Target Sites of Fungicide Action Editor Wolfram Koller Assistant Professor Department of Plant Pathology Cornell University New York State Agricultural Experiment Station Geneva, New York Boca Raton London New York CRC Press CRC Press is an imprint of the BocTaay lRor a&t Fornan ciAs Gnronu pA, anr bionfro rmLao bnudsinoenss First published 1992 by CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 Reissued 2018 by CRC Press © 1992 by CRC Press, Inc. CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright. com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not- for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Target sites of fungicide action/editor, Wolfram D. Köller, p. cm. Includes bibliographical references and index. ISBN 0-8493-6855-3 1. Phytopathogenic fungi--Control. 2. Fungicides--Physiological effect. 3. Phytopathogenic fungi--Physiology. I. Koller, Wolfram D. SB733.T37 1991 632’ .952--dc20 91-22650 A Library of Congress record exists under LC control number: 91022650 Publisher’s Note The publisher has gone to great lengths to ensure the quality of this reprint but points out that some imperfections in the original copies may be apparent. Disclaimer The publisher has made every effort to trace copyright holders and welcomes correspondence from those they have been unable to contact. ISBN 13: 978-1-315-89798-1 (hbk) ISBN 13: 978-1-351-07708-8 (ebk) Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com PREFACE Fungi are not only the most important class of plant pathogenic organisms, but they are also the causal agents of animal and human mycoses. Fungal diseases of crops can be devastating, and disease symptoms have been described since farming became the major source of our food and fiber supply. 1 Although the incidence of human diseases caused by fungi is less than that of bacterial and viral infections, they do occur and can be life- threatening.2 Major weapons in the combat of fungal diseases have been, in both cases, chemicals with antifungal activities. Interestingly, two different names have emerged in the past. The term "fungicide" describes antifungal compounds developed for agriculture, whereas "antifungal agent", "antifungal drug" and "antimycotic" are more commonly used in medicine. From a historic point of view, this distinction is not surprising. The first generation of agricultural fungicides had no application in medicine, and the first antifungal drugs introduced in the late 1950s, amphotericin B and 5-fluorocytosine, were useless for farmers. Over the last two decades, however, the distinction between the two classes of antifungal compounds has become less pronounced. In particular the discovery and devel- opment of azoles with their broad field of application in agriculture and medicine has triggered increasing cooperation between the mycological disciplines concerned with either control of plant diseases or antifungal chemotherapy. While the recognition of mutual scientific interests was on the rise, the future perspectives for fungicides and antifungal drugs began to drift apart. Although the common goal remained the same, agricultural fungicides and medical drugs are viewed differently. What has happened? It had been the progress in chemistry and chemical engineering in the first half of the 20th century that made the reliable control of insects, weeds and plant diseases a reality. The initial acceptance of early organic pesticides was overwhelming, and it is worth re- membering that P. Muller, the inventor of DDT, was honored with the Nobel Prize in 1948. This positive perception has vanished and given place to a global awareness of pesticide threats to our environment. Undoubtedly, the chemical control of plant diseases, although still irreplaceable, has become part of our current "chemophobia". The issue has become increasingly emotional, and public concerns are not always justified from a scientific point of view. Moreover, the tremendous progress in safety and application rates made over the last four decades is not always fully recognized and appreciated. Regardless of many existing misperceptions, however, some of the fungicide-related problems are real, and scientists engaged in fungicide research will have to answer a question of considerable importance: Was the development of chemical fungicides a dead-end street with low expectations for the future, or will it be feasible to apply our scientific progress to the development of novel and effective chemical control agents with extremely specific targets and thus without side effects on non-target organisms and the environment? Many scientists at the forefront of plant disease control appear to have answered this question. They have dismissed chemical measures from their arsenal of future strategies, and the term "innovative" has become a synonym for "non-chemical". For example, future prospects for chemical control are no longer part of a recent book on innovative approaches to plant disease control. 3 Expectations are high of finding widespread and durable solutions by recombinant DNA technology, be it through the engineering of disease-resistant crop cultivars, or through the construction of greatly improved biocontrol agents. Undoubtedly, the exciting potential of biotechnology will have beneficial impact in the future. But will it make chemical control entirely obsolete, and will it always lead to entirely "nontoxic" means of plant disease control? Zehf'l concluded in his recent reflections on innovation in plant disease control: "We might even find that new chemical pesticides carefully regulated by government agencies and applied by informed users may be among the safest of the mechanisms available to control many plant diseases and pests." It certainly would be unwise to abandon innovative research on novel fungicides before safe and lasting ''non- chemical" alternatives are in sight. Most likely, the breeding of disease-resistant cultivars, the development of biocontrol agents, and new chemical fungicides will co-exist in the future as they have in the past. In contrast to chemical fungicides, the public demand for novel antifungal drugs has increased over the last decade. The incidence of fungal infections is on the rise since an increasing number of immunocompromised patients became prone to opportunistic and sometimes life-threatening fungal infections. 56 The treatment of deep-seated and systemic • mycoses is difficult with the currently prescribed arsenal of antimycotic drugs, and a number of new antimycotics are under clinical development. 7 Mandatory for the treatment of many opportunistic fungal infections is the availability of highly specific antifungal drugs without side effects on the patient. Progress toward high levels of fungal specificity has already been made with the group of systemic fungicides developed for agriculture, and newer devel- opmental antifungal drugs are becoming increasingly similar to these site-specific fungicides, in particular within the group of sterol biosynthesis inhibitors. In general, the systemic treatment of any patient, be it plant, animal or human, is only possible with inhibitors of fungal-specific targets. Specificity, however, also has its short- comings. As the experience with specific agricultural fungicides has shown, these inhibitors are more prone to the development of resistance. Although fungal resistance has not been a serious problem for the current antimycotics, this might change if immunocompromised patients with mycotic diseases have to be maintained under long-term treatment. The iden- tification of suitable and novel antifungal targets, the modification of already existing in- hibitors, and the prediction of inherent risks of resistance will require profound data on the molecular mode of inhibitor action. The contributions in this book are a comprehensive summary of our current knowledge, but they are also meant to raise questions and to stimulate future research. The first two chapters are dedicated to the carboxamides and hydroxypyrimidines. They represent groups of relatively old systemic fungicides with a remarkable level of specificity and selectivity. The benzimidazoles described in Chapter 3 with their broad spectrum of plant disease control have been severely affected by the development of resistance. The more recent cloning and sequencing of the target gene has not only provided insight into the molecular specifics of resistance, but it also explained the mode of action of compounds with exclusive activity against resistant genotypes. Edlich and Lyr (Chapter 4) describe the mode of action concepts that emerged only recently for a group of rather unrelated fungicides, the dicarboximides and the aromatic hydrocarbons. The class of specific fungicides available for the control of oomycetes is described in depth in Chapter 5. All antifungal inhibitors covered in the first five chapters are exclusively used in agriculture. This restriction is not always apparent from a mode of action point of view, and the better understanding of molecular principles might lead to closely related inhibitors with activities against a broader or different spectrum of fungal pathogens. The melanin biosynthesis inhibitors (Chapter 6) are different from all other antifungal agents and represent a novel and desirable trait in fungal disease control. They are neither fungitoxic nor fungistatic but interfere with the penetration of fungal pathogens into their plant hosts and, thus, are the first representatives with an indirect mode of action. The inhibitors of fungal sterol biosynthesis described in Chapters 7, 8 and 9 comprise the currently most successful group of antifungal compounds. Several different enzymes of the sterol biosynthetic pathway are already antifungal targets, and interesting inhibitor lead structures for additional enzymes are promising. Moreover, the sterol biosynthesis inhibitors have crossed the border of disciplines and are successfully used in both agriculture and medicine. Several specific fungicides with known modes of action did not warrant a separate chapter. They are described, however, in Chapter 10, which is dedicated to the role of molecular studies in fungicide research. Finally, the question is asked in Chapter 11 whether it might become feasible to use industrial enzymes as disease control agents. The contributions as a whole are a mirror of the tremendous progress made over the last two decades. This progress becomes immediately evident from the contents of a mon- ograph dedicated to the same subject, written by Lukens in 1971.8 There are no reasons to believe that progress will slow down in the future. Hopefully, this book will stimulate ideas and encourage research in the field of antifungal inhibitors. Wolfram Koller Geneva, New York I. Agrios, G. N., Plant Pathology, 3rd ed., Academic Press, San Diego, 1988. 2. Rippon, J. R., Medical Mycology, 2nd ed., W. B. Saunders, Philadelphia, 1982. 3. Chet, 1., Innovative Approaches to Plant Disease Control, John Wiley, New York, 1987. 4. Zehr, E. 1., Reflections on innovation in plant disease control, Plant Dis., 73, 372, 1989. 5. Bodey, G. P., Fungal infections in cancer patients, Ann. N.Y. Acad. Sci., 544, 431, 1988. 6. Spencer, P. M. and Jackson, G. G., Fungal and mycobacterial infections in patients infected with the human immunodeficiency virus, J. Antimicr. Chemother., 23 (Suppl. A), 107, 1989. 7. Saksena, A. K., Girijavallabhan, V. M., Cooper, A. B., and Loebenberg, D., Recent advances in antifungal agents, Annu. Rep. Med. Chern., 24, Ill, 1989. 8. Lukens, R. J., Chemistry of Fungicidal Action, Springer-Verlag, New York, 1971. EDITOR Wolfram Koller is assistant professor of plant pathology at the Geneva Campus of Cornell University, where he joined the faculty in 1986. He was trained in chemistry and biochemistry and received his Ph.D. in chemistry from the Philipps University of Marburg in 1979. He spent two postdoctoral years as a recipient of a DFG fellowship at the Institute of Biological Chemistry of Washington State University in Pullman. Thereafter he became the head of a biochemistry laboratory in fungicide research at Bayer in Leverkusen. His research interest is focused on the biochemistry of plant disease control. He has authored more than 60 scientific contributions and is co-author of a textbook on undergraduate chemistry training. He has given more than 30 invited lectures and seminars. His current appointments are on the editorial board of Phytopathology and on several national and international committees in pesticide resistance. CONTRIBUTORS Pierre Benveniste, D.S. Hideo Ishii, M.Agr. Professor Laboratory Head Institut de Biologie Moleculaire des Division of Plant Protection Plantes Fruit Tree Research Station, MAFF Institut de Botanique Tsukuba, lbaraki, Japan Universite Louis Pasteur Strasbourg, France Paul A. J, Janssen, Dr. President and Director of Research Amanda Davis, B.Ag.Sci. Janssen Pharmaceutica Research Scholar Beerse, Belgium Department of Biochemistry University of Melbourne Wolfram Koller, Ph.D. Parkville, Victoria, Australia Assistant Professor Cornell University Wilfried Edlich, Dr. Department of Plant Pathology Research Scientist New York State Agricultural Experiment Insitute of Plant Protection Research Station Kleinmachnow Geneva, New York Kleinmachnow, Germany Yasuyuki Kubo, Ph.D. Assistant Professor S. G. Georgopoulos, Ph.D. Laboratory of Plant Pathology Professor Faculty of Agriculture Department of Plant Pathology Kyoto University Agricultural University of Athens Kyoto, Japan Athens, Greece Horst Lyr, Dr. Bruce Grant, Ph.D. Professor and Department Director Reader in Biochemistry Institute of Plant Protection Research Department of Biochemistry Kleinmachnow University of Melbourne Kleinmachnow, Germany Parkville, Victoria, Australia A. J. Poulose, Ph.D. Julia M. Griffith, Ph.D. Research Director Research Fellow Exploratory Research Department of Biochemistry Genencor International University of Melbourne South San Francisco, California Parkville, Victoria, Australia Alain Rahier, D.S. D. W. Hollomon, Dr. Directeur de Recherche Department of Agricultural Sciences Centre National de la Recherche University of Bristol Scientifique AFRC Institute of Arable Crops Research lnstitut de Biologie Moleculaire des Long Ashton Research Station Plantes Bristol, United Kingdom Strasbourg, France Hugo Vanden Bossche lsamu Yamaguchi, Ph.D. Department Head Senior Scientist and Professor Department of Comparative Biochemistry Microbial Toxicology Laboratory Janssen Research Foundation The Institute of Physical and Chemical Beerse, Belgium Research (RIKEN) Wako-shi, Saitama, Japan G. A. White, Ph.D. Research Scientist Agriculture Canada Research Centre London, Ontario, Canada TABLE OF CONTENTS Chapter 1 Target Sites of Carboxamides ............................................................ 1 G. A. White and S. G. Georgopoulos Chapter 2 Target Sites of Hydroxypyrimidine Fungicides .......................................... 31 D. W. Hollomon Chapter 3 Target Sites of Tubulin-Binding Fungicides ............................................. 43 Hideo Ishii Chapter 4 Target Sites of Fungicides with Primary Effects on Lipid Peroxidation ................. 53 W. Edlich and H. Lyr Chapter 5 Target Sites of Fungicides to Control Oomycetes ....................................... 69 J. M. Griffith, A. J. Davis, and B. R. Grant Chapter 6 Target Sites of Melanin Biosynthesis Inhibitors ........................................ 101 Isamu Yamaguchi and Yasuyuki Kobo Chapter 7 Antifungal Agents with Target Sites in Sterol Functions and Biosynthesis ............. 119 Wolfram Koller Chapter 8 Target Sites of Sterol Biosynthesis Inhibitors in Plants ................................. 207 P. Benveniste and A. Rahier Chapter 9 Target Sites of Sterol Biosynthesis Inhibitors: Secondary Activities on Cytochrome P-450-Dependent Reactions ............................................................ 227 Hugo Vanden Bossche and Paul A. J. Janssen Chapter 10 Target Research in the Discovery and Development of Antifungal Inhibitors .......... 255 Wolfram Koller Chapter 11 Biotechnology and Fungal Control ..................................................... 311 A. J. Poulose Index ................................................................................... 319