Growth, fruiting body development and laccase production of selected coprini Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultäten der Georg-August-Universität zu Göttingen vorgelegt von Mónica Navarro González (aus Cuernavaca, Mexiko) Göttingen, den 18.03.2008 D 7 Referent: Prof. Dr. Gerhard Braus Korreferent: Prof. Dr. Andrea Polle Tag der mündlichen Prüfung: 30.04.2008 To my parents: Othón and Isabel and to my nieces and nephews: Samy, Marco, Claus, Aldo, Andy, Lucero, and Montse …for bringing me always motivation, joy and love If you think you are a mushroom, jump into the basket Russian proverb Acknowledgments Since performing Ph. D is more than experiments, tables, graphs, monday seminars, conferences and so on I would like to express my gratitude: I am immensely happy to thank all those who have helped me in this hard way. My gratitude goes to Prof. Dr. Ursula Kües for giving me the opportunity to work in her group and for providing any opportunities for developing myself in many directions during my doctoral studies, and my life in Germany. I sincerely thank Dr. Andrzej Majcherczyk for his advices and direction on part of this work. My special thanks to Dr. Patrik Hoegger for his invaluable support and his endless patience during large part of my work. Furthermore, I would like to thank Prof. Dr. Gerhard Braus, Prof. Dr. Andrea Polle and Prof. Dr. Stefany Pöggeler for their readiness to evaluate my thesis and being my examiners. Víctor Mora-Pérez deserves part of the acknowledgments, without his primary motivation my interest for working with mushrooms during my bachelor studies probably would have never been appeared in my mind. Dr. Mercedes Sobal Cruz motivated me for continuing master studies in the same field. In the same way, I would like to extend my gratitude to Dr. Daniel Martínez-Carrera for making, through his motivation and support, possible this adventure called PhD. If I continue motivated to work with mushrooms is thank to you! By now, it is not easy to find the appropriate words to describe all the feelings towards all the people with whom, in different times, I have been sharing this period of my life. Easy to say, but full of experiences, happiness, disappointments, laughs, anger and even tears… All of you guys have a space in my heart and will never forget all the time we spend together. Thanks for being my colleagues, friends and family at the same time: Akiko Ono, Matthias Hoffmann, Ravi Dwivedi, Rajesh Velagapudi, Sreedhar Kilaru, Prayook Srivilai, Wassana Chaisaena, Kateřina Svobodová, Sudhakar Peddireddi, Kalyani Pemmasani, Annette Naumann, Martin Rühl, Dorothea Fragner, Banyat Cherdchim and Dong Sheng Wei. i Acknowledgments Katka, Wassana, Sudhakar, and Dong Sheng, deserve a special mention (5 stars!) for their invaluable support in the last part of the PhD. Thanks a lot! Impossible to forget Mojtaba Zomorrodi for being always ready to provide anything one ever can imagine for working in the lab, always with a smile on his face. Karin Lange and Alexandra Dolynska deserve a special place for being nice co-workers and loyal friends. I would like to take this opportunity to thank all the members of the Section Molecular Wood Biotechnology and Technical Mycology, and the Section Forest Botany and Physiology of Trees of the Büsgen-Institute, who were always very friendly and kind, especially Andrea Olbrich for their readiness to help me with microscopy stuff; Bernd Kopka for solving the endless problems with the computer and Gisbert Langer-Kettner for solving many of our technical problems in the lab and in the office. During the studies I had the support in the lab form different students, to whom I am also grateful to discover, develop and improve my abilities in teaching: Carmen Yen, Samantha Navarro, Alberto Domingo, Frauke Kleemann, Olivia Sánchez, David Ris and Marlit Arndt. I would like to thank Prof. Stefan Schütz and Pavel Plašil from the Section Forest Zoology and Forest Conservation of the Büsgen-Institute for the collaboration in the mite project. Without your interest in the topic, we would have not gone too far. Prof. Ottmar Holdenrieder from ETH Zurich is greatly acknowledged for his idea to work with coprini, for his valuable comments and his readiness to discuss about my work. I am very grateful to Prof. Susanna Badalyan and her students Lilit Melikyan and Helen Avetisyan from Yerevan State University, Armenia for the nice collaboration built during all these years, hopefully we will continue with our coprini studies! Dr. Peter Beutelmann from the Johannes Gutenberg-University of Mainz is gratefully acknowledged for providing the strains that initiated the chapter 5 of this thesis. ii Acknowledgments International and national friends gave a multicultural invaluable friendship during this time: Crina Vulpe, Tanja Dučić, and especially Artemio Carrillo and Rosa Isela Rasura from bringing a piece of Mexiko to the table every time we had opportunity to share knowledge and Mexican songs! Thank you for being there in good and bad times. I would like to thank to my cousin Miguel Cagigal Navarro, who always encouraged and supported me to continue with postgraduate studies. Special position in this list is given to František Vilčko for the time he has shared with me, his invaluable support and for giving color to my life. No words to express the immense gratitude to my parents Isabel Gonzalez-Vizcarra and Othón Navarro-Tobón, to my sister Diana, brothers Saul, Mario Alberto and Héctor for the immense support given all this years. Thank you all for being there during this time. Last but not least I am very grateful to the Mexican folk, without the money provided for my studies through the Mexican Council for Science and Technology (CONACYT, grant 118752) all this would have not been possible, and the DBU (Deutsche Bundesstiftung Umwelt) for financial support to the laboratory. iii Table of contents Table of contents Acknowledgments i Table of contents v Summary xi Zusammenfassung xv 1. Introduction 1 1.1 Fungi 2 1.2 Wood degradation 3 1.3 Laccases 4 1.4 Mushrooms as economical resources 4 1.5 Coprini 6 1.6 The model fungus Coprinopsis cinerea 10 1.7 Aims of this thesis 16 1.8 References 18 2. Lignocellulolytic activities within coprini species 27 2.1 Abstract 28 2.2 Introduction 29 2.3 Growth of coprini species on wood and other living and dead plant material 31 2.4 Brown-rot or white-rot fungi? 55 2.5 Lignocellulolytic enzymes from coprini species 61 2.6 Conclusions 67 2.7 Acknowledgments 67 2.8 References 67 3. Growth of selected coprini on lignocellulosic substrates and detection of laccase activities 87 3.1 Abstract 88 3.2 Introduction 89 3.3 Materials and methods 90 v Table of contents 3.3.1 Evaluating ITS sequences from coprini 90 3.3.2 Screening of the strains on different media 91 3.3.3 Phenoloxidase activity on solid media 91 3.3.4 Growth on lignocellulosic substrates 91 3.4 Results and discussion 92 3.4.1 Identification of the strains 92 3.4.2 Growth of the strains on artificial media (MEA) at different environmental conditions 97 A. Effect of temperature 97 B. Effect of pH 99 C. Phenoloxidase activity 100 D. Growth of selected strains on lignocellulosic substrates and laccase activities 102 E. Degradation of 14C-labelled lignin: 104 3.5 Conclusions and outlook 106 3.6 Acknowledgments 107 3.7 References 107 4. Biologically active metabolites and medicinal properties of coprinoid mushrooms 111 4.1 Abstract 112 4.2 Introduction 113 4.2.1 Biological safety 114 4.2.2 The Coprinus syndrome 116 4.2.3 Antimicrobial compounds within coprini 121 4.2.4 Hypoglycemic properties 123 4.2.5 Antitumor properties 123 4.2.6 Proteinases 129 4.2.7 Surface proteins 129 4.3 Conclusions 132 4.4 Acknowledgments 133 4.5 References 133 vi Table of contents 5. Monstrosities under the inkcap mushrooms 143 5.1 Abstract 144 5.2 Introduction 145 5.3 Material and methods 146 5.3.1 Strains, culture conditions and spore germination 146 5.3.2 Microscopy 146 5.3.3 DNA techniques 147 5.4 Results and discussion 148 5.4.1 ITS analysis 148 5.4.2 Culture and mycelial characteristics of the four newly isolated strains 149 5.4.3 Fruiting abilities of the Coprinellus sp. 1 153 5.4.4 Fruiting abilities of the Coprinopsis clastophylla 2, 3 and 4 isolates 156 5.4.5 Fruiting abilities of the Coprinopsis clastophylla type strain 159 5.4.6 Fruiting bodies and related sterile structures in Coprinopsis clastophylla, and its anamorph Rhacophyllus lilacinus 161 5.5 Conclusions 163 5.6 Acknowledgments 164 5.7 References 164 6. The course of fruiting body development in the basidiomycete Coprinopsis cinerea (Coprinus cinereus) 167 6.1 Abstract 168 6.2 Introduction 169 6.3 Material and methods 170 6.3.1 Strain and general culture conditions 170 6.3.2 Fruiting bodies dissection in fresh material 170 6.3.3 Microscopic preparations 170 6.4 Results and discussion 171 6.4.1 Time course of fruiting body development 171 6.4.2 Hyphal aggregation 173 6.4.3. Primordia development 175 6.4.3.1 Gill development 179 vii Table of contents 6.4.4. Sexual reproductive development 181 6.4.5. Fruiting body maturation 186 6.4.6. Autolysis 187 6.5 Conclusions 188 6.6 References 188 7. Effect of copper in Coprinopsis cinerea (Coprinus cinereus) development 195 7.1 Abstract 196 7.2 Introduction 197 7.3 Materials and methods 198 7.3.1 Coprinopsis cinerea strain and culture conditions 198 7.3.2 Sampling procedures 199 7.3.3 Enzymatic assays 199 7.3.3.1 Extracellular laccase activity 199 7.3.3.2 Nitrate reductase and Nitrite reductase activity 200 7.3.3.3 Ammonium determination 200 7.3.3.4 Glucose determination 201 7.3.4 Nitrate assimilation gene cluster prediction 201 7.3.5 DNA isolation, RNA isolation, cDNA synthesis and transcript analysis 201 7.4 Results and discussion 204 7.4.1 Copper, laccase activity and fruiting in liquid cultures 204 7.4.2 Fruiting pathway of etiolated stipes in liquid cultures 209 7.4.3 Copper and fruiting in solid cultures 211 7.4.4 Other effects by copper in liquid cultures at 37°C 217 7.4.5 Transcript profiles for the nitrate assimilation gene cluster 220 7.5 Conclusions 222 7.6 Acknowledgments 222 7.7 References 222 8.