). To the best of our knowledge, the genus Mrakia is the only ethanol fermentable fungi inhabiting the continental Antarctica region. In this chapter, we review ethanol fermentation by the basidiomycetous yeast genus Mrakia. We also describe brewing Japanese sake using Mrakia blollopis with sake yeast under low temperature.

Ethanol Productivity of Genus Mrakia Sinclair and Stokes (1965) first characterized the ethanol productivity of Mrakia species under low-temperature conditions. At 10–15°C, M. frigida CBS 5917 is a more efficient sugar fermenter than conventional yeast strains of Saccharomyces cerevisiae. More recently, Thomass-Hall et al. (2010) reported on homemade beers produced by Mrakia species native to Antarctica. M. frigida, M. blollopis, M. gelida, and M. robertii were tested for ethanol fermentation in a home brewing kit at 6°C and 15°C. All four of these strains fermented sucrose; however, their sucrose to ethanol conversion was incomplete and cell growth ceased when the ethanol levels exceeded 2% (v/v). Nonetheless, these reports did not investigate ethanol production by the genus Mrakia in detail. Therefore, we tested ethanol production by 27 strains of Mrakia spp. isolated from Skarvsnes ice-free area, East Antarctica. Eight out of 27 Mrakia strains could produce over 2% (v/v) ethanol when grown in a medium with 40 g/L glucose at 10°C (Tsuji et al. 2016); however, we did not know what concentration of ethanol limits ethanol production by the members of genus Mrakia. Therefore, ethanol fermentation was carried out in a medium with high glucose concentration (100 g/L or 120 g/L) using six Mrakia strains selected based on their ethanol productivity in a medium with 40 g/L glucose. Two strains (M. blollopis SK-4 and NIR-4) produced over 4% (v/v) ethanol at 10°C, and especially strain SK-4 could produce approximately 4.8% (v/v) ethanol in a medium with 120 g/L glucose at 10°C (Tsuji et al. 2013d). Mrakia blollopis SK-4 was isolated from an algal mat in lake sediment of Naga-ike, a lake in the Skarvsnes ice-free area of East Antarctica. It could produce ethanol at –1 to 20°C with optimum temperature range of 10–15°C. Further, SK-4 could produce ethanol between pH 5.0 to 10.0; at pH below 4.0 and above 10.5, it did not completely convert glucose to ethanol. For maximum ethanol productivity with SK-4, the optimum pH ranges were 4.5–5.0 and 8.0–10.0. Strain SK-4 secretes extracellular enzymes, such as cellulase, β-glucosidase, catalase, and amylase, as well as lipase under low temperature conditions. Its lipase is stable against metal

122  Fungi in Polar Regions ions and organic solvents, and the optimum pH range for SK-4 lipase is 8.5–9.0 (Tsuji et al. 2013a). Naga-ike is an oligotrophic lake, and pH of this lake water is 8.5 (Tsuji et al. 2013c). Many studies have reported the ethanol tolerance and productivity of the conventional yeast S. cerevisiae (Beaven et al. 1981, You et al. 2003). Sake yeast are known to have the characteristics of high ethanol production and low stress tolerance. Rim15p, known as PAS kinase, is positioned upstream of the stress responsive related gene Msn2P and Msn4P in S. cerevisiae. In sake yeast strains, Rim15p is truncated at the carboxyl terminus as a result of a mutation. When the full-length Rim15p gene is deleted from a laboratory yeast strain, it improves the fermentation rate, while losing the ability of stress tolerance, including ethanol tolerance. Therefore, Rim15p deletion mutants of sake yeasts have a low ethanol tolerance, but strong ethanol fermentation ability (Watanabe et al. 2012). Then, we tested ethanol tolerance ability of six Mrakia strains isolated from a site near the Syowa Station. These six Mrakia strains were evaluated for their ability to grow in media containing 2% (v/v), 4% (v/v), or 8% (v/v) ethanol. Consequently, we found that the ability of Mrakia strain to grow in media containing ethanol is correlated to their ethanol productivity (Fig. 9.1). In other words, high ethanol producing strains of Mrakia have high ethanol tolerance (Tsuji et al. 2016). Moreover, Mrakia strains isolated from a site near the Syowa Station have high osmotic pressure tolerance, such as 10% (w/v) NaCl or 50% (w/v) glucose. Ethanol is known to be cytotoxic, and wild-type fungi usually cannot produce over 4% (v/v) ethanol due to a feedback repression. In S. cerevisiae represented in sake yeast, the ethanol tolerance ability is inversely proportional to the ethanol productivity; however, we found that the characteristics of Antarctic basidiomycetous yeast genus Mrakia are opposite to those of the sake yeast, implying that the ethanol fermentation system of the genus Mrakia is different from that of conventional sake yeast.

Fig. 9.1.  Ethanol tolerance tests among Mrakia strains. Ethanol tolerance tests were performed at 10°C. Six strains were incubated with shaking at 120 rpm for 120 hours in YPD liquid medium, and diluted to 1.0 × 105 cell/ml for the assay. Serial 10-fold dilutions were spotted onto plates and grown for 4 weeks at 10°C.

Ethanol Fermentation by Mrakia blollopis 123

Ethanol Fermentation from Lignocellulosic Biomass As previously stated, Mrakia blollopis SK-4 has a superior ethanol fermentation ability under low temperature. We attempted ethanol fermentation by SK-4 under low temperature using lignocellulosic biomass hydrolysates, such as eucalyptus and Japanese cedar, as substrates. This yeast strongly ferments Japanese cedar hydrolysate, but could not ferment eucalyptus hydrolysate (Tsuji et al. 2013d). SK-4 has tolerances for both acetic acid and formic acid during fermentation. The reason for the difference in the results of fermentation using Japanese cedar and eucalyptus remains uncertain. Direct ethanol fermentation (DEF) from a cellulosic biomass was first reported by Takagi et al. (1977). In this technique, enzymatic hydrolysis and ethanol fermentation were carried out at the same time. In the presence of high concentration of glucose, cellulase activity is considerably repressed. However, when yeast is mixed with an enzymatic-hydrolysis reactor, glucose is formed from the cellulolytic biomass by the cellulase activity. Glucose is maintained at a low concentration, and is rapidly converted to ethanol by the yeast. Moreover, when a lignocellulosic biomass is saccharified and fermented at the same time, major fermentation inhibitors, such as furfural and 5-methylfolate (5-MHF) are maintained at very low concentrations compared to their concentrations in the enzymatic hydrolysate (Thomsen et al. 2009). Therefore, this technique is expected to improve the rates of saccharification and ethanol fermentation. At first, we attempted DEF from Japanese cedar and Eucalyptus using SK-4. DEF converted Japanese cedar and Eucalyptus to 12.5 g/L and 7.2 g/L ethanol, respectively. Eriksson et al. (2002) suggested that cellulase was protected from absorption by the biomass in the presence of a non-ionic surfactant. Since the non-ionic surfactant Tween 80 is thought to promote hydrolysis and fermentation, we tested DEF with Tween 80. Ethanol concentration was improved by approximately 1.1–1.6-fold by the addition of Tween 80. However, when SK-4 was fermented on mechanochemically treated Eucalyptus and Japanese cedar wood meals using the DEF technique with the non-ionic surfactant Tween 80, theoretical ethanol yields from these biomasses were 51.2% and 65.1%, respectively. Since these yields did not exceed 70%, it is necessary to improve fermentation efficiency of DEF from lignocellulosic biomass with M. blollopis SK-4. Eucalyptus and Japanese cedar trees are known to contain approximately 4% oil (Gupta et al. 1981). The cell structures of these biomasses were completely destroyed by the mechanochemical treatment (Endo et al. 2006). We speculate that the oil covers the surfaces of Eucalyptus and Japanese cedar wood meals, and that contact of cellulase with the biomass is impeded by the oil. Since it is thought that oil can be decomposed by the addition of lipase and adsorption of cellulase on biomass can be prevented by the addition of Tween 80, we conducted DEF in the presence of Tween 80 and lipase. When DEF was carried out with 5 U of lipase/g dry substrate, the yield of ethanol was approximately 1.2- to 1.8-fold higher than that without lipase. When DEF was carried out with 1% (v/v) Tween 80 and 5 U lipase/g dry substrate, the

124  Fungi in Polar Regions yield of ethanol was approximately 1.4- to 2.4-fold higher than that without Tween 80 and lipase. Theoretical ethanol yields were approximately 76.5% and 81.0% from Eucalyptus and Japanese cedar, respectively (Tsuji et al. 2014).

Japanese Sake Co-Fermented by Antarctic Basidiomycetous Yeast Mrakia and Sake Yeast Antarctic basidiomycetous yeast Mrakia blollopis SK-4 could produce approximately 5% (v/v) ethanol (Tsuji et al. 2013d), which is the highest among the genus Mrakia. First, we attempted brewing sake using only SK-4 at 15°C. The ethanol concentration estimated from the total decrement value in the sake mash (moromi) was not above 2% (v/v) (Fig. 9.2). Moromi, also known as sake mash, is a general term for the starter mixture made of steamed rice, water, and koji fungus. In moromi substrate, starch in the steamed rice is digested to glucose by the amylase activity of koji fungus. The rate of glucose production is probably faster than the growth rate of SK-4. Therefore, the low yield obtained in fermentation with only SK-4 is probably caused by osmotic pressure due to high glucose concentration. For brewing sake successfully using Antarctic basidiomycetous yeast SK-4, we thought that the osmotic pressure in the sake mash must be reduced. To reduce the osmotic pressure in moromi by consuming glucose, we performed co-fermentation using SK-4 and sake yeast strain K701. The co-fermentation was performed under various conditions with different initial cell density ratios of SK-4 and K701.

0

5 10 15 Fermentation time (day)

20

Fig. 9.2.  Total decrement in sake mash. SN0-K100 indicates sake brewed by K701 alone. S40-K60 shows sake brewed by 40% SK-4 and 60% K701 used as initial inoculum. S100-K0 indicates sake brewed by SK-4 alone.

Ethanol Fermentation by Mrakia blollopis 125

After careful consideration of various fermentation conditions, initial inoculum ratio of 40% SK-4 and 60% K701 was shown to have the same total decrement value as that obtained by brewing sake using K701 alone (Fig. 9.2). Final ethanol concentrations estimated from the values was 19.7% (v/v) using 40% SK-4 with 60% K701 as initial inoculum, and 19.5% (v/v) using K701 alone (Fig. 9.3). Addition of SK4 to sake mash increased isoamyl acetate approximately 1.25-fold than that obtained by using K701 alone. In Sake, ginjoko is the most important factor, and refers to the fruit-flower scent of sake. Isoamyl acetate imparts a fragrance similar to the banana fragrance and is known to be a type of ginjoko (Fig. 9.4). Moreover, the amino acid content in sake resulting from a co-fermentation using SK-4 and K701 was lower than that from using K701 alone. After death of the yeast cells, their contents are released in the sake mash. Proteases released from the yeast cells digest peptides and proteins in the steamed rice. Consequently, the amino acid content of sake increases. We checked cell death ratio after brewing using SK-4 with K701 or only K701. For brewing using only K701, the cell death ratio was 43.9%, whereas when sake was co-fermented using SK-4 and K701, the cell death ratio was 32.2%. The cell death ratio dramatically decreased by the addition of SK-4 in the sake mash (Fig. 9.5). This data shows that decrement in amino acid content of sake results from the improvement in the cell death ratio caused by the addition of SK-4 as an inoculum. Consequently, ethanol concentration in co-fermentation conditions was thought to be higher than that with K701 alone. Taken together, co-fermentation using SK-4 and K701 resulted in the quality sake that is similar to that obtained with K701 alone.

SN0-K100

S40-K60

Fig. 9.3.  Ethanol concentration in sake. SN0-K100 indicates sake brewed by K701 alone. S40-K60 shows sake brewed by 40% SK-4 and 60% K701 used as initial inoculum.

126  Fungi in Polar Regions

Fig. 9.4.  Isoamyl acetate concentration in sake. SN0-K100 indicates sake brewed by K701 alone. S40-K60 shows sake brewed by 40% SK-4 and 60% K701 used as initial inoculum.

Fig. 9.5. The cell death ration in sake mash. SN0-K100 indicates sake brewed by K701 alone. S40-K60 shows sake brewed by 40% SK-4 and 60% K701 used as initial inoculum.

Mrakia blollopis is a member of Basidiomycota, order Tremellales. Basidiomycota have high concentration of β-glucan, known as a dietary fiber, in the cell wall. β-glucans are considered to have an immunostimulating action and prevent allergy. Strain SK-4 satisfies the criteria of Biosafety Level l: that is, the strain is harmless to animals, plants, and humans. Thus, strain SK-4 is a strong candidate not only for alcoholic beverage production, but also for the production of health food known as amazake, which is a traditional sweet, low- or non-alcohol Japanese drink made from sake mash.

Ethanol Fermentation by Mrakia blollopis 127

Appendix Methods for Ethanol Fermentation Test The Necessary Equipment for Ethanol Fermentation Test The following equipment are necessary to conduct the test; centrifuge for 1.5 mL and 50 mL tubes, low temperature incubator, HPLC equipped with a refractive index detector, and multi position magnetic stirrer for volumes up to 50 mL. Agar and Liquid Media Potato dextrose agar (PDA, Difco) or Yeast peptone dextrose (YPD) agar (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose, and 20 g/L agar) was used for preparing fresh yeast culture before pre-cultivation of ethanol fermentation test. The growth rate of yeast isolated from polar regions is generally much lower than that of conventional yeast. For our Mrakia strains, doubling time was approximately 20–24 hours, which is approximately 10-fold of that of S. cerevisiae. When the yeast cells are transferred on fresh PDA plate, several days are required for the appearance of the colonies on the plate. This factor must be considered while starting ethanol fermentation test using the yeast. Checking Cell Number or OD600 Cell number must be equal in every initial fermentation test so as to compare the ethanol productivity among the strains. Therefore, cell number or OD600 value of the strains should be checked by hemocytometer, spectrophotometer, or other adequate methods before preparing inoculum for ethanol fermentation. Pre-Inoculation of the Cells Pre-cultivation is done at least two times in YPD broth. For first pre-cultivation, inoculate yeast cells from agar plates to YPD broth. Grow the yeast cells in YPD broth for 1 week or until well-grown at 10°C. The cells from 1st pre-cultivation are collected by centrifugation at low speed (3500 × g to 5000 × g) for 5–10 min at 4°C. The collected cells are washed by distilled water and collected by centrifugation at low speed (3500 × g to 5000 × g) for 5–10 min at 4°C. The pellet is resuspended in distilled water. These resuspended cells are used as the inoculum for the second pre-cultivation, which continues for 5 days to 1 week at 10°C. The cells from 2nd pre-cultivation are collected by centrifugation at low speed (3500 × g to 5000 × g) for 5–10 minutes at 4°C. The collected cells are washed by distilled water and collected by centrifugation at low speed (3500 × g to 5000 × g) for 5–10 min at 4°C. The pellet is resuspended in distilled water and adjusted to appropriate cell number or OD600 (i.e., 1.2 × 109 cells or OD600 = 150). The resulting suspension is used as the inoculum.

128  Fungi in Polar Regions Ethanol Fermentation Test Condition Ethanol fermentation is recommended in glass vials under sterile conditions. The ethanol fermentation mixture comprises 40 g/L or 100 g/L glucose, 5 g/L yeast extract, 5 g/L Bacto peptone, 2 g/L NH4Cl, 1 g/L KH2PO4, and 0.3 g/L MgSO4·7H2O in 20 mM citrate buffer (pH 5.0) (Tsuji et al. 2013d). If the fermentation mixture cannot be prepared, modified YPD broth (10 g/L yeast extract, 20 g/L peptone, and 40 g/L or 100 g/L glucose) can be used instead. Nevertheless, cell growth speed and ethanol concentration in the fermentation mixture is higher than those in YPD broth. The initial cell density in the fermentation medium is adjusted to an appropriate OD600 value or appropriate cell number. In our case, initial cell density was adjusted to OD600 = 2 or 1.6 × 107 cell/mL. After inoculation, Mrakia strain is cultivated at 120 rpm at 4–15°C. Three hundred μl of each sample is collected every 20–24 hours or doubling time, and OD600 value or cell number in the collected sample is checked. Then, collected samples are centrifuged at high speed (13,000–15,000 × g) for 5–10 minutes at 4°C. The supernatant is retained for measurement of glucose and ethanol concentrations. All experiments must be carried out independently in three vials. Analysis of Fermentation Products The fermentation products are identified by HPLC equipped with a refractive index detector. Ethanol and carbon sources in the fermentation medium are separated in a column of alcohol and sugar for analysis. We use an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA, USA) for glucose and ethanol detection and the HPLC equipment is operated at 65°C with a mobile phase of 8 mM H2SO4 at a flow rate of 0.6 ml/min. Please note that fast flow rate of the mobile phase reduces to the sensitivity of the HPLC analysis. Materials and Methods for Sake Brewing M. blollopis SK-4 and S. cerevisiae K701 strain were used for Japanese sake brewing test. Yeast strains were pre-cultured in 3 ml YPD at 15°C for 24 hours to 72 hours; 2 ml pre-cultured yeast was added to 100 ml YPD and cultured at 15°C for up to 1 week. The cells (OD660 = 200) were harvested by centrifugation and mixed with 60 g pre-gelatinized rice (Tokushima Seiko Co., Tokushima, Japan), 23 g dry koji (Tokushima Seiko), 200 mL water, and 44.5 µL 90% lactic acid. Dry koji is a culture of A. oryzae that is grown on steamed rice and then dried, and is a source of diastatic enzymes, such as amylase and glucoamylase. Lactic acid was added to prevent bacterial contamination, since the fermentation set-up for sake brewing is not sterile. Sake mash was incubated at 15°C without shaking. The sake mash was weighed every day and the CO2 output was estimated. After 20 days of culture, the mash was centrifuged and the supernatant was analyzed as sake. Ethanol concentration was measured by a contact combustion system with an alcohol densitometer (Yazaki, Tokyo, Japan). Specific gravity was measured

Ethanol Fermentation by Mrakia blollopis 129

using a density hydrometer (KEM, Kyoto, Japan). Acidity and amino acidity were measured with an electric potential difference autotitration apparatus (KEM), using the National Tax Administration Agency method (Okazaki 1993). Volatile aromatic compounds (ethyl acetate, n-propyl alcohol, isobutyl alcohol, isoamyl acetate, isoamyl alcohol, and ethyl caproate) were measured by headspace gas chromatography, using an Agilent 7694 Headspace Sampler and Agilent Technologies GC 6890N. Aliquots of samples (0.9 ml) were placed in 10 mL vials and 0.1 mL solutions of n-amyl alcohol (200 p.p.m.) and methyl caprate (5 p.p.m.), used as internal standards, were added. The vials were sealed with a silicon rubber stopper, covered with an aluminum cap, and then h eated at 50°C for 30 minutes. Esters and higher alcohols were separated using a DB-WAX capillary column (0.32 mm i.d. × 30 m, film thickness 0.25 µm; Agilent) after autoinjection of a headspace volume of 1 mL. The following conditions were used: injection temperature, 200°C; oven temperature, 85°C; detector temperature, 250°C; and carrier gas, He 2.2 mL/min.

Acknowledgments This work was carried out as part of the Science Program of JARE-48. It was supported by NIPR under MEXT, Japan. This work was supported by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for C Young Scientists (A) [grant numbers 16H06211]. The brewing of Japanese sake was conducted in cooperation with National Research Institute of Brewing, Japan.

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130  Fungi in Polar Regions Fonseca, Á., T. Boekhout and J.W. Fell. 2011. Cryptococcus Vuillemin (1901). pp. 1661–1737. In: C.P. Kurtzman, J.W. Fell and T. Boeckhout (eds.). The Yeast, a Taxonomic Study, 5th edn. Elsevier, Amsterdam. Gupta, P.K., A.F. Mascarenhas and V. Jagannathan. 1981. Tissue culture of forest trees—Clonal propagation of mature trees of Eucalyptus citriodora Hook, by tissue culture Plant Sci. Lett. 20: 195–201. Hoshino, T. and N. Matsumoto. 2012. Cryophilic fungi to denote fungi in cryosphere. Fungal Biol. Rev. 26: 102–105. Jones, E.B.G. and W.C. Slooff. 1966. Candida aquatica sp. nov., isolated from water scums. Antonie van Leeuwenhoek 32: 223–228. Margesin, R., V. Fauster and P.A. Fonteyne. 2005. Characterization of cold-active pectate lyases from psychrophilic Mrakia frigida. Lett. Appl. Microbiol. 40: 453–459. Liu, X.Z., Q.M. Wang, M. Göker, M. Groenewald, A.V. Kachalkin, H.T. Lumbsch et al. 2015. Towards an integrated phylogenetic classification of the Tremellomycetes. Stud. Mycol. 81: 85–147. Okazaki, N. 1993. Seishu, gousei-seishu. pp. 7–33. In: N. Nishitani (ed.). The Annotation of Official Methods of Analysis of the National Tax Administration Agency, Japan, 4th edn. The Brewing Society of Japan, Tokyo. Pathan, A.A.K., B. Bhadra, Z. Begum and S. Shivaji. 2010. Diversity of yeasts from puddles in the vinicity of Midre Lovénbreen Glacier, Arctic and bioprospecting for enzymes and fatty acids. Curr. Microbiol. 60: 307–314. Rao, R.S., B. Bhadra and S. Shivaji. 2008. Isolation and characterization of ethanol-producing yeasts from fruits and tree barks. Lett. Appl. Microbiol. 47: 19–24. Sampio, J.P. 2011. Rhodotorula Harrison (1928). pp. 1873–1927. In: C.P. Kurtzman, J.W. Fell and T. Boeckhout (eds.). The Yeast, a Taxonomic Study, 5th edn. Elsevier, Amsterdam. Sinclair, N.A. and J.L. Stokes. 1965. Obligately Psychrophilic Yeasts from the Polar Regions. Can. J. Microbiol. 11: 259–269. Singh, P., M. Tsuji, S.M. Singh, U. Roy and T. Hoshino. 2013. Taxonomic characterization, adaptation strategies and biotechnological potential of cryophilic yeasts from ice cores of MidreLovénbreen glacier, Svalbard, Arctic. Cryobiology 66: 167–175. Singh, S.M., M. Tsuji, P. Gawas-Sakhalker, M.J.J.E. Loonen and T. Hoshino. 2016. Bird feather fungi from Svalbard Arctic. Polar Biol. 39: 523–532. Takagi, M., S. Abe, S. Suzuki, G.H. Emert and A. Yata. 1977. A method for production of alcohol directly from cellulose using cellulase and yeast. Chemicals and Microbial Protein, 551–571. Thomas-Hall, S.R., B. Turchetti, P. Buzzini, E. Branda, T. Boekhout, B. Threelen et al. 2010. Coldadapted yeasts from Antarctica and Italian Alps-description of three novel species: Mrakia robertii sp. nov., Mrakia blollopis sp. nov. and Mrakiella niccombsii sp. nov. Extremophiles 14: 47–59. Thomsen, M.H., A. Thygesen and A.B. Thomsen. 2009. Identification and characterization of fermentation inhibitors formed during hydrothermal treatment and following SSF of wheat straw. Appl. Microbiol. Biotechnol. 83: 447–455. Tsuji, M., Y. Yokota, K. Shimohara, S. Kudoh and T. Hoshino. 2013a. An application of wastewater treatment in a cold environment and stable lipase production of Antarctic basidiomycetous yeast Mrakia blollopis. PLoS One 8: e59376. Tsuji, M., S. Fujiu, N. Xiao, Y. Hanada, S. Kudoh, H. Kondo et al. 2013b. Cold adaptation of fungi obtained from soil and lake sediment in the Skarvsnes ice-free area, Antarctic. FEMS Microbiol. Lett. 346: 121–130. Tsuji, M., S.M. Singh, Y. Yokota, S. Kudoh and T. Hoshino. 2013c. Influence of initial pH on ethanol production by Antarctic Basidiomycetous yeast Mrakia blollopis. Biosci. Biotech. Bioch. 77: 2483–2485. Tsuji, M., T. Goshima, A. Matsushika, S. Kudoh and T. Hoshino. 2013d. Direct ethanol fermentation from lignocellulosic biomass by Antarctic Basidiomycetous yeast Mrakia blollopis under a low temperature condition. Cryobiology 67: 241–243. Tsuji, M., Y. Yokota, S. Kudoh and T. Hoshino. 2014. Improvement of direct ethanol fermentation from woody biomasses by Antarctic basidiomycetous yeast Mrakia blollopis under a low temperature condition. Cryobiology 68: 303–305.

Ethanol Fermentation by Mrakia blollopis 131 Tsuji, M., S. Kudoh and T. Hoshino. 2016. Ethanol productivity of cryophilic basidiomycetous yeast Mrakia spp. correlates with ethanol tolerance. Mycoscience 57: 42–50. Tsuji, M., Y. Tanabe, W.F. Vincent and M. Uchida. 2018. Mrakia arctica sp. nov., a new psychrophilic yeast isolated from an ice island in the Canadian High Arctic. Mycoscience 59: 54–58. Valente, P., T. Boeckhout, M.F. Landell, J. Crestanil, F.C. Pagnocca, L.D. Sette et al. 2012. Bandoniozyma gen. nov., a genus of fermentative and non-fermentative Tremellaceous yeast species. PLoS ONE 7: e46060. Watanabe, D., Y. Araki, Y. Zhou, N. Maeya, T. Akao and H. Shimoi. 2012. A loss-of-function mutation in the PAS kinase Rim15p is related to defective quiescence entry and high fermentation rates in Saccharomyces cerevisiae sake yeast strains. Appl. Environ. Microbiol. 78: 4008–4016. You, K.M., C.L. Rosenfield and D.C. Knipple. 2003. Ethanol tolerance in the yeast Saccharomyces cerevisiae is dependent on cellular oleic acid content. Appl. Environ. Microbiol. 69: 1499–1503.

Index A Adaptation 44–46, 51, 55–57, 59, 61 Antarctic fungi 1–15, 30–43, 17–29 Antarctic yeast 120, 122, 124 Antifreeze protein 92, 95, 104 Antioxidant 92, 95, 103 Arctic 92–96, 102, 105 B Basidiomycetous yeast 110, 111, 118, 120–122, 124 Biodiversity 31 Biogeography 17–29 bipolar distribution 86, 88 Brewing 121, 124, 125, 128, 129 Bryophytes 18 Bryum pseudotriquetrum 19–22

Fungal succession 19–22, 24–27 Fungi 92–96, 98, 100, 103–105 G Glacier foreland 17, 23, 27 H High arctic 18 High-throughput sequencing 68, 71 Hylocomium splendens 19–21 Hyphal length 19, 20, 22, 25, 26 I Ice-free region 18, 27 Internal transcribed spacer (ITS) region 74 J

C

JARE 1–15

checklist 1–15 Continental Antarctica 21, 24, 27

M

D Decomposition 17–21, 23–27 Diversity 44–47, 52, 59–62 DNA metabarcoding 67–71, 74, 78, 79 E Ecological role 31, 33, 38 Effect on host and ecosystem 44, 46 Ellesmere Island 17–29 Environmental sequencing 67–82 Enzyme 92, 93, 95, 101–105 Ethanol fermentation 120–123, 127, 128 F Fungal diversity 67–70, 74, 78, 79

Microbes interaction 29–43 Microfungi 17–29 Milk fat 110–114, 118 moss 85–88 Mrakia 111–115, 120–124, 126–128 O Oomycetes 83–86, 88 Operational taxonomic units 69 P PCR 73–78 Phoma herbarum 19–22 plant parasite 85 Plant pathogenic fungi 45, 46, 48, 52, 59 Primary succession 17, 23, 24, 26 Pseudogymnoascus pannorum 19–22

134  Fungi in Polar Regions R

T

Racomitrium lanuginosum 19, 20, 22

the Arctic 44–47, 51, 52, 55–57, 59–62

S

W

Salix 23, 25 snow rot 85–88 Syowa Station 1–15

Wastewater treatment 110, 113, 114–118 Z zoospore 83, 86–88

Color Plate Section Chapter 2 (a)

(b)

Fig. 2.1.  Photographs of study areas. (a) Oobloyah Bay area in the Arctic, (b) Lützow-Holm Bay area in Antarctica. Locations are given in Table 2.1.

Fig. 2.2.  Photographs of moss profiles. (a) Hylocomium splendens in the Arctic, (b) Bryum pseudotriquetrum in Antarctica.

136  Fungi in Polar Regions (b)

(a)

Fig. 2.4.  Photographs of dead willow materials. (a) Salix arctica in the Arctic, (b) Location where Salix spp. were planted in Antarctica.

Chapter 3 A.

B.

C.

Fig. 3.1.  Typhula sp. in continental climate. Partial view of Kuujjuarapik/Whapmagoostui and the Great Whale River in Quebec, Canada (A). A river bank and communities of Sea pea (B). Sclerotia of Typhula sp. died leaves of Sea pea (C; bar = 1 cm).

Fig. 1. Hoshino et al.

Fig. 3.2.  Mycelial growth inhibition of snow mold, Typhula ishikariensis co-culture with various bacteria from cold enviroments. Bacillus simplex from Yakutsk, Sakha republic in Russia (A), Pseudomonas sp. from Fairbanks, Alaska, USA (B), Pseudomonas sp. from Svalbard (C) and Paenibacillus macquariensis subsp. defensor from Oblast Magadan, Russia.

Color Plate Section 137 B.

D.

Fig. 3.3.  Fungal symptoms of mosses in continental Antarctica. Terra Nova Bay, Ross Sea (A and B) and Skarvsnes, the Sôya Coast (C and D).

Fungi in Polar Region Figure 1

138  Fungi in Polar Regions

Chapter 4

Fig. 4.1.  Tar spot disease caused by R. polare on S. polaris on Spitsbergen Island (A; bar = 2 mm) and the ascus (C; bar = 10 μm) and ascospore (D; bar = 5 μm). Rust disease caused by Melampsora epitea on S. arctica on Ellesmere Island (D) and the urediniospore (E; bar = 10 μm). The pictures A, C, and D are adapted from Masumoto et al. (2014) and B and E from Smith et al. (2004).

Color Plate Section 139

Chapter 7

Fig. 7.1. (a) Landscape of Svalbard Arctic showing different habitats where fungal biota exists. (b) Arctic wild mushroom Lycoperdon molle collected from Svalbard, Arctic. (c) A novel species of yeast contributed from Svalbard, Arctic. (d) A novel species of filamentous fungi contributed from Svalbard, Arctic. (e) Strong enzyme (lipase) activity of an ice core yeast collected from Svalbard, Arctic. (f) A Cryoconites yeast (Rhodoturala svalbardensis sp. nov.) showing positive AFPs activity.