An Anecdotal History of Nematology

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The book is addressed to all who love to work on nematodes. It will be of interest also to historians of science and to any zoological library.

An Anecdotal History of Nematology

ematology is not only about those lovely Nobel prize winning creatures, nematodes, but also about the people who work with them, the nematologists. Your good friends Bengt Eriksson, David McNamara and John Webster have cajoled a whole galaxy of story-telling nematologists to reminisce about their loved ones, their nematodes, and to tell us how they got to know them so well. It is all disclosed in „An Anecdotal History of Nematology“. It is good nematology, but it’s different, and you will be able to read the other side of some of our nematological world’s most fascinating discoveries and about their discoverers.

An Anecdotal History of Nematology

J. M. Webster, K. B. Eriksson & D. G. McNamara Editors

An Anecdotal History of Nematology

An Anecdotal History of Nematology J. M. Webster, K. B. Eriksson & D. G. McNamara Editors

Sofia–Moscow 2008

AN ANECDOTAL HISTORY OF NEMATOLOGY J. M. Webster, K. B. Eriksson & D. G. McNamara (Editors) First published 2008 ISBN 978-954-642-324-5 (paperback) ISBN 978-954-642-426-6 (e-book) Book and cover design: Zheko Aleksiev © PENSOFT Publishers All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the copyright owner. Pensoft Publishers Geo Milev Str. 13a, Sofia 1111, Bulgaria Fax: +359-2-870-42-82 [email protected] www.pensoft.net Printed in Bulgaria, 2008

TABLE OF CONTENTS – – – 1. 2. 3.

4.

5.

6. 7. 8. 9.

10.

Contributors 7 Preface 11 Prologue 14 Our early stars 17 Bengt Eriksson Nematological nebulae in Europe and the USA 33 John M. Webster and Seymour D. Van Gundy First catch your nematode! – the development of methods for recovering nematodes from soil 59 David McNamara Through nematode diversity to living soil processes – holistic studies aid progress 67 Gregor Yeates Nematode physiology: Significant developments in the understanding of the biology of simple eukaryotic animals 80 Howard Ferris and Haddish Melakeberhan Molecular taxonomy of nematodes 98 Pierre Abad and Philippe Castagnone-Sereno A history of potato cyst nematode research 107 Ken Evans and David L. Trudgill Cereal cyst nematode complex 129 Roger Rivoal The science and art of soybean cyst nematode research 137 Terry L. Niblack and Don P. Schmitt Nematodes/Viruses/Plants: “A 32-year love affair” 152

Derek J.F. Brown 11.

12.

Horticultural hazards: In and out of hot-water baths and other transient technologies 168 Simon R. Gowen and Philip A. Roberts The spread of nematology to developing countries: A case study 187 Michel Luc

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Contributions by Latin American nematologists to the study of nematode plant disorders and related impact on crop production 191 Rosa H. Manzanilla-López, Patrick Quénéhervé, Janete A. Brito, Robin Giblin-Davis, Javier Franco, Jesse Román and Renato N. Inserra

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Quarantine nematodes 219 David McNamara The pinewood nematode: a personal view 231 Helen Braasch and Manuel M. Mota History of the development of nematodes as biocontrol agents 246 Parwinder S. Grewal and Harry K. Kaya Dynamics of nematological infrastructure 258 Roland N. Perry and James L. Starr Nematology: Dreams and visions of the future 272 A nematology dream: Miscalculations, and false prophecies? 272 Ernest C. Bernard Vision of nematology in Canada in the next 50 years 276 Guy Bélair The future of nematode systematics and phylogeny over the next 50 years 279 Virginia R. Ferris A nematologist’s dream 283 Florian Grundler My nematology dream 286 Paulo Vieira The devil’s advocate 288 Derek J.F. Brown My dream, not a nightmare 292 Forest Robinson My dream of the future of nematology and chemical communication research – 50 years from now 295 Ekaterina Riga C. elegans as a model system for space travel 297 Robert Johnsen and David Baillie

15. 16.

17. 18. (i) (ii) (iii)

(iv) (v) (vi) (vii) (viii)

(ix)

CONTRIBUTORS PIERRE ABAD, UMR Interactions Plantes Microorganismes et Santé Végétale, INRA/CNRS/Université de Nice Sophia Antipolis, BP 167, 06903 Sophia Antipolis Cedex, France. [email protected] DAVID L. BAILLIE, Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, V5A 1S6, Canada. [email protected] GUY BÉLAIR, Agriculture and Agri-Food Canada/Agriculture et Agroalimentaire Canada, St. Jean-sur-Richelieu, Quebec, J3B 3E6, Canada. [email protected] ERNEST C. BERNARD, Entomology and Plant Pathology, The University of Tennessee, Knoxville, TN 37996-4560, USA. [email protected] HELEN BRAASCH, Kantstrasse 5, D-14471 Potsdam, Germany. [email protected] JANETE A. BRITO, FDACS, DPI, Nematology Section, PO Box 147100, Gainesville, FL 32614-7100, USA. [email protected] DEREK J.F. BROWN, RD Consultants, “Penbro”, 3, Neofit Rilski Street, 2778 Banya, Razlog Municipality, Bulgaria. [email protected] PHILIPPE CASTAGNONE-SERENO, UMR Interactions Plantes Microorganisms et Santé Végétale, INRA/CNRS/Université de Nice Sophia Antipolis, BP 167, 06903 Sophia Antipolis Cedex, France. [email protected] BENGT ERIKSSON, Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Box 7026, SE-750 07, Uppsala, Sweden. [email protected]

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KENNETH EVANS, Nematode Interactions Unit, Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK. [email protected] HOWARD FERRIS, Department of Nematology, University of California, Davis, CA 95616, USA. [email protected] VIRGINIA FERRIS, Department of Entomology, Purdue University, West Lafayette, IN 47907-2089, USA. [email protected] JAVIER FRANCO, Fundación PROINPA, Casilla Postal 4285, El Paso, Cochabamba, Bolivia. [email protected] ROBIN M. GIBLIN-DAVIS, Fort Lauderdale Research and Education Center, University of Florida/IFAS, Fort Lauderdale, FL 33314, USA. [email protected] SIMON R. GOWEN, School of Agriculture, Policy and Development, The University of Reading, Reading, RG6 6AR, UK. [email protected] PARWINDER GREWAL, Department of Entomology, The Ohio State University, Wooster, OH 44691, USA. [email protected] FLORIAN GRUNDLER, Department of Applied Plant Sciences and Plant Biotechnology, Institute of Plant Protection, BOKU-University of Natural Resources and Applied Life Sciences, A 1190 Wien, Austria. [email protected] RENATO N. INSERRA, FDACS, DPI, Nematology Section, PO Box 147100, Gainesville, FL 32614-7100, USA. [email protected] ROBERT JOHNSEN, Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, V5A 1S6, Canada. [email protected]

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CONTRIBUTORS

HARRY K. KAYA, Department of Nematology, One Shields Avenue, University of California, Davis, CA 95616, USA. [email protected] MICHEL LUC, formerly Muséum National d’Histoire Naturelle, Paris, France. DAVID MCNAMARA, formerly East Malling Research Station, East Malling, Kent , UK and European and Mediterranean Plant Protection Organization, Paris, France. [email protected] ROSA H. MANZANILLA-LÓPEZ, Plant Nematode Interactions Unit, Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK. [email protected] HADDISH MELAKEBERHAN, Agricultural Nematology Laboratory, College of Agriculture and Natural Resources, Michigan State University, East Lansing, MI 48824, USA. [email protected] MANUEL M. MOTA, NemaLab-ICAM, Departmento Biologia, Universidade de Évora, 7002-554 Évora, Portugal. [email protected] TERRY L. NIBLACK, Department of Crop Sciences, University of Illinois, Urbana, IL 61801, USA. [email protected] ROLAND N. PERRY, Plant Pathogen Interactions Division, Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK. [email protected] PATRICK QUÉNÉHERVÉ, Laboratoire de Nématologie Tropicale, PRAM, IRD, BP 8006, 97259 Fort-de-France, Martinique, France. [email protected] EKATERINA RIGA, Nematology, Washington State University, Prosser, WA 99250, USA. [email protected]

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ROGER RIVOAL, UMR INRA/ENSAR, Biologie des Organismes et des Populations Appliquée à la Protection des Plantes (BiO3P), BP 35327, 35653 Le Rheu Cedex, France. [email protected] PHILIP A. ROBERTS, Department of Nematology, University of California, Riverside, CA 92521, USA. [email protected] FOREST ROBINSON, USDA-ARS, 2765 F&B Road, College Station, TX 77845, USA. [email protected] JESSÉ ROMÁN, Crop Protection Department, Agricultural Experiment Station, PO Box 21360, Rio Piedras, Puerto Rico 00928. [email protected] DONALD P. SCHMITT, 14844 Highway 5, Marceline, MO 64658, USA. [email protected] JAMES L. STARR, Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843-2132, USA. [email protected] DAVID L. TRUDGILL, Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA Scotland, UK. [email protected] SEYMOUR D. VAN GUNDY, Department of Nematology, University of California, Riverside, CA 92521, USA. [email protected] PAULO CEZANNE VIEIRA, NemaLab/ICAM, Universidade de Évora, Évora, Portugal. [email protected] JOHN M. WEBSTER, Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, V5A 1S6, Canada. [email protected] GREGOR W. YEATES, Landcare Research, Private Bag 11052, Palmerston North 4442, New Zealand. [email protected] 10

CONTRIBUTORS

PREFACE Derek Brown first had the idea of writing this anecdotal history of nematology and, when he invited us three to undertake the editorial work, we were convinced by his reasons as to why now was the ideal time to prepare such a book. Compared to other areas of science (e.g. chemistry, physics), nematology is a young subject. Even the related biological fields of entomology and plant pathology have much longer histories. Less than 150 years have passed since the true pioneers of nematology were in action. Those of us of a certain age knew the previous generation of nematologists, who themselves knew some of the pioneers. So we realized that it would be a good idea to get the stories about the “old days” down on paper before it was too late. One of the advantages of being old (and there are a few!) is that you become rather good at history – because you were part of it or, at least, you think that you remember that you were! A new generation of nematologists is now holding centre stage, and, although they know well how their technology was developed and they understand perfectly the significance of their results in the context of the nematological study, they, perhaps, do not always fully appreciate the broader biological relationships or how and by whom the existing body of knowledge was obtained. Nematology has also been, up to now, a “small” science with a limited number of adherents. Even though it has often been subdivided into different subject areas, everyone who called him/herself a nematologist knew nearly every other nematologist, no matter in which country they lived and worked. However, as the subject areas themselves have become more specialized, they have tended to drift apart, so that there is an increasing trend for nematologists involved in particular fields of the science to know only minimal amounts about areas of nematology other than their own. We believe that this trend is not good for nematology as a whole and we feel that it is a distinct advantage for those in different areas to be aware of each other’s work and to share ideas. We are all working on nematodes, after all! By presenting a history of discovery in nematology in the form of different chapters relating to different subject areas, we hope that we can, in some small measure, help to bridge the expanding gap. To appreciate fully the significance of the advances PREFACE

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in nematology, it is necessary not only to understand something of the knowledge that has been gained but also to recognize the achievements of the men and women who have been responsible for the advances. By producing this book, we hope to promote an appreciation of the subject matter of nematology and of the people involved in its discovery, within our own tiny sector of the broad front of scientific progress. We hope that this does not sound too grandiose and we hope that it does not dissuade you from reading and enjoying this book as you trip through time, technique and title! We began preparation of the book by, firstly, trying to decide what was to be covered by the name “nematology”. We agreed that we should only include the study of nematodes relevant to plant protection, as this has traditionally formed a quite self-contained area of study, and, of course, has been the particular area of expertise of the three editors throughout their careers. This definition includes all soil and plant-inhabiting species, and those soil species that are not plant parasites (whose relationships with each other and with other soil organisms contribute to the general health of the soil). Also included are the entomopathogenic species, which play an important role in controlling insect pests of plants. We have not included in our particular definition of nematology the study of marine and fresh-water nematodes which have long had their own particular specialists; nor have we included nematodes that are parasitic in humans and other mammals, which are generally covered in medical and veterinary research. The field of study surrounding the genetic elucidation of Caenorhabditis elegans, though contributing enormously to our understanding of the Nematoda and of living systems, is not included in detail as the mass of information emanating from that field would have required another volume. We then divided up the science of nematology into some of its major subdivisions, and invited experts in these areas to write a chapter on their history. We asked them to highlight the principal milestones along the road to the present, to tell us about the leading personalities who achieved these milestones (and about others who were just interesting personalities), and we asked them to make the history personal and anecdotal. And much to our surprise, very few of those experts that we approached declined to participate, despite the fact that everyone is always very busy these days (just as nematologists have always been), and despite the fact that “anecdotal” is a strange word, possibly difficult for the non-English speakers among our authors (and there are a few!). 12

PREFACE

Just as we have defined „nematology“ for our own particular purposes, so have we adopted our own interpretation of “anecdotal”. Originally, an anecdote was simply something that has not been published. The word now generally refers to the telling of some biographical incident that may or may not be true; in fact, often information that could be considered to be untrustworthy. Our intent, however, was to include, within the historical narrative, references to the personalities engaged in the progress of nematological research, to try to add stories (but true stories!) about these people that might bring the history to life. We left it to the individual authors to decide how, and how much, they should incorporate of the anecdotal elements. We also invited a number of nematologists to stick their necks out and predict how their own particular subject area will develop in future years. Predictions of any kind usually provide immense entertainment to readers in the future, who can smugly laugh and compare the way things really turned out with the totally erroneous vision of the prophets. Of course, such prophets are not usually experts in their chosen field of prediction. Our prophets, on the other hand, are true experts and visionaries and we are quite confident that the future will arrive just as they predict!!! With such a wide range of individuals contributing their own views, it is inevitable that the style and format of different chapters will vary. Nevertheless, we hope also that our readers will be enlightened and amused. We recognize that, despite our own enthusiasm, very few people outside of the field have even heard of nematodes. So, to those readers who are not already nematologists, this book might convince you of what we have always known to be true: that nematodes are delightful little creatures and that nematology is an endlessly fascinating, multifaceted subject. Finally and wholeheartedly, we thank all our contributors, and the many other nematologists who contributed in diverse ways, and hope that everyone involved will feel that this has been a worthwhile project. JOHN M. WEBSTER BENGT ERIKSSON DAVID MCNAMARA August, 2007

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PROLOGUE According to the famous Russian nematologist and philosopher A. Paramonov, nematodes are an ancient group of organisms exhibiting true biological progress, manifested by a high level of taxonomic diversification, intensive speciation, diversity of life cycles and life strategies, trophic specialization and the occupation of various habitats. The differentiation of nematology, the science about nematodes, into a great variety of branches, reflects that diversity. To look into the anecdotal history of nematology was an excellent idea, now realized through the efforts of many outstanding nematologists who have shared their personal memories, feelings, thoughts and a lot of information accumulated through many years committed to nematology. The main driving forces, both objective and subjective, the circumstances, ideas and concepts marking the development of our science and the progress of nematological research all over the world are traced. The photographs present a rich gallery of pioneer nematologists and teams – insights into early epochs and into the richness and excitement of more recent times. B. Eriksson leads us back to the beginning of the science of nematology and its “early stars”. The first nematological centres were established in Europe and North America, inspired and pushed forward by the discovery of the microscope, elucidation of the pathogenic role of plant parasitic nematodes, the social needs, and….the curiosity of the scientists (J.M. Webster & S.D. Van Gundy’s chapter). The centres had, and still have, a significant influence on the establishment of nematology on the world scene and on its continued advance. It is fascinating to follow the genealogy of the different schools and research branches with their inputs into uncovering the secrets of the nematodes, showing their economic importance and learning how to manage their control. Special attention is given to the events of nematology in Latin America where a range of remarkable scientists have studied the diversity of nematode pests under a variety of agricultural and horticultural circumstances. Development of nematology in this part of the world has often occurred in collaboration with colleagues from centres in North America and Europe. Progress in science is highly dependent on the development of research methodology. D. McNamara outlines the evolution and application of methods for recovering nematodes from soil and plant 14

PROLOGUE

tissues – prerequisites for further studies. H. Ferris and H. Melakeberhan demonstrate how research into nematode biology provoked the promotion of new methods, which was followed by discoveries in nematode physiology, adaptation strategies, life cycles, plant-nematode interactions etc. Further, P. Abad and P. CastagnoneSerrano review the modern molecular approaches used in nematode taxonomy and phylogenetic reconstructions, briefly showing the main problems and shortcomings, and prospecting the future of this quickly-evolving branch of nematology. I was touched by the stories told by G. Yeates and M. Luc – they fully confirm the concept that the history of a science is (nothing but) the summary of the personal experiences of the scientists themselves. In a treatise of this kind we must not omit the prime nematodes such as the cyst nematodes of potato, soybean and cereal, the virus vectors, the burrowing nematode, the stem and bulb nematodes, the pinewood nematode and Caenorhabditis elegans – all have their own sagas marking the different developmental phases of nematology as a whole. Most of these nematodes are quarantine objects and, nowadays, quarantine services are becoming very important for preventing the spread not only of well-known pest organisms but also of potentially invasive species, as pointed out by D. McNamara. The flourishing of nematology is intimately connected with the establishment and diversification of nematology infrastructure – journals, newsletters, conferences and societies – as distinctly shown by R. Perry and J. Starr. All these structural units help substantially to stimulate the exchange of knowledge and ideas, facilitate contact between scientists and promote international collaboration. Of special interest is the final chapter on dreams and visions of the future of nematology. Comfortingly, the majority of predictions are positive, foreseeing new horizons, new findings, and an increase in the role of nematological research; all this enthusiasm, though, perhaps somewhat linked with reality by the pessimism of the “devil’s advocate”…! Books – they have their own history. This book is dedicated to the 50th Anniversary of the European Society of Nematologists (ESN) which was celebrated at the 28th Symposium of the ESN in Bulgaria, in 2006. A memorable congress, and one which I was honoured to be allowed to organize. We have to say a big THANK YOU to our colleagues who have contributed the various chapters and PROLOGUE

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have shown us the many-sided face of our science in a vivid and inspired manner. We are very much obliged also to the editors J. M. Webster, K. B. Eriksson and D. G. McNamara and to D. Brown who came up with the idea for this book, and finally to PENSOFT publishers. This work has particular value for the young and future generations; it shows the long road that has been traveled from the first discoveries and inventions to present-day nematology. I leave you, dear Reader, with a wonderful history of nematology presented with a sense of humour and true love for those little but very powerful creatures – the nematodes. VLADA PENEVA, Past-President, European Society of Nematologists. January, 2008

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PROLOGUE

1. OUR EARLY STARS BENGT ERIKSSON Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, SE-75007 Uppsala, Sweden.

There is a saying that the history of a country is the history of its rulers or sovereigns. So, who are the excellencies, the “stars”, in the history of nematology? Before we identify some of the more obvious figures and pioneers, let us describe in historical terms the development of the study of these ‘beautiful little beasts’, commonly called nematodes or roundworms, and so poetically described by B.G. Chitwood, as cited: “The grace of movement of some lowly soil-inhabiting forms finds little equal among other living organisms, being comparable to the gliding of snakes, which Solomon noted as one of the four mysteries of life. The complex patterns of their body markings and of the head and other parts might well be used in designing ladies’ dresses. None of their graces and beauty is suggested by a name that carries the stigma ‘worm’”.

From ancient times… In their classic book the Chitwoods (1974) traced nematological history back to parallel the history of any field of zoology. If not mentioned specifically as nematodes, or even roundworms, the phenomena associated with them were well-known from the ancients in scriptural times. Often cited are the biblical passages in Numbers 21, where Moses “fiery serpent” made from brass is supposed, rightly or wrongly, to be the Guinea worm (Dracunculus medinensis) causing cutaneous lesions on the extremities of man. To remove the parasite the adult female is rolled out of the ulcer and onto a forked stick, the so called ‘Indian barber’ technique (Maggenti). The way it is so figuratively described in being extracted from the body tissues has a OUR EARLY STARS

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striking resemblance to the rod of Asclepius, the symbol of medicine. Human-parasitic nematodes (e.g., Ascaris, Dracunculus), or at least the diseases they cause, seem to have been known since before the times of Moses, as indicated in an Egyptian papyrus dating from the 1550s B.C., and published in 1889 by the German Egyptologist, G. Ebers, as “Der Papyrus Ebers”. Probably the oldest reference to parasitic nematodes is found in the Yellow Emperor’s Classic of Internal Medicine from China, some 2700 B.C., where a disease is accurately described that thousands of years later is known as an Ascaris infection. A giant of these ancient times is, of course, the Greek philosopher and natural scientist, Aristotle (384–322 B.C.), also known as the Father of Zoology. In his “Historia Animalium” he refers to nematodes, especially Ascaris and the pinworm, and states that “these intestinal worms do not in any case propagate their kind”. Aristotle was a strong believer in abiogenesis, “spontaneous generation”, a theory that was not refuted until the 19th century by Louis Pasteur, and that we were again confronted with in nematology in the 1700s. In his historical parallelisms Chitwood considered that after the ancient times there followed the many hundred years of the “dark ages” of science, nematology included, followed by a “blank period” that was terminated with the Arab philosopher and physician, Avicenna (ca. 1000 A.D.), who had an enormous influence on human medicine. In his classic “Canon Medicinae” Avicenna refers to parasitic worms, notably ascarids and dracunculids. The “dark” and superstitious medieval period was followed in the eighteenth century by the Age of Enlightenment in science, literature, culture etc. In that enlightened period are some prominant figures that we, perhaps somewhat generously may enrol, in the cadre of nematologist, most notably Linnaeus and Needham.

…to the age of enlightenment As we see from the above synopsis, nematology in its broad sense has a long history in parts of the Old World. It was the invention of the microscope, in the 1600s that opened the eyes of Needham and Linnaeus and all those scholars, scientists and curious naturalists of the time. Thanks are due to the Dutch draper and official Antonie 18

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van Leeuwenhoek, who later in his career as a naturalist (died 1723 at 91 years old), started to build microscopes. Science began to flourish, and nematologists now had the means to study also the lesser creatures in soil and plants. Probably the first person ever to study nematodes under a microscope was Borellus, who in 1656 published the first information on a free-living nematode, viz. the ‘vinegar eelworm’ (Turbatrix aceti). I personally, remember how the local vinegar producer helped me each year by providing these nematodes for demonstration in the zoology courses. Even before Borellus, William Shakespeare who, in his play ‘Love’s Labour’s Lost’ (1594), may have been referring to a plant parasitic nematode, the wheat gall nematode, Anguina tritici, with the line, “Sowed cockle, reap’d no corn”. However, those of us nematologists who are more or less lenient with the possible nematological connection in this statement, may surrender to the fact that these cockles might have contained the “longitudinal fibres” that an English clergyman discovered some 150 years later. Hence, it was the poet who unwittingly was the first to point at a plant parasitic nematode, later named Anguina tritici, causing the wheat “cockles”. It was as a clergyman that Turbevill Needham in 1743 described, before a probably startled audience of the Royal Society of London, what he saw when he examined shrunken and blackened wheat kernels under the microscope: “I dropped water upon it…when to my great surprise these imaginary fibres…took life, moved irregularly…”. It is not surprising, therefore, that this report contributed to the prevailing theories on spontaneous generation (generatio spontanea). An “all category star” whose 300th anniversary we celebrated in 2007 and who ought to be mentioned in this context is the Swedish polymath, botanist, physician and natural scientist, Carl Linnaeus (1707–1778), who was raised to the nobility with the name Carl von Linné. In his ‘Systema Naturae’ (1758) he pioneered a practical classification of organisms by his binomial system with a genus and a descriptive species attribute – “Deus creavit, Linnaeus disposuit” (God created, Linnaeus ordered), as he so proudly declared. Linnaeus named several animal and human parasitic nematode species, including Ascaris lumbricoides and Dracunculus medinensis, which he recorded under the group Vermes (“worms”). He was first to give a scientific name to a free-living nematode, the vinegar eelworm, which he named Chaos redivivus (“the resurrected chaos”), with the comment: “…reviviscit ex aqua per annos exsiccaOUR EARLY STARS

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tum” (“raised from the dead by water after years of desiccation”). Had he heard of Needham or did he make the same observation? Linnaeus, who seemed cautious towards generatio spontanea, also knew that the wheat seed gall nematode was, at that time, known as Vibrio tritici.

Modernists in Europe In his attempts to identify historical epochs Chitwood chose 1870 as the beginning of recent nematology, “because following this time there was a marked uptrend in the amount and average quality of the work”. Publications by O. Bütschli (1875), J.G. de Man (1884) and R. Leuckart (1876) are typical of the time. A few years earlier (1865) H.C. Bastian had brought new ideas into the field of nematology through his famous “100-new-species-of-free-living-nematodes” paper, and Thorne (1961) appears to consider these publications as the beginning of the science of nematology. Raski (1959) defines the period of “early modern nematology” as the years 1845–1907, beginning with the great work of F. Dujardin, “one of the major taxonomic works in early nematology”. With the upswing in nematology after the Second World War the writer dares to suggest that the period of “contemporary nematology” perhaps began with the introduction, in 1943/44, of the soil fumigants DD and EDB for the control of plant parasitic nematodes. Various monographs published during the latter half of the 19th century were milestones in the development of nematology. They were primarily concerned with nematode taxonomy and the differentiation of forms living in fresh-water, soil and marine habitats. Plant-parasitic nematodes came suddenly and dramatically into the public eye when H. Schacht (1859) identified the beet cyst nematode (Heterodera schachtii) as a threat to the beet sugar industry in Europe. Investigations into the control of this nematode dominated the literature of much of European nematology during this period, with researchers such as Schmidt, Strubell, Liebscher and Kühn. In 1829, root galling, caused by Subanguina radicicola, was reported from Norway, where the entomologist W.M. Schoeyen (1885) illustrated and described it as Tylenchus hordei, without knowing that it had already been described by Greeff in Germany. Another significant event during these years was the discovery of the root-knot 20

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nematode, described as “Vibrios” by M.J. Berkeley in 1855, and named, Meloidogyne, by E.A. Goeldi in 1892. The increased interest in plant parasitic nematodes was excellently summarized by Kati Marcinowski (1909) in her book “Parasitisch und semiparasitisch an Pflanzen lebende Nematoden” (Parasitic and semiparasitic nematodes living on plants). Let us linger on three particular figures in Europe that stick out as “stars” during this time. Henry Charlton Bastian (1837–1915) has a reputation as an outstanding taxonomist (see above) and was prominent in the classification of the Nematoda. He contrasted the animal parasitic nematodes with the free-living, and subdivided the latter into continental (soil and fresh water nematodes) and marine forms. Johann Adam Otto Bütschli (1848–1920) was not only a nematologist, in the modern sense of the word, but also a pioneering histologist and professor in Heidelberg, Germany. His embedding methods for thin tissue sections paved the way for all biological research, and proved that nematodes could be useful study objects in embryology and genetics. With his excellent, detailed drawings Bütschli set the standard for nematode illustration. Many morphological details that he discovered are still used in nematode taxonomy. Johannes Govertus de Man (1850–1930), the “Altmeister der Nematodenkunde” (The old master of nematode science) (Micoletzky, 1925) and “one of the most interesting personalities ever engaged in nematology” (Thorne), completes this remarkable triumvirate of founders of the science of nematology. De Man’s first nematological paper (1876), dealing with soil nematodes, was the beginning of nematology in The Netherlands and Belgium (Coomans). Financially independent, and an all-round zoologist, he felt free to pursue any problem and did much of his later research in private life. His devotion to nematodes resulted in nearly 50 papers among a total of about 170 scientific publications. Andrassy (1976) grades him as “the first modern nematologist” and recommends his beautifully illustrated monograph (de Man, 1884) on nematodes in soil and fresh water as “the bible of nematologists”. The versatile German plant pathologist Julius Kühn was a leading figure in the campaign against the beet cyst nematode. Kühn (1871) probably was first to use soil fumigation against nematodes when he applied carbon disulfide to nematode infested beet fields. He also tried trap crops, although with limited success, and crop rotation, which became the principle control method in the sugar OUR EARLY STARS

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beet industry. Kühn has his “star” status also for his discovery of Ditylenchus dipsaci, found in teasel (Dipsacus fullonum), and the causal agent of “Stockkrankheit” in rye and several other crop plants. Another celebrity is described as follows: “…somewhat oldfashioned in appearance with his round-lensed glasses…full of vitality…for a scientist he was unusually smartly dressed (!) …travelling to and from work on his high stepping bicycle with its large front basket…and upon his arrival sustained himself with a piece of cheese!”(Hooper, 1994, Ann. Rev. Phytopathol.). “He” is the “Father of Nematology” in Britain, Tom Goodey (1885–1953). In his early scientific career Tom Goodey interested himself in soil protozoa. In his forties he started as a parasitologist in St. Albans, and from 1926 he specialized in free-living and plant-parasitic nematodes. In 1947, he was transferred to Rothamsted as head of the new Nematology Department where he stayed until a year before his death. His major research contribution was on the biology of the stem nematode, but he published also on nematodes that cause galls on plants. Tom Goodey was a real ambassador for nematology and instigated training courses in nematology, the second one of which, in Harpenden in 1951, was followed by an international symposium. This marked the beginning of regular, international nematology symposia in Europe, eventually under the auspices of the European Society of Nematologists. His famous textbook, “Plant Parasitic Nematodes and the Diseases they Cause” (1933) was a landmark in the development of the science, and a couple of years before his sudden death he completed his well-known book, “Soil and Freshwater Nematodes” (1951), which was revised and enlarged in 1963 by his son, J. Basil Goodey. Hooper points at Tom Goodey as a person with many interests, most notably as a singer and actor. His theatre and concert hall performances were so professional and successful that he adopted a stage name, Roger Clayson, so as not to conflict with his profession as a scientist! Imagine our symposia banquets with Tom Goodey performing!

A nematological troika In the Russian nematological starry sky I see three fixed stars that represent very different personages with a profound influence on modern Russian nematology and with worldwide impact. Ivan 22

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Nikolaevich Filipjev created a new scientific school in Russia – nematology, and is looked upon as the founder of Russian nematology. He was born in 1889 in St. Petersburg and died in 1940. He came from a wealthy family and had a good education. As a student, he was advised to study free-living, marine nematodes, and had the opportunity to make a short visit to Naples, Italy. At the age of 21 he published his first nematological paper, which was about the nervous system of nematodes. During the years 1923–1933 Filipjev had a position in Petrograd (St. Petersburg) where he lectured on entomology and evolutionary theory. He even wrote on evolutionary aspects of modern genetics, the first of its kind in Russia. Filipjev’s entomological interests took him to the USA where, in 1928, he presented a paper on the general classification of nematodes which was of fundamental importance for nematode taxonomical research. Just as important was his book on systematics, “Nematodes that are of importance for agriculture”, published 1934 in Russian. A year later he asked the Dutch parasitologist, S.J.H. Schuurmans Stekhoven Jr., to cooperate in editing a second edition of his book, in English. An intimate cooperation between the two followed, and resulted in the co-authored classic, “A Manual of Agricultural Helminthology”, published in 1941 (after Filipjev’s death). The book – with the motto “Concordia parvae res crescent” (“Collaboration fosters small undertakings”) – was “dedicated to the memory of the pioneers in the field of agricultural helminthology, Cobb, Filipjev, de Man, Micoletsky and Ritzema Bos”. Filipjev’s epoch-making contribution to systematics is his classification of the class Nematoda, in which he grouped free-living and parasitic families into a total of eleven orders. At the same time as his international reputation increased, being elected a member of scientific societies in various countries, Filipjev experienced increasing hardships in his own country. In 1931 he was arrested and charged with counter-revolutionary, subversive activity. The charges, however, were dropped, but in 1933 he was again arrested and exiled to Alma-Ata, Kazakhstan, from where he was prohibited to leave. He became persona non grata, but was allowed to continue his research. It was during these years that he wrote all his major works on the Nematoda and prepared his fundamental revision of nematode taxonomy. In 1937, Filipjev was arrested a third time and charged as an “enemy of the Soviet people”. His correspondence with colleagues abroad continued until the end of OUR EARLY STARS

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1937 when “the sendings suddenly stopped” (Schuurmans Stekhoven). He was taken to court and sentenced in March, 1938. Though the exact particulars remain obscure, Filipjev’s death is officially reported as being on the 22nd October, 1940. According to his biographers, S.Ya. Tsalolikhin et al. (Russian J. Nematol. 8:173–9), a fellow-prisoner was an eyewitness to Filipjev’s execution, but official documentation is lacking. His good name and scientific recognition were officially restored in 1956. His two sons also perished in the war. A full bibliography of Filipjev numbers 51 entries. When reading the biography and memorial sketches by S. Tsalolikhin et al. one gets the impression of Filipjev as a purposeful and hard-working naturalist with wide biological interests, climbing to the summit of Vesuvius rather than moving in society, when in Naples. He did not give in to the charges made against him. He appears as being of an independent nature, kind to his friends and colleagues but uncompromising and even sarcastic with scientific opponents. Deeply committed to his science he sometimes appeared lost in his thoughts and was absentminded – found to buy tickets twice for the same train and tried to get his watch repaired when he had forgotten to wind it. (How many of you, fellow nematologists, have not been frenetically searching for your specs until your colleague tells you that there is something on your nose…!) While I.N. Filipjev was held in high esteem internationally as a taxonomist, Alexander Alexandrovich Paramonov (1892–1970) became renowned for his evolutionary concepts and hypotheses on nemic relationships and nematode evolution. He had wide biological interests, which ranged from studies of fur-bearing animals to mammalian skulls and entomology to general biology. From 1925 onwards, his research involved also free-living, soil and phytoparasitic nematodes. His main interest was evolutionary theory and the major principles of phylogeny. After 1945, he concentrated his studies on plant nematodes in the broad sense, and also on saprozoic forms. He drew up an ecological classification of soil and plant nematodes, considering their (biocoenotic) interrelationships with fungi, bacteria and other organisms. His particular interest was the evolution of parasitic adaptations of plant nematodes. Paramonov, who was known for his almost encyclopaedic knowledge, held various positions at museums and academies in Moscow and Leningrad, where he lectured on zoology, entomology, general biology and 24

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Darwinism. Among his 130 publications on various biological subjects, about 50 deal with nematodes. Paramonov summarized his research in the internationally recognized three-volume monograph, “Fundamentals of Phytohelminthology” (1962, 1964 and 1970), which was also translated into English. In his student days A.A. Paramonov was arrested and charged with active participation in the revolutionary student movement. He was expelled from the university but continued his studies in Heidelberg in Germany, where he was taught nematology by Professor Otto Bütschli. The frosty connections between Russia and Germany forced Paramonov to return to Russia where he in spite of personal restrictions, managed to finish his undergraduate studies in 1922. During the following years he held academic positions in Moscow. In 1948, he was dismissed from the Academy because of his criticism of the biological concepts of Lysenko. He was restored to favour in 1952, and devoted his time until his death to develop phytohelminthology as an independent scientific discipline in Russia. Although Paramonov deprecated political and scientific trends of opinion that appeared iniquitous or totally wrong he treated those that he considered to be offenders with indulgence, and was an attentive listener in discussions. Eino Krall, Estonia (Fig. 1), who Fig. 1. Eino Krall (right), Estonia, fraternizing with Vicente Campos, met Paramonov and even visited his home in Moscow and experienced the Brasil. officialdom of the 1950s, found him to be a very nice and humanitarian person, who treated his students and colleagues with respect and amiability. “A brilliant lecturer and teacher he was always surrounded by young people to whom he was a sincere friend”, to cite his biographer. If Filipjev and Paramonov were the prominent founders of Russian nematology, Ekaterina Sergeevna Kirjanova (1900–1976) could be considered “the Mother of Soviet Plant Nematology” (W.R. Nickle). Certainly, she had a widely recognized impact on Soviet nematology. Dr. Kirjanova was born in Alma Ata and graduated in OUR EARLY STARS

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1928 at the Middle Asian University in Tashkent. She was awarded a post-graduate studentship and moved, in 1930, to Leningrad and the Zoological Institute of the USSR Academy of Science, where she worked until her death. There she worked with I.N. Filipjev for a couple of years. She even cooperated with Filipjev and Schuurmans Stekhoven in their co-authored book. Ekaterina Sergeyena (Fig. 2), as she was commonly called, was concerned mainly with the systematics and ecology of free-living and plant parasitic nematodes but interFig. 2. Ekaterina Sergeevna Kirjanova chatting up ested herself also in insect Ben Chitwood at the Warsaw ESN Symposium parasitic nematodes and hairin 1967. worms. A life-time achievement of hers was to put together, as completely as possible, a nematode collection for the USSR. Nematologists visiting her were expectantly greeted with, “did you bring any specimens for the collection!” (according to A. Ryss). Her ambition resulted in a collection of 50,000 slides and several thousand fixed nematode samples, together with 3,000 hairworm samples. Throughout, she worked untiringly on the identification to species level of her samples, with detailed recording of various aspects pertaining to their taxonomy and to the damage they caused. A colleague commented on her sedulous work that “not even a bomb-hit could remove her from the microscope”. She published some 170 scientific papers, several books and guided 25 dissertations. Only days before her sudden death, 76 years old, she was planning the details of her forthcoming participation in an expedition to the wild mountains in Tajikistan….!

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A star-spangled nematological banner The “versatile and ingenious”, “illustrious and influential” but also “adventurous” are but some of the epithets that have been used in historical overviews to characterize one of Nematology’s most distinguished individuals, Nathan Augustus Cobb (1859–1932) – “The Father of Nematology”, not only in the United States but worldwide. He introduced the term “nema”, though “nematode” has been the commonly used word , and also the name “nematology” (first in 1914) as a new branch of study in science and distinct from helminthology. Cobb’s name is connected with various methods (Cobb decanting and sieving) and techniques (Cobb aluminium slide). For many of us he is also well-known for his magnum opus, “Contributions to a Science of Nematology”, a monumental compilation of his greatest works in nematology through the years 1914–1935. In this context we must not forget the name W.E. Chambers, who illustrated the papers with unsurpassed, beautifully detailed drawings, “under the author’s personal supervision”(!), as Cobb self-confidently(?) pointed out in his paper “The Mononchs”. Was he adventurous – or maybe, rather restlessly always on the move? At least that is the impression one gets when reading about Cobb travelling all over the world, Europe (to complete his doctorate in Jena, Germany), America, Asia, Australasia, Oceania, finally in 1915 to settle down in North America, where he became recognized as the founder of nematology in the United States. His family, with six children, followed him, probably suffering some hardships, finding dad at times flogging soap and watches to survive. Certainly, he was a versatile and talented man! Preceding his last 15 or so years as a full-time nematologist he held positions as a plant pathologist and studied fungal and bacterial diseases of plants. He even developed an interest in the breeding and cultivation of wheat, selection of wheat varieties and the handling of grain in commerce, the cultivation of sugarcane and won approval for his standardization of cotton grading! He was influential, as exemplified by the legacy he left, the Division of Nematology (in the United States Department of Agriculture), which was officially established in 1929, and ensured the continuation of his work. In the group of dedicated workers who belonged to the staff or were influenced by Cobb we find many of the names of contemporary nematologists, OUR EARLY STARS

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such as G. Steiner, B.G. Chitwood, G. Thorne, J.R. Christie, and A.L. Taylor, all of whom rose to stardom. If N.A. Cobb, as the first full-time nematologist in the United States, made the most of his talents, Benjamin G. Chitwood (1907–1972), supervised by Cobb in the late 1920s, is alternatively referred to as “the greatest systematist of Nematoda”, “likely the most outstanding nematologist of all time” and “one of the early architects of the Science of Nematology”, whose career had a profound influence on nematology. As a professional, after he received his Ph.D. degree at the age of 24, B.G. Fig. 3. Herman Nilsson-Ehle, Chitwood held positions as nematologist Swedish geneticist, renowned for his pioneer breeding work and zoologist in the U.S. Department of on cyst nematode resistance in Agriculture, and later held short term posithe early 20th century. tions as university professor, chief nematologist in Florida, consultant in the Kaiser Foundation Research Institute and various short term assignments until he retired, in 1964. Difficult to get on with and apparently a poor mixer, occasionally involved in controversy, his personal life was marred by adversity. In his later years he abandoned nematology and took up other interests. We know him best for his numerous contributions to zoology, parasitology and nematology, and for his tremendous classic “An Introduction to Nematology”, co-authored with his wife, M. B. Chitwood. Apparently, students and collaborators found Ben Chitwood not only a brilliant scientist, but also exigent as a teacher and often a complex character – would that be a correct definition of a genius? But, “Ben was also willing to put himself out for his students and give unlimited amounts of his time to them”, as Father R.W. Timm observed in a personally kept obituary. Father Timm has given us some vivid, on-the-spot accounts of what it was like to be with this remarkable man, who excelled not only at theorizing but was also eminently practical. Students were taught how to collect “spaghetti worms” in the slaughter-house and from bear faeces in the Zoo, not that pleasurable or even as innocuous, as it may seem. Ben’s comment was, “if you can’t stand smell you will never be a 28

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parasitologist” (it reminds me of the smell from a dissected seal in a small lab in St. Albans during my training years). Students being examined were urged to gather and classify all nematodes to be found in a snake from a road kill, from cow manure or retrieved from plant tissues. If you have available the SON Nematology Newsletter of March, 1973 I recommend that you read Father Timm’s most amusing anecdotes! I met or rather saw Dr.Chitwood once at a symposium in the late 1960s – and, sadly, that was a person, as I remember him, sitting alone on a sofa, smoking cigarettes, going downhill…. Cobb, as the prominent “Father of Nematology”, surrounded himself with several prospective nematologists who, as time went by, became the founders and architects of the science in its modern shape. One of them was Gerald, “Jerry”, Thorne (1890–1975), whose impact on nematology developed after the Second World War. Influenced by Cobb and making acquaintance with the sugar beet cyst nematode in 1917, he joined the Nematology Section of the Bureau of Plant Industry of the U.S. Department of Agriculture. During his 38 years in this organization his official headquarters was the Regional USDA Laboratory at Salt Lake City, Utah, his native state. After his mandatory retirement, in 1956, he was an indefatigable professor and lecturer at several universities, and a consultant and expert, advising widely in North America and worldwide. What an enviable “otium cum dignitate” (leisure with dignity), to cite Cicero, or perhaps more appropriate in this case, to put it “otium est pulvinus diaboli” (“leisure is the devil’s cushion”)! With unimpaired energy and dedication, Thorne continued working until a few weeks before he died. Again, the name of a nematologist is inevitably associated with a book, “Principles of Nematology” (1961), a classic that had a profound influence on nematology and nematologists in the latter half of the 20th century. I met professor Thorne once, during an ESN symposium, and was very surprised (flattered) when this world famous, nematological giant approached me, an embryo nematologist in the street, and asked me about my training, research interests and place of work. Actually, my instructor and supervisor, Professor Sven Bingefors, was one of those who had part of their training with Thorne in Utah, in the 1950s. Another “star”, old enough considering its origins long before all Chinese and Egyptian notations, but probably not as brilliant as the OUR EARLY STARS

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nematological heroes we have remembered thus far, would be our co-operative friend, Caenorhabditis elegans. “Rundmask får Nobelpris” (“Roundworm receives Nobel Prize”) was the announcement in empathic letters in Swedish newspapers in the autumn of 2002. Handsomely, our gracile study object gave its collaborators, Sydney Brenner, Robert Horvitz and John E. Sulston, quite a bit of the cash flow we experience each year when the Prize is bestowed on those who (hopefully) deserve it. With its 959 cells and somewhat 20,000 genes, C. elegans – oh yes, kindly assisted by the three laureates(!) – has helped us to sniff at the secrets behind plagues like cancer, AIDS and Alzheimer’s. Caenorhabditis, as benefactor of mankind, is no doubt a unique star in the nematological sky. By 1998, C. elegans had already placed itself at our disposal and had become “a landmark in biology: determination of the essentially complete DNA sequence of an animal genome” to cite Science, 1998, 282: 2011.

Reflections Who are the real “stars”, the “old” and the brilliant – I repeat my question from the introductory lines. Who was most important: the “collector” who surveyed and described the manifoldness of nematode species and tried to open our eyes to their biology, the ingenious one who invented and constructed apparatus, techniques and equipment for laboratory work, or the brave and imaginative enthusiast who entered the untrodden paths, launching and defending new ideas and theories (Fig. 3). I think it was Einstein who maintained that “fantasy is more important than knowledge” – but isn’t knowledge, acquired by the assiduous microscopist and observer, the necessary sound and solid platform on which to gain the fruitful results of our imaginations! Most important, though, are the vital discussions where concepts and ideas are brought face to face, without which there would be no progress in science. Some of us have vivid memories from symposia sessions of such “fights” between stars and giants of all ages and extraction. Among all the giants we must also remember the patient laboratory assistants and technicians who with skill and care extracted the soil samples, prepared the solutions and mounted the nematodes to be studied by the star. Remember Chambers who helped Cobb 30

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with the incredible, artistic drawings that have stimulated teachers and students for almost a century to try to gain a closer aquaintance with the “nems”. How many of us have not stared, at the full, en face view of the grisly Mononch head “about to seize its victim”, sending thrills of fascination along the backbone of students? A tribute also to those, not mentioned above, who welcomed “nematologists in embryo” to their institutes and laboratories to be trained and to gain Fig. 4. J.W. Seinhorst receives the insignia (hat, ring & diploma) as Doctor Honoris Causa at the first hand experience Swedish University of Agricultural Sciences, 1983. from the master. The writer remembers vividly the friendly and helpful atmosphere at Rothamsted in the early 1960s, with Fred Jones and all his colleagues chatting at the morning coffee-breaks in the cosy little lab prep room, with Basil Goodey who devoted much of his time to teach us beginners nematode taxonomy based on his newly issued book, with David Hooper so kindly teaching us the “Seinhorst two-flask method”, and making life-long bonds of friendship between staff and more or less occasional visitors and researchers. Some of the giants from ages ago live on with their names tied to techniques and methods. Wim Seinhorst (Fig.4) is one of them. We know too about de Man indices measures, the Demanian system named by Cobb in tribute to de Man, Cobb’s slide and Peter’s 1 ml nematode counting slide, the Fenwick can, Seinhorst’s and Oostenbrink’s elutriators, just to mention a few. And during a two months’ stay and training in Wageningen, The Netherlands, I found my humble self accommodated in a room on OUR EARLY STARS

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Fig. 5. Professor Sigurd Andersen (in shirt-sleeves in the middle) demonstrating his cereal cyst nematode field plots for a group of Nordic nematologists. Far left: Wim Seinhorst (visiting Denmark) and Knud Lindhardt; to the right: Gunnar Videgård (with hat), Osmo Roivainen, Sven Bingefors and Mrs. Kirsten Andersen.

Ritzema Bos weg… – I found myself as at home with my Ditylenchus and Aphelenchoides pets. Many others, besides myself, have similar precious memories and experiences from the labs at Rothamsted, Wageningen, Gent and elsewhere. It has been my privilege to select and pause at some of the “early stars”, and I think you will agree, dear reader, that the choices I made were not too controversial. I would not be surprised, however, if on similar reflection, you miss one or two or several names that certainly reached the status of grandeur and have left behind a lot of anecdotes (Fig. 5). Therefore, stop a minute in your hustle and bustle, and remember what they meant to you; I know they deserve it! In preparing this article I acknowledge the help I had from historical reviews as indicated in the text, obituaries etc, as well as from stimulating talks with Eino Krall.

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2. NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA JOHN M. WEBSTER Department of Biological Sciences Simon Fraser University, Burnaby, Vancouver, Canada &

SEYMOUR D. VAN GUNDY Department of Nematology, University of California, Riverside, USA

Research into plant parasitic nematodes evolved steadily throughout the first part of the twentieth century and then entered a rapid phase of expansion in mid-century as clusters of nematologists with significant technical and financial support became established in research centers in many parts of the world. The collaboration and productivity of researchers in these emerging and expanding nematological research centers tended to overshadow the excellent research done by scattered, lone research nematologists and their teams. This may have been inevitable as the thrust of the collective expansion focused on increased food production rather than on curiosity driven research. The trigger for this expansion was, undoubtedly, a combination of the discovery, in the 1940s, of chemical nematicides and the increased awareness by both agricultural and government agencies of significant nematode-induced loss of crop yield during a period of projected, worldwide, food shortage and major replant problems in perennial crops. This period of expansion of nematological research led, concurrently, to a wider recognition in plant pathology and agriculture of the economic importance of plant parasitic nematodes. Nematology, as it became commonly known, expanded to include the study of insect parasitic and free-living, soil nematodes because of their close association with agriculture. Inevitably, through association and extensive networking and exchange of ideas, nematology came to include also free-living nematodes of freshwater and marine habitats as well as nematode pests of forest trees. NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

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The “enhanced food production” mandate worldwide in the 1950s and 1960s triggered different approaches to solving nematode problems in different countries due to socio-political and cultural differences, variation in climatic and soil conditions in the agricultural areas and to the contemporary level of nematological expertise in different jurisdictions. Europe and the USA already had a history of nematological research, although its organization and management had developed along different lines. Europe, due to its longer history in zoological research, had its nematological foundations scattered in the laboratories of eminent, lone, research zoologists in established institutions. There was rarely more than one or two per country, they were usually located in universities and their research was eclectic and individualistic. The change in momentum and the expansion in nematology to one with a distinct agricultural focus resulted in the establishment in several European countries of large centers of nematological research each with a distinct utilitarian focus. In the USA, nematologists had for many years been located in the laboratories of the US Department of Agriculture (USDA) in its network of regional centers and through the Land Grant University system of Agricultural Experimental Stations. It was within the USDA research framework that, early in the twentieth century, N.A. Cobb not only established a strong and influential nematological research reputation, but trained what was to become the next generation of nematological researchers and teachers. These well-trained researchers either remained in the USDA or relocated to some of the major universities across the country, where they were well-positioned to lead the development of major nematological research centers during the rapid growth phase of the 1950–1960s. This lends itself to a genealogical approach to the history of nematology in the U.S.A. It is upon this array of different, evolving centers of nematological research in Europe and the United States that this chapter focuses. It will help to identify the role and contributions of these research centers, and of the associated nematological personalities, in those formative years of change.

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Early research centers in Europe As the ravages of the Second World War faded low agricultural productivity was manifest, in part due to several years of monocropped land and to widespread crop diseases. The potato cyst nematode and beet cyst nematode, together with some local, severe outbreaks of stem eelworm and lesion nematode, were the major nematode pests. Unfortunately, and sadly, there were virtually no young, trained nematologists. There were, however, a few older, senior nematologists in countries across Europe who recognized that the control of nematode pests of potatoes, sugar beet and fodder crops could significantly help to improve Europe’s struggling agricultural renewal. They had the foresight to realize the necessity of moving away from the traditional approach of “lone star” nematologists, working diligently but undervalued and in relative isolation, and moving towards the concept of larger, well-funded, collaborative research teams with international links. Significant initiatives were taken to establish research units in nematology at some of the major research centers in crop production and protection. Within the space limitations of this chapter the emergence and development of some of these nematological units during this critical period of change are addressed. Unfortunately, the particular focus in time and structure of the chapter results in omitting reference to many of Europe’s nematologists of the day, in particular those that worked alone or in smaller centers. In England, a new Department of Nematology was established in 1947 at one of Europe’s largest centers of agricultural research, Rothamsted Experimental Station. The already distinguished nematode taxonomist, Tom Goodey, was brought in as Head from a nearby research outpost of the University of London, namely the Institute of Agricultural Parasitology at St. Albans. He and his initial staff of eight, including his son J.Basil Goodey, D.W. Fenwick, M.T. Franklin and B.G. Peters provided substantial taxonomic expertise plus ecological and control interests. This department was to grow into one of the world’s most famous nematological research centres with particular emphasis on cyst nematodes, their taxonomy, biology, population genetics and control. The department flourished, and it attracted and spawned many distinguished nematologists during the headships of F.G.W. Jones (1956–1979) and A.R. Stone (1979–1986). In addition to the four NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

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Fig. 1. Staff of the Nematology Department at Rothamsted Experimental Station in the late 1970s.

founding members of the Department these early years included C.C. Doncaster (nematode behaviour), K. Evans (potato cyst nematode), J.J. Hesling (nematode populations), D.J. Hooper (taxonomy), B.R. Kerry (biological control, and current Head), J.E. Peachey (chemical nematicides), R.N. Perry (physiology), A. Shepherd (cyst nematode hatching; ultrastructure ), D.L. Trudgill (Heteroderidae), H.R. Wallace (nematode ecology and locomotion), J.M. Webster (nematode host-parasite relationships), T.D. Williams (cereal nematode management), A.G. Whitehead (nematode control) and R.D. Winslow (cyst nematode populations). This remarkable department prospered (reaching a complement of about 45 nematologists) (Fig.1) at a time when agricultural research focused on maximizing crop yields, on the search for new nematicides, on the development of resistant cultivars, and when new pest problems ensured the allocation of increased resources for research. The increasing concerns worldwide, in the 1970s, over the use of nematicides stimulated the search for alternative methods of nematode pest management and increased support of research into basic nematode biology and ecology. However, the early 1980s marked the beginning of a marked decline in resources for nematological research. This was mainly the result in some countries, including the UK, of decreasing direct government support for agricultural research in favour of the concept that agriculture should be treated like any other industry by financing its own research. This trend was 36

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compounded by the overproduction of European agriculture and the lack of development of new, safer nematicidal products. Unfortunately, many research centers were unable to find adequate, alternative sources of funds so the previous level of research was unsustainable, and some smaller research groups were disbanded. Rothamsted Research (as it became known), like several other research centers, moved away from discipline-based structures and the Nematology Department was closed in 1987. Currently, a smaller but active group of nematologists (about 21 including visiting researchers from around the world), led by B.R. Kerry, conduct their nematological research within a program on rhizosphere biology, and so encompass nematode-host recognition processes, nematode interactions with the rhizosphere microbial community and nematode management. This multifaceted approach to plant nematode control involves extensive collaboration with researchers in the public and private sectors in the UK (e.g., CABI Bioscences and the University of Reading) and abroad (e.g., The Netherlands, Norway, Sweden, Cuba and the USA). A smaller research group of nematologists (R.S. Pitcher, D.G. McNamara and J.J.M. Flegg), working at the East Malling Research Station, were focused on interactions between nematodes and other pathogens, in particular, the virus vectors Xiphinema and Trichodorus. Concurrent with these activities were those of C. Ellenby (The University, Newcastle-on-Tyne) on potato resistance to cyst nematodes, R. Cook (Welsh Plant Breeding Station) on resistance to stem eelworm in clovers, H. Howard (Plant Breeding Station, Cambridge) on commercializing resistant potatoes to cyst nematode, J.F. Southey (ADAS, Harpenden) on plant quarantine, M.R. Siddiqi (Commonwealth Institute of Parasitology) on tylenchid taxonomy and H.J. Atkinson (University of Leeds) on biochemistry and physiology. To help train nematologists in the UK for the expanding research needs a graduate diploma programme was established in 1958 at Imperial College, University of London. B.G. Peters headed the programme, the first of its kind in Europe. Through funding from Shell Research, the marketers of D-D, one of the most successful nematicides, a customized research and training facility was built to house the twelve-month diploma programme and the increasing number of researchers. A prominent research group assisted Peters and N.G. M. Hague (cyst nematode control) to develop research excellence in nematicides, especially soil fumigants, (F. Call, NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

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O.F. Lubatti and A.B. Page). Later, with the evolving research emphasis, N. Croll (behaviour), A.A.F. Evans (ecology and survival), W. Hominick (insect nematodes), and D.J. Wright (physiology and control) joined the center. Within the Scottish Horticultural Research Institute, subsequently renamed the Scottish Crops Research Institute (SCRI), a major nematological research group was established by C. E. Taylor in the Zoology Department. Its focus was on the nematode vectors of viruses causing disease in raspberry and strawberry crops. He was soon joined by P. Thomas (trichodorids) and W. Robertson (virus retention in longidorid and trichodorid vectors). At that time chemical control of virus vectors was an important research endeavour. In the 1970s, Taylor was appointed Director of the Institute, and D. Trudgill became Head of the Department, and was joined by T. Alphey, B. Boag and D. Brown. The mandate of the Department expanded to include research on potato cyst nematodes (PCN) because Globodera pallida was increasingly recognized as a serious threat to UK potato production, replacing G. rostochiensis which by then was beginning to be controlled by resistant potato cultivars. In the mid 1970s, the Scottish Plant Breeding Station (SPBS) in Edinburgh was closed and relocated at the SCRI, bringing J. Forrest and M. Philips to further develop PCN research while Brown led research on nematode vectored potato viruses. Later, V. Blok was appointed to help expand the application of molecular techniques, and J. Jones, B. Griffiths and R. Neilson to expand research on nematode-plant community interactions (Fig. 2). Subsequent internal reorganization and restructuring at SCRI, in the 1990s, resulted in closure of the Zoology Department and the nematologists retired or moved into several newly created departments. In The Netherlands, the long tradition of Wageningen as a centre for plant pathology and agricultural research provided the foundation for one of the world’s great centres of nematology. M. Oostenbrink and J.W. Seinhorst, “nematological giants” of the 1950s and 60s, began to build the nematological reputation of the Agricultural University of Wageningen and of the independent Instituut voor Plantenziektenkundig Onderzoek (I.P.O.), respectively. The research excellence of these two centers evolved over subsequent years based in part on the major contributions of Oostenbrink (nematode control) and Seinhorst (nematode population dynamics). Oostenbrink, as Reader in Nematology, became Head of the Nema38

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tological Section in 1956 but it did not become a Department of Nematology within the University until 1972. Within it P.A.A. Loof became a world authority in nematode taxonomy and T. Bongers supervises a large, international collection of nematodes. The other “giant”, Seinhorst, focused his attention, initially with H. den Ouden, developing mathematical models of nematode populations to express their growth in relation to plant host growth. He retired from Head of Department in 1983. The intensity and emphasis of agricultural cropping in The

Fig. 2. Nematology staff, students and associates at the Scottish Crops Research Institute in 1998.

Netherlands resulted in large population increases of potato cyst nematode and, on flowers and onions, of stem eelworm. This led to the first Dutch, commercial advisory service with a focus on nematodes in soil and on crops (P. Kleijburg). The research focus in Wageningen on plant parasitic nematode populations and their control continued until about 1980. Thereafter, changing methods of nematode control and increasing environmental pressures led to changing research emphases as can be seen in the major contributions of J.J. s’Jacob, J. Kort, K. Kuiper, in the early days and, more recently, of T. Bongers, H. Hoestra, P.W.Th. Maas, A. Mulder, J. van Bezooijen, L. den Nijs, T. Been, C. Schomaker, and F. Gommers at the University. The application of molecular genetics for the identiNEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

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Fig. 3. Research staff at the Wageningen laboratories with a visiting group. Numbering from the left the names are 1. van der Wout, 2. van Berkum, 3. M. Oostenbrink, 4. P.A.A. Loof, 5. J.W. Seinhorst, 6. J. Kort, 7. J. van Bezooijen, 8. K. Kuiper, 9. J.J. s’Jacob.

fication of nematode species and of species within populations became a major focus with the appointment of J. Bakker, A. Schots, G. Smant and A. Goverse (Fig. 3). The vigour of nematological research at Wageningen attracted worldwide attention also through its international nematology training programme, led by J. van Berkum. It operated very successfully over several years within the Agricultural University of Wageningen together with adjunct programmes in India and Venezuela. Another major European center of nematology emerged at the University of Gent, Belgium, under the leadership of L.A.P. de Coninck. This center developed into one of the world’s largest and most successful groups of nematode taxonomists. The laboratory had the good fortune in its early years to have a series of astute leaders in L.A.P. de Coninck, A. Coomans (dorylaimids, fresh water nematodes), and E. Geraert (plant parasitic tylenchids) who successfully built the nematode taxonomy reputation and maintained it into the twenty-first century. The Department’s research activities, and those in collaboration with colleagues in Brussels, extended beyond that of light-microscope studies of the morphology and systematics of free-living and plant-parasitic 40

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nematodes and into the biochemistry, phylogeny, ecology and (recently) embryology (G. Borgonie) of nematodes. W. Decraemer (desmoscolecids, trichodorids, general morphology), N. Smol (marine nematodes), P. De Ley (rhabditids, phylogeny), G. Gheysen (biotechnology), J. van Fleteren (biochemistry, molecular phylogenetic research) and M. Vinx and A. Vanreusel (marine nematodes, ecology) played leading roles in the success of this department. They developed also a graduate degree programme in nematology, and it continues to this day as the only remaining programme of its type in Europe. They were greatly helped by cooperation with A. De Grisse (criconematids, electron microscopy) and M. Moens (applied nematology, biochemical identification), in the Faculty of Agriculture, and with J. Coosemans, D. de Waele and A. Elsen (applied pest management) at Leuven. Nematologists from other European countries have contributed to the success of this programme which has graduated almost 150 Masters (mostly from African and Asian countries) and almost 40 Ph.D.’s in nematology. Although nematology in the Nordic countries dates back to the earliest record of a plant parasitic nematode, Subanguina radicicola in Norway in 1849 (studied by W.M. Schoeyen), the occurrence of cyst nematodes became the economic driver of nematology in Denmark and Sweden. In the 1930s, I. Wåhlstedt mapped the occurrence of cereal cyst nematode (Heterodera avenae s.l.) in Sweden, and P. Bovien led research in plant and insect-associated nematodes in Denmark. The 1950’s expansion of nematology in the Nordic countries is very much linked to the names of O. Ahlberg, S. Bingefors (stem nematode) and J. Mühlow in Sweden, K. Lindhardt, C.O. Nielsen (ecology) and S. Andersen (cereal cyst nematode) in Denmark, M. Stoen in Norway and O. Roivainen in Finland, all of them more or less specializing in plant breeding and plant protection research. In 1962, they formed the Nordic Working Party which was instrumental in developing nematology in Scandinavia within the modern sense of the science. One of the activities emerging from this group was the 1962 handbook “Nematoder på Växter” (Nematodes on Plants) which has been widely used in teaching and advising. The prime mover in nematological research was Bingefors. From the late 1950s, he also laid the basis for teaching nematology at the Swedish University of Agricultural Sciences, Uppsala. He was supported by B. Eriksson and C. Magnusson in Uppsala (Ultuna), and S. Andersson and A. Banck in Alnarp (near Lund) who subsequently established specialized laboratories (Fig. 4). The Alnarp department NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

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Fig. 4. At the Swedish University of Agricultural Sciences, Uppsala in 1982. Front row, sitting, Christer Magnusson, Bengt Eriksson, Pompilio Preste; standing from the left MarjaLeena Magnusson, Sven Bingefors, Anita Banck, Sven Boström, Sigrid Bingefors, Violeta Insunza, Stig Andersson, Riita Hyvönen, Lucyna Wasilewska (visiting), Björn Sohlenius.

that originated in the former Plant Protection Institute became the laboratory for routine testing of soil and plant material for nematodes. Seinhorst claimed that Andersson had made this laboratory “one of the best equipped and fitted for its purpose in Europe”. At the Swedish Museum of Natural History, Stockholm, B. Sohlenius and S. Boström developed international recognition in nematode ecology and taxonomy, respectively, while at Lund University B. Nordbring-Hertz and H.B. Jansson focused on nematophagous fungi. In Norway, nematology was nurtured initially by M. Stoen, and later by C. Magnusson who moved from Ultuna to the Norwegian Institute for Agricultural and Environmental Research. In Finland, nematological research was pursued by K. Tiilikkala, S. Kurppa and J. Tomminen, and in Denmark by J. Jacobsen and C. Holm Nielsen. Marked reductions in financial support and personnel in recent years have restricted nematological teaching and research throughout the Nordic region. Consequently, there remains S. Manduric, (potato cyst nematode) managing nematology in Sweden, and C. Magnusson (plant-nematode interactions; pinewood nematode) R. Holgado (cereal cyst nematode) and S. Haukeland (foliar nematode; entomopathogenic nematodes) in Norway. In southern Europe, M. Ritter established, within INRA, a major 42

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Fig. 5. Staff at the Ministere de l’Agriculture et de la Foret-INRA, Antibes. Front row, left to right, J. de Almeida Engler, M. Bongiovanni, P. Abad, R. Voisin, P. Castagnone, P. Lecomte, C. Djian-Caporalino. Second row, left to right, C. Van-Ghelder, J. Lozano, C. Francois, B. Favery, F. Deau, K. Mulet, A. Fazari, L. Pijarowski. Third row, left to right, D. Esmenjaud, S. Paillard, T. Taconnet, G. Engler, C. Castagnone, M.-N. Rosso, N. Mateu.

nematological research center at Antibes to help solve agronomic problems due to nematodes in France. At that time, there was a wide array of research interests, from plant parasitic nematodes to entomopathogenic nematodes and from chemical nematicides to biocontrol strategies maintained by J.B. Berger, G. de Guiran, C. Laumond, K. Netscher and C. Scotto La Massese. During the 70s, A. Dalmasso introduced biochemical approaches to nematode taxonomy and by the end of the 1980s the new molecular techniques were incorporated into the nematode research programme. As well, the research focus changed from identification of root-knot nematode subspecific groups to nematode interactions, and this group now has a staff of 20 including eight scientists. In 2003, the INRA center was moved from the Cap d’ Antibes area to Sophia Antipolis, located 15 km to the north. Ritter’s leadership was followed by that of Dalmasso and then P. Abad, and the department evolved into a unique group of researchers with strong molecular approaches to nematological research. It included P. Castagnone-Sereno, M.-N. Rosso, B. Favery, C. Djian-Caporalino and D. Esmenjaud (Fig. 5). Their research focus was on understanding the molecular dialogue between root-knot nematode and the plant, and on the key steps leading to the develNEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

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opment of the pathogen (compatible interaction) or its rejection by the plant (incompatible interaction). Another focus was on the genetic variability of root-knot nematodes and on the identification of natural resistance genes in plants in order to develop durable resistance management of this nematode pest. The INRA research group was the first to initiate a complete genome sequencing of a plant parasitic nematode, Meloidogyne incognita. M. Luc, at the Muséum National d’Histoire Naturelle in Paris, led a major taxonomic effort on plant parasitic nematodes as well as leading the ORSTOM nematology research centers in Senegal and Cote d’Ivoire. He and G. Merny also were responsible for starting the Revue de Nématologie for the publication of international articles on nematodes. Since the early 1950s, much of the nematological research in the former West Germany has been centered at the Institut für Hackfruchtbau, Biologische Bundesanstalt für Land- und Forstwirtschaft (BBA), the present-day Institut für Nematologie und Wirbeltierkunde, Münster. Initially it was under the directorship of H. Goffart, and successively since then under the leadership of W. Steudel, B. Weischer and J. Müller. It has maintained a permanent research staff including F. Burckhardt, H.J. Rumpenhorst, J. Schlang, M. Schauer-Blume, D. Sturhan and, more recently, E. Grosse, J. Hallmann and B. Niere. The BBA institute was (and still is) responsible for basic and applied nematology related to German agriculture, horticulture and forestry, with the main focus being on cyst, stem, foliar and virus-transmitting nematodes. Being the main nematological center in West Germany, numerous diagnostic courses and more than 40 nematological workshops have been organized through this federal German research institute since its foundation. Two significant centers of nematology emerged later at the Institut für Phytopathologie, Christian Albrechts Universität, Kiel and at the Institut für Pflanzenkrankheiten, Universität Bonn where U. Wyss and R. Sikora, respectively lead small, vibrant teams that trained the next generation of nematologists. In East Germany, important nematological research related to agriculture was centered at the Institut für Phytopathologie und Pflanzenschutz, Universität Rostock under H. Decker and with A. Dowe among the long-term members of the permanent staff. Since the early 1960s, the Institute has been known also for organizing annual nematological meetings. At the Ernst-Moritz44

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Arndt-Universität in Greifswald, L. Kämpfe researched nematode physiology and behaviour. Germany, like other European countries, also has been fortunate in having many internationally recognized nematologists and their research teams working at institutions throughout the country. Through the latter part of the twentieth century, nematological research followed a pattern somewhat similar to that elsewhere in Europe, namely a period of rapid growth followed by a period of rationalization and contraction. At the turn of the century, a relatively wide, stable situation seemed to prevail in which there were fewer research nematologists, and those that remained were integrated into collaborating research groups rather than in identifiable nematological research centers. Interestingly, some of the first nematological activities in Italy were by N.A. Cobb! After completing his doctorate in Germany he spent some time at the Zoological Research Station, Naples where he collected nematodes, mounted them in balsam (some of the first permanent slides of nematodes) and described his first genus, the marine nematode, Tricoma. Agricultural nematology in Italy began in the 1950s at the Entomology Station of the Ministry of Agriculture in the laboratory of A. Marinari. Other research, undertaken at the Osservatorio per le Malattie delle Piante, Pescara, was oriented towards extension nematology under the guidance of A. Scognamiglio, and at the Plant Pathology Institute, Bari University, on fumigant nematicides and nematode virus vectors in viticulture by A. Ciccarone. By the early 1960s, plant parasitic nematodes were recognized as being serious crop pests in Italy and, in 1964, G. Martelli and F. Lamberti reported on the distribution of Xiphinema index in declining vineyards in Southern Italy. However, it was not until 1970, that the Istituto di Nematologia Agraria was established, in Bari, with Lamberti as Director. The main focus of the Institute was to carry out national surveys of the nematode fauna in cultivated and uncultivated areas and research on nematode taxonomy, biology and epidemiology. The Institute grew and developed strong research collaborations with the private sector. In 1973, the journal “Nematologia Mediterranea” was founded and, in 1974, Lamberti linked up with C.E. Taylor and J.W. Seinhorst to organize a NATO Workshop on Nematode Vectors of Plant Viruses. This was the first of a series of outstanding workshops initiated by Lamberti. G. Zacheo was one of the European pioneers who contributed to an understanding of the biochemical changes that occur folNEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

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lowing nematode infection, and as techniques changed, ultrastructure, cytochemistry and immunocytochemistry were used (T. Bleve-Zacheo and M.T. Melillo) to confirm the temporal, spatial localization and function of proteins and enzymes in nematode pathogenesis. Nematological research started in Poland in the 1950s. At the Institute of Plant Protection in Poznan´ , A. Wilski (who was succeeded by S. Kornobis) focused on nematodes on crops, their biology and control and especially on mechanisms of resistance of the potato to G. rostochiensis (J. Giebel). The Laboratory of Applied Entomology of the Department of Ecology in the Polish Academy of Sciences (PAS) in Warsaw, was headed by H. Sandner, a specialist in entomopathogenic nematodes. In 1971, the Institute of Ecology replaced the Department of Ecology and was moved to Dziekanow Lesny, near Warsaw. In the 1960s, this research group (H. Sandner, J. Kozlowska, A. Fedorko. K. Domurat and L. Wasilewska) was mainly faunistic, concentrating on nematodes parasitizing different crops, and over time, developing a strong ecological interest. By the 1970s, A. Fedorko, S. Stanuszek and M. Kamionek had extended their research activities to entomopathogenic nematodes and biological control. In 1975, Kamionek joined E. Pezowicz and A. Bednarek at the Warsaw Agricultural University under Sandner, to concentrate on entomopathogenic nematodes. The Institute of Ecology, comprising L. Wasilewska, J. Kozlowska, E. Dmowska, K. Domurat and S. Stanuszek, continued as a major research center and, after 1990, K. Ilieva-Makulec, focused for many years on the abundance, diversity, structure and function of nematode communities in different habitats – natural or man-transformed. M. Brzeski, concentrated on nematode taxonomy and systematics at the Research Institute of Vegetable Crops, Skierniewice and, later, at the Museum and Institute of Zoology of PAS. Research of A. Szczygiel and A. Zepp, at the Fruit Experiment Station, Research Institute of Pomology and Floriculture Brzezna, was focused on nematodes of fruit crops, especially strawberries. Unfortunately, these research groups split up as many personnel retired or left nematology. The nematological research interests of Poland remain now in the hands of a few e.g., K. Ilieva-Makulec (nematodes in the soil food web) and M. Tomalak (pinewood nematode). Spain, like other countries, had isolated nematological contributions in the early part of the twentieth century and then, starting in the 1950s, 46

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E. Gadea, at the University of Barcelona, initiated a stronger national focus on nematode research. However, it was F. Jimenez-Millan who, in the 1960s, pioneered the creation of a nematological research centre at the Instituto Español de Entomologia, Madrid, a center that later moved to the Instituto de Edafologia y Biologia Vegetal, and more recently called the Centro de Ciencias Medioambientales. Whilst there, JimenezMillan trained a generation of very successful nematologists, namely A. Bello (criconematids, biocontrol and quarantine), M. Arias (longidorids and trichodorids), M.D. Romero (cyst nematodes) and A. GomezBarcina (biology and control). Jimenez-Millan moved to the University of Granada in 1970 and, together with Gomez-Barcina, established another generation of nematologists. Except for A. Ocaña (freshwater nematodes), they eventually radiated to other Andalusian institutions; D. Jimenez-Guirado (free-living dorylaimids and mononchids), R. PeñaSantiago (plant parasitic nematodes) and P. Castillo (plant nematodemushroom interactions). Since then, the “academic tree of Spanish nematologists” has continued to assist growers facing nematological problems and to research on aspects of nematode biology. There are some exceptional examples of international collaboration in nematological research, often funded through the European Union, that involve plant parasitic nematodes (e.g., molecular basis of root-knot nematode interactions) and insect parasitic nematodes (entomopathogenic nematodes as biological control agents of insect pets) International collaboration among nematological researchers has not been confined to Europe but has extended to, among others, Israel (e.g., Volcani Institute) and the USA and to key research centers in countries worldwide. The extent of the research interaction between Europe and the USA has included not only focused research projects and exchange studies but also the relocation of several distinguished scientists e.g., R. Sikora to Germany from the USA and P. De Ley to the USA from Belgium. Their insight, energy and expertise has been infectious among fellow nematologists, and their legacy, through the printed word and direct influence on others, exemplifies the value of international flow of people and ideas in nematology.

Genealogy of nematology in the USA, 1907–1980 In the United States the first organized nematology research program was in the U.S. Department of Agriculture (USDA) Bureau of Plant NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

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Industry, Washington D.C. led by the “Father of Nematology”, N.A. Cobb, who joined in 1907. It is Cobb’s professional genealogy at the USDA that is the platform for the current day history and science of nematology and the nematology research and training centers in the U.S.A. as well the theme of this section of the chapter. Cobb developed a nucleus of scientists who became the architects of nematology in the U.S.A. (Genealogy Chart 1, Fig. 7) and to whom most nematologists for the next seven decades could trace their professional lineage. Some of the early U.S. nematologists (including Cobb) had training in Europe, but most were trained as plant pathologists, entomologists, and parasitologists who subsequently trained themselves in plant nematology. Many of his colleagues and students became the leaders and teachers of nematology during the 1930s, 1940s and 1950s. By 1980 it becomes difficult to continue this genealogical approach to the history of nematology in the U.S.A. Cobb’s 1889 PhD thesis focused on nematodes from whales and on some free-living nematodes at the University of Jena, Germany while studying with Haeckel, Hertwig, Lang and Stahl. His interest in free-living nematodes was derived from the publications of Bütschli and de Man. He became familiar also with marine nematodes at the Naples Zoological Station. In 1889, he became a plant pathologist in the New South Wales Department of Agriculture, Australia. After joining the USDA in 1907, he published his first nematology paper in the United States, in 1913, and became the undisputed leader of the USDA Nematology Division until his death, in 1932. Early in his career he started the movement to remove the free-living and plant parasitic nematodes from the science of helminthology (Helminthological Society of Washington DC), and eventually established them in a separate field of “Nematology”. During his career Cobb served as President of the American Society of Parasitology, Helminthological Society of Washington DC, American Microscopical Society, and Washington Academy of Sciences. He contributed major discoveries in nematode taxonomy, morphology, and methodology. His laboratory manual “Estimating the Nema Populations of the Soil” (1918) formed the basis for many of the methods and apparatus used in nematology today. As an aside, he and Chambers, an artist and microscopist, were intensely interested in gadgets (Fig. 6) and they produced some of the finest illustrations of nematodes that have ever been made. In administrative style, “his way” was the only way to do 48

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things and it may have been his quirks of personality that resulted in his having only two employees who remained, at field stations outside Washington DC, Thorne (see Fig. 11) in Salt Lake City (1918) and Courtney (1919) in Long Island.

Fig. 6. N.A. Cobb at his microscope turntable gadget used for observing nematodes.

After Thorne and Courtney, Steiner (1920), Christie (1922) and Chitwood (1929) joined Cobb’s team at the USDA. After Cobb’s death, in 1932, Steiner became the director of the USDA Division of Nematology. He led the nematology crusade and hired and directed the next generation of nematologists who joined the USDA; Allen (1937) (see Fig. 10), McBeth (1937) (see Fig. 11), Taylor (1936), Reynolds (1937) and Tyler (1932). Many of these early nematologists eventually moved on to universities to provide formal training of future nematologists. Some early students desiring to obtain training from Steiner, Chitwood and Christie did their research under the direction of USDA scientists at the University of Maryland. Their genealogy reflects the “Eastern Branch of Nematology” (Genealogy Chart 2, Fig. 12) which had a broad focus on taxonomy and morphology of plant parasitic and freshwater nematodes. The Thorne genealogy reflects the “Western Branch of Nematology” (Genealogy Chart 3, Fig. 13) which focused primarily on taxonomy, morphology and control of important plant parasitic nematodes. Thorne not having the artistic talent of Cobb relied on “scratchboard” for his nemaNEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

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tode drawings as did many of his students. Chitwood’s approach to nematology was very broad, in many ways that of a parasitologist, but he also knew plant parasitic nematodes especially pests like the potato cyst nematode.

Fig. 7. Geneology Chart 1. Early nematologists employed by the USDA from 1907–1964. Dates in parenthesis are the periods they worked for USDA and then their moves to other employment.

Beginning in the 1950s some major academic training centers began to develop; Mai (see Fig. 8) at Cornell University, Sasser (see Fig. 17) at North Carolina State University, Christie and Tarjan at University of Florida, Cairns at Auburn University, Courtney at Oregon State University, Thorne at University of Wisconsin and Allen (see Fig. 10) at University of California. The first formal academic nematology class in the USA was taught by Allen at the University of California Berkeley, in 1948, and then by Chitwood, at the Catholic University in Maryland, from 1949–1952. These early nematology training centers were associated with Departments of Plant Pathology, Entomology or Biology, and continue today as departments that offer PhDs in nematology. Detailed histories of nematology along with some general international history of nematology with photographs of nematologists can be found at the University of Florida website http:flnem.ifas.ufl.edu/history/nem_history.htm 50

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Fig. 8. From left to right, standing: V. G. Perry, A. C. Tarjan, W. F. Mai, G. C. Smart, J. A. Meredith, J. H. O’Bannon, D. W. Dickson, R. A. Dunn; sitting: K. B. Nguyen, R. N. Inserra at University of Florida, 1985.

and at the University of California website http://plpnemweb.ucdavis.edu/nemaplex/HISTNEMCALIF.htm.

Fig. 9. UCR Nematology Department 1962. Top Row, Left to Right: John Radewald, R.C. Baines, Skip Sher, S.D. Van Gundy, Oscar Clarke, Charles Castro, Rodriguez Tarte, Robert Small. Middle Row: unknown, S. Fromer, M. Papp, D. Milam, N. Nobles. Bottom Row: Arnold Bell, Ivan Thomason, Ron Mankau. NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

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Fig. 10. UCD Nematology Department. Left to Right,Top Row: Bert Lear, Armand Maggenti, Win Hart. Seated: Dave Viglierchio, Merlin Allen, and Dewey Raski.

Fig. 11. Dewey Raski, Gerald Thorne, and Clyde McBeth.

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Fig. 12. Geneology Chart 2. The “Eastern Branch of Nematology” that originated at the USDA beginning with the Steiner-Chitwood era.

Genealogy Charts 2, 3, 4, have attempted to trace the origin of nematology training, dates that individuals joined the training centers as faculty members and dates that their students graduated and the institutions at which they were first employed, from 1922 to 1980. We apologize for omissions or misrepresentations. Chart 2 continues from USDA Chart 1, representing the genealogy of Steiner and Chitwood who conNEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

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Fig. 13. Geneology Chart 3. The “Western Branch of Nematology” that originated at the USDA beginning with the Thorne era.

tributed to the nematology training centers at the University of Maryland, Auburn University, University of Florida, North Carolina State University, Rutgers University, University of Illinois and Texas A&M University. Chart 3 represents the Thorne Genealogy that gave rise to the nematologists at Oregon State University, University of Arizona Field Station, Kansas State University and the University of 54

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California. Genealogy Chart 4 (Fig.14) represents the genealogy of most of the independent nematologists at Cornell University, University of Nebraska, University of Iowa, University of Illinois, University of Kentucky, Purdue University and Louisiana State University. During this early period of USA nematological development, the European Society of Nematologists was formed, in 1954-55, followed by the Society of Nematologists, in 1960-61, and the Organization of Nematologists of Tropical America, in 1967. Many USA nematologists belonged to all three societies. The Society of Nematologists grew rapidly reaching a total of 680, in 1980. Other forms of nematological training during the 1950s included a series of Shell Chemical Workshops throughout the USA aimed primarily at educating farmers on the importance of nematodes. Federal funding supported the formation of Regional Nematology Research Groups (Northeast, Central, Southern and Western) to encourage collaboration and communication between

Fig. 14. Geneology Chart 4. A collection of independent nematology training centers not directly related to either the Eastern or Western branches of nematology. NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

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Fig. 15. Victor Dropkin, Ron Mankau, Virginia Ferris, Grover Smart, Morgan Golden, Robert Esser at Society of Nematologists meeting in 1965

nematologists in each region. For example in the Southern Regional Nematode Project (S-19) Sasser (see Fig.17) and Cairns formed and held nematology workshops at North Carolina State University, in 1954, and at Alabama Polytechnic Institute, in 1955, for professional nematologists. In the Northeast Region, Mai held nematology workshops at Cornell University in the late 50s. Sasser and Jenkins conducted a summer course in 1959. Since 1968, California has held annual, statewide nematology workshops for faculty students, extension advisors, regulatory and industry nematologists to improve communication and to foster collaboration. These workshops and research groups helped promote the importance of nematodes and the need of funding for research and new faculty positions. Some of the other early driving forces that contributed to the rapid growth of nematology in the USA were, 1) the discovery of nematicides (D-D, EDB) in the 1940s that demonstrated to farmers the crop losses caused by nematodes, 2) the designation of nematodes subject to quarantine and regulation: stem and bulb nematode in 1926, golden nematode in 1941, the burrowing nematode in 1954, 3) the discovery of crop losses due to ectoparasitic nematodes and 4) the importance and role of nematodes in plant disease interactions. These driving forces spawned a soil fumigation industry, federal and state regulatory programs, and the attention of the agricultural industry to 56

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provide special funding for nematology teaching, research, and agricultural extension programs. For example, the discovery of the burrowing nematode in Florida and its potential threat to California’s citrus industry stimulated the California agricultural industry and the California Department of Agriculture to propose legislation to recognize nematology as a separate and distinct science from entomology, plant pathology and parasitology. Funds were provided in the state legislation to support the establishment of a separate Statewide Department of Plant Nematology at University of California Davis, in 1954, under the direction of Raski. Within 5 years the department had grown to 12 faculty members on the Davis and Riverside campuses. The name was changed to the Department of Nematology in 1962 . In 1965, Davis (headed by Raski) (see Fig. 17) and Riverside (headed by Sher) (see Fig. 9) became separate departments of nematology and have continued as such up to the present time. By 2000, the two departments had hosted 90 visiting scientists and postdoctoral scientists and trained 140 graduate students. Another significant advancement of nematology in the USA was the development of the International Meloidogyne Project (1975–1984) by Sasser at North Carolina State University. It was funded by USAID, and the project was given a mandate to focus on root-knot nematodes of economic food crops in developing nations. It

Fig. 16. Western Region Research Project W-157 (1966). Back row left to right, Ed Jorganson, Idaho, O.J. Hunt, Nevada, Lin Faulkner,Washington, L.A. Ayres, Wyoming, Harold Jensen, Oregon. Middle Row left to right, Olie Holtsman, Hawaii, C.P. Wilson, Hawaii, Jack Altman, Colorado. Bottom Row left to right, Ben Lownsbery, California, Ed Nigh, Arizona and E.C. Dallimore, Idaho. NEMATOLOGICAL NEBULAE IN EUROPE AND THE USA

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attracted scientists from 60 countries and brought with it collaboration with many scientists in the USA. In the process of conducting the project North Carolina State University had the unique opportunity to assemble a vast amount of informational observations and research on Meloidogyne species which led to important publiFig. 17. Dewey Raski and Ben Lownsbery, cations. University of California Davis and Joe Sasser, Currently, applied nematolNorth Carolina State University at APS meetings ogy in the USA during the in Estes Park, 1954. 1990s and 2000s has fallen on a period of reduced funding from state and federal resources, resulting in reductions in faculty positions, graduate students and support staff in the universities as well as other trained researchers working in the field of nematology. There also has been a mass retirement of senior nematologists who started in the 1950s and 1960s and have not been replaced. Concurrent with these changes there has been a consolidation in chemical companies along with a reduction in the use of nematicides and a search for new nematicides for the control of nematodes in important agricultural crops. These reductions in applied nematology are reflected in the December, 2005 SON membership of 604, which was down from its peak membership of 680 members, in 1980. So even though the crop losses due to nematodes in agriculture continue to be as important or even more important than in the 1950s, 60s, and 70s, the emphasis and breadth of nematological research has shifted from a strong, applied agricultural emphasis to a strong biological emphasis on systematics/evolution/morphology, ecology/biodiversity and classical and innovative genomics along with an administratively driven increased emphasis on teaching in the biological sciences. This biological research shift in nematology was highlighted, in 2002, by the Nobel Prize in medicine being awarded for research on the Caenorhabditis elegans model system suggesting that basic nematode research is important, strong and growing.

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3. FIRST CATCH YOUR NEMATODE! – THE DEVELOPMENT OF METHODS FOR RECOVERING NEMATODES FROM SOIL DAVID MCNAMARA Formerly: East Malling Research Station, Kent, UK and European and Mediterranean Plant Protection Organization, Paris, France.

Introduction It has often been said that there is no such thing as a fat plant nematologist. And this is strikingly obvious if you attend a nematology symposium, where you will be surrounded by fit and athletic looking people. The reason for this is obvious: unlike their colleagues in plant pathology or entomology who need only to pick a leaf or two, or wave a collecting net around for a few seconds in order to obtain samples of their organisms, nematologists must dig up quantities of heavy soil, carry it back to the laboratory and then engage in complicated and difficult procedures involving lots of water, before they can get a sight of their little creatures. This is all heavy work. (Of course, the above description of life style does not apply to nematologists who have reached a higher position of authority and who have a team of assistants to do the heavy work for them (Fig.1). Nor does it apply to the group of younger molecular nematologists who work only with tiny samples of macerates and extracts of nematodes, presumably given to them by the slim, fit nematologists.)

Finding nematodes in soil The earliest success at seeing soil nematodes was performed by direct observation of plant roots using magnifying glass or microscope. This method was used in the second half of the 19th century to demonstrate the presence and the impact of cyst nematodes and root knot FIRST CATCH YOUR NEMATODE!

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nematodes. It could also be used to find free-living ectoparasitic nematodes but was a very inefficient means of determining their presence or population densities. In the early years of the 20th century, two men provided crucial insights into the physical relationship of nematodes with other soil components. This led to the development of a range of efficient extraction methods which enabled the science of nematology to advance our understanding of the quantitative effects of parasitic soil nematodes on plant growth. The first insight, on the extraction of nematodes, was the exploitation of the fact Fig. 1. R.S. Pitcher (right) instructs that nematodes are aquatic David McNamara on the methods for animals and that they are capturing the world’s largest soil nematode, (usually) mobile; this led the Ersatzonema gargantua. Dutch physician, G. Baermann, working in Java in 1917, to develop the method, now known as the Baermann funnel technique. By putting soil contained in a muslin bag into a funnel filled with water for several hours or days, he discovered that nematodes (e.g. larvae of Ancylostoma) tended to migrate downwards out of the soil and through the muslin, and then could be seen in the water at the stem-end of the funnel. Unfortunately, the water resulting from this method was generally rather murky (because of the presence of small soil particles that would leach out of the soil through the muslin, for example, colloidal clay) and the nematodes could only be seen with difficulty – but they could be seen, and counted, and transferred to microscope slides for more detailed examination. Later, several improvements were made to Baermann’s method. For example, the muslin bag was replaced by a sieve, so that only a 60

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narrow layer of soil needed to be traversed by the nematodes; the sieve is, preferably, not constructed of metal, in order to avoid the release of toxic ions into the water. Furthermore, a flat dish is generally used instead of the funnel so that the nematodes do not stay for many hours in the narrow base of the funnel where the oxygen supply is limited (Whitehead and Hemming tray). A major advantage of the Baermann funnel method is that it can also be used for extracting nematodes from parts of plants (e.g., roots, leaves, wood). The second insight was made by the American nematologist, Nathan Cobb who, in 1918, recognized that nematodes in suspension in water would sink more slowly than soil particles of a similar size, based on the fact that the organic material of which the nematodes are composed has a lower specific gravity than the rock which constitutes the inorganic fraction of the soil. By setting the soil sample into suspension in water, waiting for a predetermined period of time (i.e., a period just less than the time needed for biological material to sink), and then passing the supernatant liquid through a sieve of appropriate size, he was able to separate the nematodes, and other organic matter, from the smaller soil particles still in suspension. The pore size of the sieve was chosen so that the small soil particles would pass through, whereas the nematodes would not pass and could be collected from the surface of the sieve. Sieves of different pore sizes can be used in order to selectively extract species of different body size. Cobb’s decanting and sieving method produced a relatively clean suspension of nematodes for examination under the microscope, provided that the original soil contained little organic material, otherwise it would be necessary to search through much distracting debris in order to find the nematodes. This problem could be partly solved for samples from richly-organic soils by combining the method of Cobb with that of Baermann; a very clean sample could be obtained by putting the product of decanting and sieving onto a sieve suspended in water, thereby allowing the nematodes themselves to move away from the other organic debris. However, not all nematodes can, or will, move out of the organic debris, so that many nematodes (species or individuals) will fail to be detected. Good advice to anyone who wishes to become a successful field nematologist is to choose to do your research in an area where the soil has little or no organic material! FIRST CATCH YOUR NEMATODE!

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These two methods, of Baermann and Cobb, contained the essential principles of nematode extraction from soils, on which the vast majority of later methods would be based. These principles ranked nematodes as being just one of many components of the soil which could be considered separately for the purposes of nematode extraction, and which could be separated from each other by their speed of sedimentation, their size and, in the case of nematodes, by their motility The methods that refined these principles and mechanized the extraction of nematodes were invented in the second half of the 20th century. For example, so-called “elutriators” used an accurately controlled, upward current of water in place of simple sedimentation. The force produced by the upward current could be precisely fixed depending on the diameter of the tube through which it flowed and the rate of water flow into the tube. The earliest example was the elutriator developed by Mike Oostenbrink, in 1954 in The Netherlands. This was an upright metal funnel filled with water, into which a fixed flow of water entered from below. The soil sample was washed into the top of the funnel. The heaviest soil particles sank to the bottom of the funnel while lighter particles were held in suspension at different levels of the funnel (depending on the diameter at these levels). The upward flow of water was so established that nematodes would be carried up and over the top of the funnel to be concentrated onto a sieve. Even the cysts of Heteroderidae species can be recovered by this method by increasing the water flow rate. Oostenbrink’s compatriot, Wim Seinhorst, invented a simpler system of two glass Erlenmayer flasks, connected to each other but with one inverted above the other. Both flasks are filled with water and the soil sample is contained in the upper flask. As the soil particles fall from one flask into the other, an upward current of water is produced that retains the nematodes in the upper flask. Seinhorst also invented a more sophisticated elutriator, in 1956, which was composed of an upright glass column with several sections along its length of different diameters, therefore allowing nematodes of different sizes to be separated at different levels. After a defined period of operation, the contents of the different sections could be drained off through side tubes, to be directly examined or to be further separated by the Baermann funnel technique (depending on the organic content of the soil sample). 62

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It was not surprising to other nematologists that Seinhorst and Oostenbrink should be competing to produce the most effective extraction procedure. They were known to be deadly professional rivals and, even though they started their university education together and they worked in the same town in The Netherlands, Wageningen, throughout their careers, they certainly could never be described as true good friends! Other versions of the elutriator were subsequently invented. For example, the elutriator often called the “Trudgill Tower”, developed by David Trudgill and his colleagues in 1973, is a plastic cylinder, without any variation in diameter, in which different sizes and types of nematodes can be extracted by varying the flow rate coming from the base of the cylinder. Byrd and his colleagues produced semi-automatic elutriators, in 1972. Although, cysts can be recovered with the elutriators discussed here, they are very often extracted by means of specialized cyst extraction techniques. The most commonly used apparatus is that developed by Fenwick in 1940, the so-called “Fenwick Can”, which extracts cysts most effectively from air-dried soil. The effect of the air-drying is to cause the cysts to float to the surface where they overflow the can and are collected on a sieve. The Schuiling centrifuge, developed in 1982, is a more sophisticated technique which is semi-automated and can process soil samples very rapidly. Centrifugal flotation is a technique, used in other areas of biology, which was adapted by Caveness and Jensen, in 1955, for the extraction of nematodes from soil. The principle of the method is that nematodes will be held in suspension in a solution whose specific gravity is greater than that of the nematodes, whereas soil particles will sediment. In practice, solutions of sucrose, zinc sulphate or magnesium sulphate are used as the suspending solution. It is interesting to note that, in the different publications concerning this technique, the specific gravity of the suspending solution (particularly sucrose) is given either as 1.15 or 1.18. This might suggest that researchers had calculated different values for the specific gravity of the different nematodes they were studying, but, in fact, it rather indicates that nematologists are not very skilled as laboratory chemists: the original recommendation was that a solution of 484 g sucrose/litre should be used, which would produce a specific gravity of 1.18, but some nematologists misunderstood this to mean 484 g added to one litre of water! FIRST CATCH YOUR NEMATODE!

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Centrifugal flotation is considered by many to be a quick and efficient method of extraction, possibly more efficient than decanting/sieving or elutriation, and it has the advantage that it can recover non-motile stages, including eggs, but the disadvantage is that it only operates on small soil samples. In addition, as with other methods, it is not suitable for soils rich in organic matter. In the 1990s, efforts were made to use DNA probes to provide information on the presence of certain nematode species in soil samples, but, so far, such probes can only be used to detect but not yet to quantify. When examining the history of the development of extraction methods, the first, and most striking thing, is the number of these methods that retain the names of their inventors when being discussed (e.g. Fenwick Can, Seinhorst elutriator, Oostenbrink elutriator, Trudgill Tower, Baermann funnel etc.). The take-home message is: “If you want to be remembered as a nematologist, invent an extraction technique”! The second noteworthy observation is that, since the early years of the 20th century, the methods have become more sophisticated, more automated, less time-consuming and, presumably, more efficient. You would, therefore, think that different nematological laboratories would move towards using the same, and most efficient, methods. This does not seem to be the case; when you visit different laboratories, you notice that every one has a different, favourite extraction method. Every nematologist tells you that the method they use is the one that works best for them. As my late Ph.D supervisor, R.S. (“Pitch”) Pitcher once said: “Extraction of nematodes is like sex: everyone does it and everyone is confident that they are quite good at it, but very few comparative tests have been carried out”. In fact, a few comparative tests have been carried out (on extraction, not sex), usually when laboratories are collaborating on a particular project and need to align their extraction techniques, so they compare the extraction of nematodes from similar soil samples. The results of such tests, if ever published, usually reveal significant differences in the numbers of nematodes recovered. Clearly, more comparative tests are needed.

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Collection of field samples As the methods to extract nematodes from soil became more efficient, it became obvious that the sampling methods for obtaining the soil also should be improved. There is little point in employing laborious methods to give an accurate picture of the types and numbers of nematodes in soil samples, if the soil samples themselves do not represent the total soil in the field. The first efforts to try to get an understanding of the accuracy or otherwise of field populations were related to potato cyst nematodes (Globodera spp.). In post-war Europe, many countries were applying legal measures to try to reduce the damaging effects of these nematodes (by, for example, prohibiting the growing of potatoes on infested land) and it was, therefore, important to try to learn whether they were present in fields and, if so, at what population density. B.G. Peters and Freddy Jones, in the UK, were probably the first, in the early 1950s, to publish statistical analyses of the errors inherent in taking samples to detect or estimate populations. Other statistically-minded nematologists followed. It was recognized that a Poisson distribution of nematodes could be assumed when trying to detect whether a field contained cysts and, therefore (and rather counter-intuitively), the probability of finding an infestation depended more on the quantity of soil in the sample than on the number of sub-samples. To try to determine the population density, on the other hand, it was shown that cyst distribution in the field conformed more closely to a negative binomial distribution. This is because nematode populations are generally patchily distributed, for a number of reasons (e.g., plant root distribution, spread due to agricultural activities, distribution of initial soil contamination etc.); in this case the number of sub-sampling points becomes of more importance. These principles are still used to decide soil sampling strategy for most types of nematodes for research purposes as well as for statutory reasons.

Microscope examination of nematodes Once they have been extracted from soil, it becomes necessary to examine the nematodes, mainly for the purpose of correct identification. For this, nematologists have taken advantage of the progress FIRST CATCH YOUR NEMATODE!

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of development of microscope technology, not only different types of electron microscopy but also the improvements in light microscopy throughout the 20th century. Examples of development are the higher quality optics and light sources of high power microscopes, and such methodologies as phase contrast, interference contrast, camera lucida and drawing tubes, microphotography and video, computer aided imaging, electronic measuring. To take full advantage of microscope technology, it was necessary to ensure that the nematode specimens were in the best condition for visualization. They first needed to be “fixed” to avoid decay, and Courtney, Polley and Miller, in 1955, developed a chemical mixture of formalin and triethanolamine (TAF) which did not distort or discolour the nematodes and which has become the most widely used fixative. In 1949, Franklin and Goodey published a method for mounting the specimens in lactophenol and glycerol which “cleared” the optical interference from the contents of the intestine, and also allowed for long-term storage of the specimens on microscope slides. Baker, in 1953, and Seinhorst, in 1959, produced alternative methods for mounting and clearing in glycerol. Such methods, with some later modifications, continue to be used for preparing nematodes for both direct observation and for permanent slide collections. With the arrival of molecular methods to identify nematodes, it will be interesting to see whether molecular technology will completely replace current slide preparation methods for nematode identification.

Conclusion The developments in molecular technology seem to be advancing towards the day when it will no longer even be necessary to examine nematodes visually; will this mean that the laboratory techniques of slide preparation and microscopy will become redundant? And when a method is developed to detect and quantify nematodes in the field by means of a probe of some kind inserted into the soil, will nematologists finally lose their much admired svelte appearance?

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4. THROUGH NEMATODE DIVERSITY TO LIVING SOIL PROCESSES – HOLISTIC STUDIES AID PROGRESS GREGOR YEATES Landcare Research, Private Bag 11052, Palmerston North, New Zealand.

My first awareness of nematodes came from my father’s attempts to control Pratylenchus in his beds of Lilium auratum. I was more formally introduced to them in 1963 when Wallie Clark visited the University of Canterbury from DSIR in Nelson. He gave us a few nematode lectures and labs in invertebrate zoology. I was hooked. The next year I spent more time in botany labs looking at, and treasuring, nematodes among algal filaments than at the algae themselves. Marine zoology field trips yielded ironids from intertidal sands, and Wallie was on hand to classify them. The zoology department had a strong ecological base and a 1965 honours course in limnology (with a class of one) introduced an isotopic method of measuring processes. In 1964/65 and 1965/66 there was the opportunity for summer work in the Antarctic, assessing the breeding success of Adelie penguins; it was a change from working in the slaughter house. Although my supplementary efforts to collect juvenile stages of nematodes from seals were unsuccessful I did manage to recover Plectus and Eudorylaimus from clumps of moss; an ecosystem with low nematode diversity. [Plectus murrayi continues to cause taxonomic discussion but “sinking” Antholaimus into Eudorylaimus is accepted.] February 1966 saw me starting my PhD studies with Wallie Clark in the Zoology Department at Massey University, New Zealand. In 1966, Wallie was striving to get nematology launched at Massey. A Leitz Ortholux arrived and Wallie suggested that Judy Killick, who had earlier emigrated from Rothamsted to work for him in Nelson, come over at lunchtime and view it. At lunchtime Wallie was nowhere to be seen so I met Judy over an Ortholux; THROUGH NEMATODE DIVERSITY TO LIVING SOIL PROCESSES – HOLISTIC STUDIES AID PROGRESS

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nematology has since taken us to many places together. However, I have been converted to Zeiss optical systems. Wallie Clark considered sand dunes to be a relatively simple system with a simple nematode fauna (some 40 years later, in 2006, these would be hypotheses to be tested, and rejected). I sampled a site at Himatangi Beach extensively. That over 50 species were recorded, including 41 proposed new species from a range of sites, was an early lesson in the richness of nematodes and in our level of ignorance. Everything that moved in samples was counted, leading to a classic illustration of the relationship between Nygolaimus and their enchytraeid prey [see: Pedobiologia 8: 173–207, 1968]. Later, similar counts showed the relation between predacious tardigrades and their nematode prey in Hestehave, Denmark. In 1967, Wallie returned to the University of Canterbury and I moved with him. The work on three bacterial-feeding species in culture on agar was completed, including observations on the facultative predation by Diplenteron (diplogasterid) and initial attempts at reviewing nematode feeding types. The thesis was completed right on the two-year minimum. In their wisdom, the New Zealand University Grants Committee provided a “no-strings” postdoctoral fellowship of £650 that enabled me to work at Rothamsted where, in particular, F.G.W. Jones and David Hooper influenced my work. Judy worked in the Biochemistry Department, partly for John Clarke. Although Fred Jones “required” me to look at the possibility of Heterodera spp. interbreeding he was also emphatic that I should be allocated new plastic bags for sampling at Wicken Fen; many samples were still being collected in cloth bags and there was a washing machine in the header house dedicated to cleansing them. Wicken Fen yielded 37 species of nematode. Access to the Nematode Collection, lovingly cared for by David Hooper, enabled a comparative study of dorylaimid morphology that, in turn, led to reflection on passage of food through the oesophago-intestinal junction. Ideas on nematode diversity and feeding types were committed to paper and sent to Pedobiologia – the main reaction being a concern that I was publishing behind the “iron curtain”. In late 1969, I accepted an amanuensis position in the Danish IBP (International Biological Programme) beech forest programme as one of the group assessing energy flow through soil animals. This was based in the thatched farmhouse at the Mols laboratory (Denmark) where Christian Overgaard-Nielsen had done his pioneering work 68

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that launched nematode ecology. In the previous year, at Rothamsted, I had picked up Alan Whitehead’s tray extraction method and used it in the Hestehave beech forest. It yielded good numbers of tardigrades and, according to an expert in their ecology group, proved reliable also for enchytraeids. Once it was realised that much of ecology was concerned with “active’” nematodes Whitehead & Hemming trays became widely applied around the world. Although the energyfocussed approach proved to be inappropriate, the intensive 12 month sampling programme in Hestehave provided good basic information on nematode populations and biomass, linked to a range of other biological studies. There were at least 76 nematode species found in this habitat. Mounting, with Judy’s paid assistance, and measuring over 100 specimens from each of three depths each month gave me an appreciation of differences between successive developmental stages. This, together with the wide range of taxa described during my PhD, still underpins my work with nematodes. Further, the IBP established links between dedicated workers that still continue today, even though some are now third generation practitioners. For example, I currently do much of my work with David Wardle who trained under Denis Parkinson, a member of a Canadian IBP group, and with David’s students. Denmark also provided my first contact with Nordic nematologists including Knud Lindhardt, Bengt Eriksson and Björn Sohlenius. In late 1970, DSIR paid my fare to return to New Zealand – for the second time in Judy’s case. As in 1969, I chose an “ecological” rather than “taxonomic” position – not that I have ever actually been formally interviewed for a job – and went to the Soil Bureau, the national soil survey organisation. The Soil Bureau not only used a genetic (processed-based) soil classification but also had sections for agronomy, soil analysis, soil biochemistry, soil biology, soil chemistry and mineralogy, soil engineering, and soil physics. Soil surveyors and colleagues collected samples from soils of known properties and some samples came from overseas, so I had plenty of help! In December 1970, my Director advised me “Gregor, you are a second generation scientist. Go and get on with your science”. I followed my nose, but it was not until 1987 that I was able to address one of the questions on the list I drew up in 1970 (collecting drilonematid nematodes from the body cavity of 30 cm long native New Zealand earthworms – but only after the earthworms had festered in the fridge at home for several days). THROUGH NEMATODE DIVERSITY TO LIVING SOIL PROCESSES – HOLISTIC STUDIES AID PROGRESS

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In national terms my efforts were justified when, in the early 1970s, we identified and later unravelled the problems with and impact of Meloidogyne and Heterodera on white clover in New Zealand pastures. These nematodes significantly reduce symbiotic nitrogen fixation by Rhizobium in nodules on white clover – the nitrogen supply fundamental to the nation’s pastoral industries. In 2006, Chris Mercer and colleagues are continuing efforts to incorporate resistance to these nematodes in commercial white clover cultivars. Little do the plant breeders realise that clover roots without Heterodera or Meloidogyne will be heaven for Pratylenchus! One of the greatest ecological advances came after I had collected monthly samples from normal, grazed pastures for 13 site-years (seven sites, some with different treatments or for 3 successive years). Total nematode abundance was positively correlated with pasture herbage production (i.e., plant growth that could be used by sheep or cows). While this was contrary to conventional wisdom, the counts included bacterial and fungal-feeding nematodes and predators as well as plant-feeders, and were recognised as, in some way, reflecting below-ground processes. The effects of irrigating pasture or of year-to-year differences at a site were of lesser influence than was the underlying soil. Also, the diversity of the nematode assemblage differed among the seven soils – the greatest diversity was found in a soil derived from material that had erupted from a volcano about 100 years earlier. Soil was more important than month, year or management practices in determining the composition of the nematode fauna. All this was achieved with nematodes essentially being identified to genus (sensu J.B. Goodey, 1963) [see: Soil Biology and Biochemistry 16: 95–102, 1984]. I was fortunate to be in a country with fairly uniform, grazed pastures on a diverse range of soil types and at a time when soil fertility was still driven by biology rather than by what comes out of a bag. As one who was ploughing through numerous samples not knowing what would result, I was amazed when Diana Freckman telephoned and asked me to talk at the Society of Nematologists meeting in August, 1977. Apart from giving “A view of nematode populations and their role in ecosystems” [see: Journal of Nematology 11: 213–229, 1979] there was a fantastic pre-conference tour. During this I was amazed to hear “nematode” “nematode” “nematode” mentioned in the bus – for many years only Judy and I had used the word conversationally. Visits to laboratories in Riverside, Davis, Fort 70

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Collins and Laramie were just great, and I am still in touch with people I met on that visit. The positive correlation of plant yield with nematode populations was integrated into the wide ecological literature [see: Advances in Ecological Research 17: 61–113, 1987]. Also, my interest in morphometrics, which began during my PhD, was strengthened during ecological studies because in some way it helps explain how so many nematode species can co-exist in a single soil. Earthworms, through their effects on decomposition pathways and on soil structure have been found to influence nematode abundance and diversity. Two laboratory studies are critical in understanding how the positive correlation arises. It must be remembered that as animals, nematodes are ultimately dependent on energy fixed by primary producers. In the soil food web nematodes either feed directly on plants (“grazing foodweb”) or in the “detritus foodweb” (as bacterialfeeders, fungal-feeders, predators, omnivores, etc). Firstly, at NREL (Natural Resource Ecology Laboratory), Colorado State University, Fort Collins Russ Ingham, together with Dave Coleman and others, produced a classic paper in which they demonstrated that, under nutrient-poor conditions, grazing of nematodes on bacteria increases the turnover of plant nutrients and thus increases plant growth [Ecological Monographs 55: 119–140, 1985]. We had to wait until 1998 before the Howard Ferris” group at Davis demonstrated why fungal-feeding produces a lesser response [see: Plant and Soil 203: 159–171, 1998]. The loop was completed when I, transmuted to Landcare Research in Palmerston North, with Chris Mercer, Richard Bardgett and others, and applied Surinder Saggar’s pulse labelling technique. We grew white clover plants with various nematode species infecting the roots, let the plants photosynthesize with 14C labelled CO2 and determined the distribution of the 14C label after 14 days. In the presence of nematodes, more of the new photosynthate was found in the soil microbial biomass, being available for plant uptake as it cycled in the rhizosphere. The degree of “leakage” varied, generally reflecting the degree of root damage by the nematode [see: Nematology 1: 295–300, 1999]. Thus even plant-feeding nematodes can make a positive contribution to soil nutrient cycling; this may be a mechanism by which small populations stimulate plant growth, but large populations may lead to pathogenicity. We now knew that the soil type controlled the composition of the nematode fauna and that the activities of the nematode fauna as a THROUGH NEMATODE DIVERSITY TO LIVING SOIL PROCESSES – HOLISTIC STUDIES AID PROGRESS

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whole contributed to the productivity of a site. Several other questions were worrying nematode biologists and ecologists. What resources do nematodes actually use? How many nematode taxa occur at a site? How does the composition of the nematode assemblage at a site respond to various stresses? How does all this fit into the global ecological picture? Such questions are neither trivial, as nematodes are the most numerous animals on the earth, nor simple, as the limited progress since Overgaard Nielsen’s 1949 work has shown. At the Second International Congress of Nematology in Veldhoven (1990) a group met and agreed to pool their combined knowledge on resources used by plant and soil nematodes. The widely cited “state of knowledge” synthesis on nematode feeding types arose from this meeting [see: Journal of Nematology 25: 315–331, 1993]. Although there has been some skirmishing around the edges, the only significant subsequent advance has been the confirmation that several Tylenchidae reproduce well as fungal-feeders [see: Soil Biology & Biochemistry 35: 1601–1607, 2003]. We remain profoundly ignorant. Conventional wisdom is still that mononchids are predacious. In 1984, I was talking to Carol Morley, a student at NREL, about mononchids and casually mentioned that I had one in culture apparently feeding on bacteria. So did she! [See: Ecology 70: 1127–1141, 1989]. Around 1992, I was working through hundreds of tubes of samples collected from a field trial some months earlier when I noticed that the technician had failed to fix one of the samples – it contained solely mononchids and protozoa (at 50 xs). These, and similar, observations have been reported but still people regard mononchids as solely predacious. The similarity of the behaviour of their populations to those of omnivores is not coincidence – it is real, reflecting food resources and our ignorance. Our marine colleagues, such as John Tietjen and John Lambshead, lead the way in assessing nematode species diversity in samples. For soils, most of us have a great “taxonomic impediment” (that is, we do not know what species we have) and must work at genus level, although some nations have species lists. The added complication of compensating the “taxon count” for the effort (i.e. specimens identified) is too often forgotten, but Brian Boag and I tried to bridge the gap in carrying out a global review [see: Biodiversity and Conservation 7: 617–630, 1998]. Mike Hodda has probably been the most successful in looking at diverse habitats and allocating nematodes to nominal species by working on an English 72

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grassland and tropical Cameroon forests. Anton Šály’s study in Slovakia counted species in a mosaic of habitats. The latter two studies add to the complication in that they looked at a series of sites in a region, determining regional or ψ diversity rather than α or community diversity. While the diversity of nematodes in adjacent cores from the same locality can be seen as a sampling problem it does show habitat patchiness, and is very useful in comparing patchiness in neighbouring habitats with similar underlying soil types. Christien Ettema and I looked at this, comparing adjacent pasture and forest, on a soil in New Zealand [see: Soil Biology and Biochemistry 35: 339–342, 2003]. It was good to have someone else come up with the same list of genera as I would have (except her Aporcelaimellus was more up to date than my Aporcelaimus). It was also entertaining to be told by the farmer’s wife how she dug under rows of potatoes to “tickle” potatoes from the tenant’s crop. I think that there is now an appreciation among nematologists that all the various diversity measures are valid; the problem is finding the right framework with which to interpret the results. Christien Ettema’s working with me was the latest in a series of exchanges that began when Tom Bongers had me talking about nematode ecology in Wageningen in 1987. I was amazed to be taken halfway around the world to the home of nematology, and to find that my eight lectures, “Resource utilization by nematodes”, which included the ecology of plant, soil and marine nematodes, went down well. In Wageningen, Ron de Goede and Gerald Korthals were eager learners. When he was in New Zealand in 1990-91, I asked Marco van Étagère to measure specimens of Longidorus taniwha and sort out the juvenile stages. [Wallie Clark had described this nematode earlier, taniwha being the native name for a monster.] A very concerned student approached me next morning to say that he could find only three juvenile stages rather than four. We looked at his tabulation and confirmed his interpretation. I commented that the nematodes had not read the textbook and that Marco went home wiser on those days when he had more questions after working than before. This was the first record of only three juvenile stages in Longidorus, it had previously been found in Xiphinema; now it is also known in some diplogasterids. In the late 1950s, Harry Wallace and others did excellent work on movement of nematodes through sand etc., packed into rings, finding differences with texture. Consensus was that nematodes were THROUGH NEMATODE DIVERSITY TO LIVING SOIL PROCESSES – HOLISTIC STUDIES AID PROGRESS

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best able to migrate when soils are at field capacity. By 1980, we knew that changing the structure of a soil of a given texture affected the nematode fauna. With soil physics colleagues I set up a laboratory trial using cores of undisturbed soil with differing degrees of compaction. Hypotheses were clear, replication good. However, the three nematode species had not read Wallace (1963). Populations of all three bacterial-feeding species increased at all moistures until everything died out at around wilting point. There was a suggestion that population increase was greater when the water films were thinner – presumably bacterial concentrations were higher and nematode feeding easier [see: European Journal of Soil Science 53: 355–365, 2002]. When I first presented these results in an invited paper at Callaway Garden, Athens, GA some American colleagues were delighted – they had had to assume this situation to get their model to fit. Conferences are great places to exchange information. In New Zealand, Wallie Clark was instrumental in setting up the New Zealand Society for Parasitology and it serves as a meeting place for nematologists sensu lato. In the 1980s, when scanning electron microscopes became simpler he used to spin yarns to complement images of the magnificent copulatory spicules of nematode parasites of insects. Initially, I had to curtail my “free-living” interests and talk about plant-pathogenic nematodes. However, at a joint meeting with the Australian society in Adelaide I was asked to look at the possible effect of Duddingtonia flagrans (= Arthrobotrys flagrans to some) on non-target soil nematodes. Duddingtonia flagrans is perhaps the first nematode-trapping fungus to have been shown to have a significant economic impact [see: Veterinary Parasitology 126: 199–315, 2004]. When fed to sheep or cattle it can pass through the gut to be deposited in the dung pat. The chlamydospores germinate and the hyphal network effectively traps the bacterial-feeding juveniles of trichostrongylid nematodes that would otherwise crawl up blades of grass to be ingested by, and infect, livestock. Having identified perhaps 100,000 soil nematodes from various field trials in Australia, Wales, The Netherlands, Denmark, Sweden and, just free of embargo, New Zealand, it can be said that adding the fungus to stock food has no effect on the abundance and diversity of soil nematodes, and thus no effect on nematode-mediated soil processes. Whew, what a relief. However, the soil nematodes had not read our site plans. Near Uppsala, the trials were on a “flat” area adjacent to a river. There was a barely detectable slope towards the river; soil texture changed down 74

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this slope and so did the nematodes [see: Acta Agriculturae Scandinavica – Section A, Animal Science 53: 197–206, 2003]. With intensive sampling the nematodes were able to show us that the soils differed. This subtle soil effect was greater than any effect of the nematode-trapping fungus. Isn’t habitat diversity marvellous! Largely in response to a requirement of a funding agency, Tom Bongers, in 1990, proposed the Maturity Index that weights nematodes as an indicator of environmental disturbance [see: Oecologia 83: 14–19, 1990]. It is not a diversity index, rather an index of the perceived population reproductive strategies of the various taxa present. It seemed to be a convenient way to summarise (i.e., index) typical data found after various disturbance events – yes, even adding powdered cow dung to soils! Although widely cited [318 times by 10 March 2006, according to ISI if you care for citation rates] and highly influential, I am on record as doubting that the postulated c-p groups are robust. In an effort to move indices to more reflect ecosystem function Howard Ferris and others proposed the Structure and Enrichment Indices [see: Applied Soil Ecology 18: 13–29 , 2001]. To me these confound the “ignorance” embedded in the feeding group paper with the doubts about c-p classes. Questioning is the basis of science. I get to referee many papers that use Tom’s and Howard’s indices – as long as the sites and soils are well described and the work generally well planned, as applications of current tools, they pass. In a way, these indices are a return to the IBP philosophy in which the flow or flux through nematodes was the important thing; we have moved on from the IBP philosophy. Ecosystem ecologists, as opposed to nematologists, seem to be getting strong relationships between the abundance of nematode functional groups and measures of ecosystem processes. It reminds me of a colleague who, 20 years ago, conceded that rotifers were much more abundant than bacteriafeeding nematodes in a set of samples, but they were “ignored”; another colleague said that, as he worked in a nematology department, he could not count the enchytraeids in his samples. I hope that we have moved on from there. Both bacterial-feeding nematodes and rotifers share the same, bacterial resource; enchytraeids, if nothing else, are prey for Nygolaimus. Analyses that I have done with both David Wardle and Wim van der Putten indicate that abundance of “predacious” and “omnivorous” nematodes correlate with the same variables; they both use similar, diverse resources. THROUGH NEMATODE DIVERSITY TO LIVING SOIL PROCESSES – HOLISTIC STUDIES AID PROGRESS

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In 2006 we know that: 1) Nematodes are diverse and their populations in soil respond to changing conditions, such as cropping cycles, grazing by herbivores, forest rotation, fertilisation and excretion by farm animals. 2) It is relatively simple (after some months of experience, preferably with Goodey 1963 or Bongers 1988 at hand) to identify soil nematodes to useful taxa and calculate indices – even some indices that ecologists as a whole use and understand. 3) 50 years after the establishment of ESN (European Society of Nematologists), soil nematodes as a whole are being treasured and there is a belief that a soil with greater nematode diversity is a better soil. In the coming years we must be outward looking and use a range of techniques to better understand: 1) Nematode diversity (with 100+ nematode taxa, each with 4 juvenile stages, females and sometimes males, in each parcel of land). 2) Nematode use of, and interactions with, food resources to provide ecosystem services (aggregating nematode information by some functional groups and across patches). 3) Nematode biology by drawing on morphological, developmental and ecological information from all habitats that the various stages of nematodes utilise. Fig. 1. David Hooper and Judith Killick (now Judy Yeates) in their respective rooms in the Nematology Department at Rothamsted Experimental Station, about 1961. This was the size of their space. Technicians worked on Saturday mornings, with one of their tasks being to blacken the benchtops with boot polish. About this time they were helping Basil Goodey with his book, and David was doing his work on Longidorus in Great Britain.

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Fig. 2. Coffee (?tea) break on the lawn of the brick building which was the first home of the Nematology Department at Rothamsted Experimental Station, about 1961. Left section: second from left Sybil Clark; at rear with glasses Bertie Winslow; on right Judith Killick Right section: John Moore, Chris Doncaster with head high and George Rao; extreme right Harry Wallace

Fig. 3. Converted house occupied by Nematology Section, Entomology Division, DSIR, Nelson (New Zealand) in 1962. The room in the left corner was the lab; that in the right darkened for microscope use. THROUGH NEMATODE DIVERSITY TO LIVING SOIL PROCESSES – HOLISTIC STUDIES AID PROGRESS

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Fig. 4. The wet lab for Nematology Section, Entomology Division, DSIR, Nelson in 1962. To left of bench is a vibromixer; along shelf flasks with rubber adaptors for the Seinhorst 2-flask method and ground glass fitting for a Seinhorst’s elutriator. Above bench, an Endocott’s test sieve for recovery of Heterodera cysts. To the right of bench, a Baermann funnel. Fig. 5. Gregor Yeates sampling nematodes beneath marram grass in coastal sand dunes at Himatangi Beach (New Zealand) in 1967. Bags of samples to left of shovel; in trench up to his knees as the corer was used horizontally for sampling at up to 90 cm depth. (Courting was a subsidiary activity).

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Fig. 6. Russ Ingham sampling in the prairie while doing his PhD at NREL Colorado State University. Russ comments “Too bad the amphibian in my hand does not show up better. We were in the field sampling nematodes when a nema toad came by”.

Fig. 7. Conference dinners can be great for exchange of ideas. Diana Wall and Tom Bongers at such a dinner.

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5. NEMATODE PHYSIOLOGY: SIGNIFICANT DEVELOPMENTS IN THE UNDERSTANDING OF THE BIOLOGY OF SIMPLE EUKARYOTIC ANIMALS HOWARD FERRIS Department of Nematology, University of California, Davis, Clifornia, USA

& HADDISH MELAKEBERHAN Agricultural Nematology Laboratory, College of Agriculture and Natural Resources, Michigan State University, East Lansing, Michigan, USA

Early insights “They occur in arid deserts and at the bottoms of lakes and rivers, in the waters of hot springs and in polar seas where the temperature is constantly below the freezing point of pure water…enormous depths in Alpine lakes and in the ocean…sometimes the eggs and larvae are so resistant to dryness that if converted to dust they revive when moistened…diversity of habitat…inconceivably abundant”. Cobb’s (1914) (Fig. 1) assertions, the validity of which has stood the test of time, demand reflection on the physiological amplitude and unique characteristics that would facilitate such a range of habitats and activities. Nematodes parasitize vertebrates, invertebrates, and plants; many are not parasites but are sustained by feeding on other organisms. Yet, there are recent discoveries of nematodes from the benthos that have no mouths but a rudimentary gut filled with chemoautotrophic bacteria (Ott et al., 1982; Miljutin et al, 2006). Even in some soil nematodes, a reduced mouth and esophagus has led to speculation of diffusion across the cuticle of nutrients in solution (Bongers, 1990, Fig. 2). Early developments in nematology were strongly driven by recognition of the causal organisms of the maladies of man and 80

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Fig. 1. Nathanial A. Cobb

Fig. 2. Tom Bongers

Fig. 3. H.C. Bastian

Fig 4. Emile Maupas

Fig. 5. Ellsworth Dougherty

Fig. 6. Warwick Nicholas

domestic animals. Commanded by their size and obvious human impact, vertebrate-parasitic nematodes have been, for centuries, a focus of investigation (W.P. Rogers, Theodor Von Brand, Donald Fairbairn and many others). Consequently, Ascaris and other vertebrate-parasitic nematodes have been models for understanding nematode physiology, embryology, and development (Wright, 1998). Advances in microscopy enabled observation of the micro- and mesofauna of soil and water. Fascination with the biology of the bacterial-feeding nematodes led to an understanding of their life cycles, including the existence of an alternative life stage by such 19th century keen observers as Schneider, Perez and Bastian (Fig. 3). Consider the likely amazement of a reincarnated Emile Maupas (Fig. 4), pioneer of protozoology, were he made aware of the scienNEMATODE PHYSIOLOGY: SIGNIFICANT DEVELOPMENTS

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tific endeavors and advances surrounding Caenorhabditis elegans, the nematode that he described from organic soils in Algeria. Pioneering work on nematode nutrition in the 1950s and 1960s, independently and/or collaboratively, by Ellsworth Dougherty (Fig. 5) in California and Warwick Nicholas (Fig. 6), Sydney Brenner (Fig. 7) and others in England, particularly the recognition of C. elegans as a potential tool for unravelling the mechanisms of gene expression, are the basis for the enormous importance of C. elegans in recent advances in biology (Riddle & Bird, 1985). A landmark in genomics was the completion of sequencing of the genome of C. elegans in 1998 by the cumulative and collaborative efforts of scientists in laboratories worldwide, particularly by Jonathan Hodgkin (Fig. 8), the Nobel Prize winning Cambridge cartel of Brenner, John Sulston (Fig. 9), Robert Horvitz (Fig. 10), and the many others whose activity they spawned. Incredibly painstaking celllineage studies, mainly through observation of cell division and activity in the nematode egg, by Sulston and colleagues, revealed that 671 cells resulted from divisions that take place in the egg, and most of the rest of the divisions necessary to make up the 959 cells of this nematode occur in later juvenile stages. Among the insights fueled by these studies is a better understanding of the mechanisms and importance of apoptosis, programmed cell death, in development (Riddle, Fig. 11). The tissue and organ functions associated with movement, sensory functions, and reproductive functions are better understood because of the structural and mechanistic insights from nematode model systems. The genome information on “the worm” has stimulated investigations on its plantand animal-pathogenic relatives. The classic studies on nematode ecology and migration through soils (Harry Wallace, Fig. 12), chemosensory attraction to mates, food (Noel Greet, Cliff Blake, Chris Doncaster), moulting (Donald Lee, Fig. 13; Ken Davey; Alan Bird, Fig. 14), survival and adaptation (Adrian Evans; Chris Womersley; Seymour Van Gundy, Fig. 15), and on pathogenesis and cellular changes (Alan Bird; Glenn Bergeson; Victor Dropkin, Fig. 16), of the 1940s to the 1970s, provided an important platform for the advances in understanding of plant-nematode interactions. In this chapter, we will highlight, somewhat anecdotally, some of the important advances made in energetics, vertical gene transfer, moulting and osmoregulation, physio-molecular interactions of nematodes and plants, and ecophysiological adaptation for fitness and survival. While recognizing the names of the contributors and pioneers, we have not been exhaustive in the completeness of our 82

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Fig. 7. Sydney Brenner

Fig. 8. Jonathan Hodgkin

Fig. 9. John Sulston

Fig. 10. Robert Horvitz

Fig. 11. Donald Riddle

Fig. 12. Harry Wallace

review or in providing citations to sources of the subject matters discussed. In some cases, we are guilty of resorting to familiarity by citing our own papers rather than providing an extensive list of references; we intend no slight and wish to be judged as expedient rather than arrogant in our economy of approach.

Energetics and function The C. elegans developmental studies illuminate the resource conservation dilemma of the plant-feeding nematodes. Assuming that the cell numbers of most plant-feeding nematodes are within a reasonable range of that of C. elegans, all of the metabolic energy assoNEMATODE PHYSIOLOGY: SIGNIFICANT DEVELOPMENTS

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ciated with those cell divisions, the differentiation of tissues and most organ systems, hatching, movement, detection of a host, and, in the case of the endoparasites, penetration of the host before feeding commences, is fueled by the resources deposited in the initial single egg cell as it progresses through the oocyte development process. Those resources are finite and must be substantially depleted when partitioned among several hundred cells by the time of emergence from the egg. Thus, energy management strategies that cause the nematode egg, or hatched juvenile, to remain metabolically active prior to access to its food may reduce infectivity. The metabolic and respiratory energetics of soil-inhabiting nematodes have been studied for populations of several species. Nematode respiration rate per individual decreases with size according to the power dependence of basal metabolism and body weight observed in many organisms, R = a Wb, where R is the respiration rate, W is the fresh weight of the individual, and a and b are regression parameters such that b is close to 0.75 for nematodes and other invertebrates (Klekowski et al., 1974; Nicholas, 1975; Apple & Korostyshevskiy, 1980; Atkinson, 1980, Fig. 17). The formula provided by István Andrássy (1956, Fig. 18), in one of the most frequently cited but least read papers in all of nematode ecology, allows calculation of the weight of a nematode as a function of its width and length. It has been an invaluable tool for stepping from the individual to the population and community in studies of nematode energetics. Estimates and measurements of the coefficients of the metabolic power function have been adopted, with some modifications, for calculating growth and energetics requirements in plant-parasitic nematodes (Melakeberhan & Ferris, 1988; Melakeberhan &Webster, 1992; Reversat, 1987). The calculations highlight the issue of feeding rates and resource partitioning. Unless they have a reliable and sustained food source, as in modified plant host-cell structure, large bodied nematodes often have a smaller gonad:body volume ratio than smaller nematodes. More energy resources are committed to growth and metabolic activity of the somatic tissues than to production of oocytes and eggs. Consequently, populations of larger nematodes that do not have a specialized feeding site grow at a slower rate than those of smaller-bodied organisms. Respiration rates of adults range between 1.25 and 8.80 nl O2 h-1 at 20°C among several species studied. Metabolic rates of adults 84

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Fig. 13. Donald L. Lee

Fig. 14. Alan Bird

Fig. 15. Seymour Van Gundy

Fig. 16. Victor Dropkin

Fig. 17. Howard Atkinson

Fig. 18. István Andrássy

range from 1.15 nl O2 µg (f.w.)-1 h-1 for Rhabditis cucumeris to 4.43 nl O2 µg (f.w.)-1 h-1 for Mesorhabditis labiata, at 20°C. At each temperature, metabolic rates of nematodes of similar size vary with thermal adaptation of the species. Metabolic rates of Cruznema tripartitum and Cephalobus persegnis were more sensitive to temperature change than were those of Acrobeloides bodenheimeri, A. buetschlii and Panagrolaimus detritophagus. Cephalobus persegnis exhibited the greatest total metabolic activity across a range of temperatures, and P. detritophagus the least. Observed differences in thermal adaptation may contribute to the predominance of species in the nematode community at different times during the year or at different depths in the soil (Ferris et al., 1995). That nematode species endemic, and apparently successful, in NEMATODE PHYSIOLOGY: SIGNIFICANT DEVELOPMENTS

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the same environment may have different thermal optima (Ferris et al., 1995) is in concurrence with the suggestion of Anderson & Coleman (1982) that temperature-niche breadth mediates competition among species. Differences in temperature-niche breadth determine the predominance of coincident species at different times during the year, or at different depths in the soil. Temporal predominance patterns determine the relative contribution of the coincident species to nitrogen mineralization in managed agricultural systems.

Vertical gene transfer and life history A.C. “Tasso” Triantaphyllou (Fig. 19) at North Carolina State University has made pioneering contributions to nematode cytogenetics (Evans, 1998). Vertical gene transfer in nematodes is achieved through several mechanisms, including mitotic parthenogenesis, meiotic parthenogenesis, amphimixis, and hermaphroditism. Significant advances have been made in our understanding of how genetic information is transferred and which taxa are amenable to mating experiments that will allow inference of gene function. Mechanisms of sex determination in both parthenogenic and amphimictic species, and sex reversal, were revealed and explained through the studies of Triantaphyllou. It seems likely that sex is determined by the effect of environmental conditions on the degree to which genes from the X chromosome regulate genes on other chromosomes. This would allow for environmentally-mediated sex determination, which is frequently observed. For example, since in sexually reproducing species females are XX and males are XO, one might infer that more X product is required to up- or down-regulate the genetic pathways that result in females than for those that result in males. Consequently, if the signal strength from the X chromosomes is suppressed at high temperature, more males might result. Current studies in the laboratory of Charles Opperman, one of Triantaphyllou’s successors, may shed some light on the genetic and molecular basis of sex determination in nematodes http://www.cals.ncsu.edu/plantpath/. Hermaphroditism, an interesting alternative in vertical gene transfer in some members of the family Rhabditidae, was another revelation of the in-depth studies on C. elegans. In sequential hermaphroditism, the gonad first produces sperm, which are stored in a spermatheca. The gonad then produces oocytes, which become fer86

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tilized eggs as they pass through the spermatheca. In C. elegans, about 150 sperm are produced in each arm of the gonad and stored in each spermatheca. The number of sperm produced apparently limits the number of offspring produced by the nematode to around 300 (Gems & Riddle, 1996). True males also occur in a population, but are rare (around 1:1000). However, the frequency of males is enhanced at elevated culture temperature. When a hermaphroditic female is mated with several males, as many as 1400 progeny may be produced (Kimble & Ward, 1988), suggesting that productivity in this form of hermaphroditism, is sperm-limited.

Excretion and osmoregulation Excretory products of metabolic activity differ in animals of different habitats. Nitrogenous waste products, usually in the form of ammonia, result from metabolic pathways that involve proteins and amino acids. Since ammonia is toxic, terrestrial animals, including arthropods and vertebrates, generally bind the –NH3 group into either urea or uric acid, which are accumulated prior to excretion (Campbell, 1973). Such organisms are termed uricotelic. Nematodes are aquatic organisms, inhabiting marine and fresh water and the water films of soil environments. Like most aquatic organisms, they continually excrete ammonia into the environment as it is produced, thus avoiding the toxic storage problem. Such organisms are termed ammonotelic (Perry, Fig. 20 and Wright, 1998). The excretion of waste nitrogenous products into the soil environment may be a significant contribution to nitrogen availability to plants (Ferris, et al., 1998; Chen & Ferris, 1999). A major difficulty in studying osmotic and ionic regulation of soil-inhabiting and plant parasitic nematodes is their small size, and larger nematodes like ascarids are often used as model systems (Wright, 1998).

Physio-molecular interactions of nematodes and plants In an era of increasing environmental awareness, it is necessary to find economically and ecologically sustainable nematode management alternatives through an understanding of the physio-molecular and genetic bases of plant nematode interactions from the sub-celluNEMATODE PHYSIOLOGY: SIGNIFICANT DEVELOPMENTS

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Fig. 19. A.C. Triantaphyllou

Fig. 21. Richard Hussey

Fig. 20. Roland Perry

Fig. 22. Jaap Bakker

Fig. 23. Valerie Williamson

lar to the ecosystem level. Studies of the physiological changes and formation of specialized feeding sites associated with sedentary plant parasitic nematodes by Alan Bird in Australia and of cellular changes by S.G. Myuge, Glenn Bergeson and Victor Dropkin in the 1960s and 1970s have been the foundation and inspiration for many host parasite studies at the cutting edge of science. The recent activities of several research groups merit high recognition. All are important research programs that are integral to longer-term trajectories from which will emerge novel approaches in nematode management. First, research spearheaded by Richard Hussey (Fig. 21) at the University of Georgia has characterized the molecular and functional nature of glandular secretions of the root-knot and cyst nematodes, which have very specialized and complex feeding relation88

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ships with their host plants. In collaboration with Rick Davis, North Carolina State University, Thomas Baum, Iowa State University, and Jaap Bakker (Fig. 22) and Arjen Schots at Wageningen University, comprehensive profiles have been developed of genes for parasitism that are expressed in the esophageal gland cells of the nematodes (Davis et al., 2000). The potential application of these studies is to target the genes in intervention strategies that will disrupt the host parasite relationship http://www.plant.uga.edu/faculty/hussey.htm. Second, the pioneering work of the Plant Nematode Genetics Group comprising David Bird, Charlie Opperman and collaborators at Rothamsted Research (Rothamsted Experiment Station) in England, which sequenced the Pasteuria penetrans (nematode-parasitic bacterium) http://www.cals.ncsu.edu/plantpath/. An important goal of that group is to understand the molecular basis of nematode-plant interactions, using Meloidogyne, Heterodera and Globodera spp. as models and employing cellular, genetic and genomic approaches. Third, Valerie Williamson (Fig. 23) at the University of California, Davis, has, over several decades, made major contributions to our understanding of the molecular and genetic basis of the Mi-gene (present in most commercial tomatoes) and other forms of resistance against root-knot nematodes and to the associated host and parasite recognition mechanisms. Isgouhi Kaloshian, now at UC Riverside, and Kris Lambert at the University of Illinois, did their postdoctoral and/or graduate work in Williamson’s laboratory and have expanded and extended her studies. Sterols are among the specific nutritional components that nematodes need from their hosts, and identifying ways to disrupt the sterol supply has been a major focus in the research of US Department of Agriculture’s David Chitwood (Fig. 24). Recently, Chitwood & Skanter (2006) identified two genes in Heterodera glycines that code for products similar to the 17â-hydroxysteroid dehydrogenases and are involved in the synthesis of steroid hormones in mammals. Harry Wallace’s (1973) soil-nematode interface and Alan Bird’s (1974) and Victor Dropkin’s (1980) nematode feeding behaviour analyses have led to more applied studies. Starting from his graduate research with John Webster, the second author of this chapter acknowledges that the classic work of Harry Wallace on the soil-nematode interface provided the basis for his own work on the manipulaNEMATODE PHYSIOLOGY: SIGNIFICANT DEVELOPMENTS

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tion of soil nutrients to adversely affect nematode infective and developmental behaviour (Melakeberhan, 1999). Moreover, Victor Dropkin’s (1980) categorizing of plant parasitic nematodes into destructive (host cells killed, Pratylenchus), adaptive (cells modified, Heterodera), and neoplastic (cells modified and undergoing new growth, Meloidogyne) feeding behaviors generated interest in whether nematodes of different feeding behaviour affect host physiology differently. Similarity of effects leads to the possibility that the damage of several nematode species may be offset by a single management option (e.g., nutrient amendment). If effects are different, more situation-specific options will be required (Melakeberhan, 2006).

Ecophysiology: physiological adaptations for fitness and survival Nematodes occupy many trophic levels and perform many services in the soil food web (Bongers & Ferris, 1999). Plant and soil nematodes have evolved a suite of adaptations that confer fitness in a variety of spatio-temporal niches and that enhance their probability of survival under adverse conditions. The physiological basis and mechanisms of nematode survival, including omnivory, dauer stages, cryptobiosis and dormancy, are reviewed by Womersley et al. (1998). Plant-feeding nematodes are primary consumers of incoming resources. They, in turn, constitute a resource for many other organisms in the soil food web through predation by fungi, bacteria, and a diversity of mesofauna. Besides providing ecosystem services of nutrient cycling, the predation involved in the transfer of carbon through the soil community may result in top-down regulation of the primary consumer species, particularly where food resources are limited due to seasonal host phenology or competition at high nematode densities. A brief review of some mechanisms of nematode survival and fitness provides insight into the observational and inferential powers of the scientists who have studied these aspects of nematode biology. OMNIVORY: Although the feeding habits of many plant and soil nematodes have not been determined, a great deal of information is available from experiments, observations, inferences based on feeding structures, and on organism associations. The classic paper of 90

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Gregor Yeates (Fig. 25) and colleagues (Yeates et al., 1993) summarized feeding habit information available to that time. They categorized six feeding types among soil and plant nematodes: feeding on vascular plants, feeding on fungal hyphae, feeding on Fig. 24. David Chitwood Fig. 25. Gregor Yeates bacteria, feeding on animals, feeding on unicellular eukaryotes and omnivorous feeding. They also recognized two other categories, the ingestion of substrate incidental to feeding by open mouthed morphotypes such as bacterial feeders and certain predators, and dispersal or Fig. 26. David Viglierchio Fig. 27. Neil A. Croll infective stages that may not be feeding, often in phoretic relationships with insects. The term “omnivore“ is usually applied to certain nematodes of the Dorylaimida for which omnivory has been observed or for which feeding habits are unknown. Clearly, some dorylaims are plant feeders (e.g., Xiphinema) and some are predators (e.g., Labronema). True omnivory is an adaptation to unreliable food sources that may be seasonally or spatially sparse. Besides its occurrence in the Dorylaimida, it has been observed in Mononchida where juveniles may be sustained on bacteria (Yeates, 1987) in certain Aphelenchina (e.g., Aphelenchoides spp.) and Tylenchina (e.g., Ditylenchus spp.) where survival between plant hosts by feeding on fungi is of obvious adaptive significance.

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DAUER STAGES: Many soil nematodes, particularly bacterial feeders of the Rhabditidae, Panagrolaimidae and Diplogasteridae, have a metabolically-suppressed specialized survival stage. Schneider (1866) reported the existence of a life stage of rhabditid nematodes with a cuticle differing from that in other stages; he considered this form to be a moulting stage but was uncertain of its role. According to Maupas (1899), Pérez (1866) recognized an “encysted” stage in Rhabditis teres and indicated that larvae easily encysted at the end of the second stage. Experimentally, Maupas (1899) determined that always the same life stage entered encystment when nutrients were lacking. He showed that emergence from the encysted stage occurred with enrichment and noted that encysted nematodes survive for weeks and are often a dispersal stage. Later, Fuchs (1916), in his description of rhabditids associated with bark beetles, coined the term “dauerlarva” for the persistent or enduring stage of these nematodes. Many of the nematodes that have phoretic relationships with insects are in a dauer stage during the phoresy. Likewise, entomopathogenic rhabditids await their insect hosts in a dauer stage. Dauerlarva induction in C. elegans is mediated by the ratio between a dauer-inducing pheromone, which is constantly produced by the nematode, and the magnitude of a carbohydrate signal from the bacterial prey (Riddle, 1988). The ratio provides a measure of population size in relation to food availability. When the dauer-inducing pheromone is significantly greater than the food signal, dauer formation commences (Ferris & Bongers, 2006). CRYPTOBIOSIS: An attribute (literally, hidden life) of certain nematodes that enables their survival without detectable metabolic activity. The most commonly recognized forms of cryptobiosis include anhydrobiosis, cryobiosis, anoxybiosis and osmobiosis in response to dehydration, cooling, low oxygen, and osmotic shock, respectively (Womersley et al., 1998). The first record of anhydrobiosis, although not recognized as such at the time, was that by Needham (1744), when he opened the seed galls of Anguina tritici on wheat. Anhydrobiosis is a common attribute of nematodes that are successful in habitats subject to seasonal drying and to those that feed on the above ground parts of plants. For example, fourth stage juveniles of Ditylenchus dipsaci enter anhydrobiosis, usually in large masses, on or below the surface of plant tissue, and the term “eelworm wool” describes the appearance of these dried nematodes. 92

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Similarly, the second-stage juveniles of Aphelenchoides besseyi enter anhydrobiosis under rice hulls. Many nematode species are capable of anhydrobiosis when subjected to slow drying. However, if the drying is rapid, there is insufficient time for the necessary physiological and membrane structural changes to take place. DORMANCY: The term is applied to the condition of lowered metabolism and various categories have been recognized in nematodes (Womersley et al., 1998): Facultative quiescence: dormancy under unfavorable conditions with development readily resumed as conditions become favorable. Obligate quiescence: required dormancy for a life stage with development readily resumed under favorable conditions. Facultative diapause: dormancy initiated by environmental factors with delayed resumption of development under favorable conditions. Obligate diapause: dormancy initiated by endogenous factors with delayed resumption of development under favorable conditions after specific requirements are satisfied (e.g., in Meloidogyne naasi). Zheng & Ferris (1991) described the delayed development of some eggs in Heterodera schachtii despite favorable conditions. They recognized eggs in four categories: the non-dormant condition of eggs that hatch rapidly in water; eggs that hatch rapidly in host root diffusate; eggs that hatch slowly in water over a long period of time; eggs that hatch slowly over a long period of time in host root diffusate. The combination of these categories of egg development results in distribution of hatch over a considerable period and enhances the probability that some of the emerging juveniles will encounter a host plant under conditions conducive to infection, and thus species survival. HOST RANGE AND HOST RECOGNITION: Many plant-feeding nematodes that are successful in annual crop agriculture have wide host ranges. There is a high probability that they are able to feed on a variety of the plants provided in cropping sequences. However, there are other successful strategies. Some nematodes with quite narrow host ranges are successful because they remain in a dormant state until stimulated to emerge by root exudate signals recognized from a host plant (e.g., Globodera rostochiensis). The non-feeding dormant stage might technically be considered a dauer stage without the morphological features of extra cuticle and a closed mouth (Bird & Opperman, 1998). In some cases, the dormant stage is the second NEMATODE PHYSIOLOGY: SIGNIFICANT DEVELOPMENTS

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stage juvenile retained in the egg (e.g., Heteroderinae) whereas, in other cases, it may be a pre-adult juvenile (e.g. Paratylenchus spp.). RESPONSE TO HOST STIMULI: The soil-root interface and nematode sensory and response behaviour have been the subjects of many investigations over the past several decades (Viglierchio, 1961, Fig. 26; Klingler, 1965; Prot & Van Gundy, 1980; Riddle & Bird, 1985; Pline & Dusenbery, 1987; Robinson, 1995). However, as the always ebullient and insightful Rolo Perry (1996; 2006) of Rothamsted Research points out, many information gaps remain when explaining the physiological basis and mechanism of the interactions. In a career cut short by his untimely death, the debonair Neil Croll (Fig. 27) synthesized the available information on a range of physiological aspects across the Nematoda in several books. With regard to nematode behavior, Croll recognized that taxes, directed movement towards or away from a stimulus, and kineses, change in the rate of activity or frequency of turning in the presence of a stimulus, are both observed in nematodes (Croll, 1970; Perry, 1996). Resource-locating behavior in nematodes probably consists of a combination of taxes and kineses (Lee, 2002; Rodger et al., 2003; Young et al., 1998), and electrophysiological analyses indicate that reduced activity or more frequent turning can result in aggregation near the stimulus (Perry & Riga, 1995). Taxis and kineses are characterized according to the nature of the stimuli, which may include CO2, pH, temperature gradients and root diffusates. CO2, expected to be in higher concentrations in the rhizosphere than in bulk soil, is a strong attractant in a certain concentration range to some nematodes (Klingler, 1965; Pline & Dusenbery, 1987; Robinson, 1995). Interestingly, given the choice of plant roots and insect larvae in an olfactory tube, bacteriophagous entomophilic nematodes moved to plant roots (Boff et al., 2002). However, prior to invading the host, nematodes must sense additional factors to differentiate between the sources of general signals (Rühm et al., 2003). CO2 may provide a directional stimulus and stimulate a taxis response. However, once the nematode is near the resource, plant signature compounds, such as flavonoids or alkaloids, may precipitate kinesis responses by the nematode resulting in their localization of individuals around food sources. Although both attraction and repellency of host plants to nematodes have been the subject of several investigations, only a few host- or nonhost-specific compounds have been identified that 94

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mediate the responses (Chitwood, 2002). In a critical assessment of the spatial and temporal nature of the chemoattraction and nematode orientation in soil, Perry (2006) concluded that CO2 and root diffusates followed by temperature are major nematode behaviour modifying factors.

Outlook In a relatively short period, significant advances have been made in our understanding of the biology and physiology of nematodes, and we are on an exciting trajectory. Three of the driving forces are evident. First, the success of each new set of researchers in stepping off from the platform erected by earlier workers, in effect embodying the Chinese concept of “standing on the shoulders of the great man”. Second, the wonderful advances in technology that have allowed the scaling-down of sensors and the amplification and conversion of the signals necessary for the equipment (developed for rats and guinea pigs) to measure the physiological processes and secretions of the nematodes. Third, the serendipitous selection of Caenorhabditis elegans, of all the organisms in the world, as the model system for developmental biology and for genomic characterization. If, as asserted by Lorenzen & Platt (1994), four out of every five multicellular animals on the planet are nematodes, they provide the potential for providing model and assay organisms for advancing science at a multitude of levels, from subcellular biology to the monitoring of global climate change.

References and citations ANDERSON, R.V. & COLEMAN, D.C. 1982. Journal of Nematology 11: 69–76. ANDRÁSSY, I. 1956. Acta Zoologica Academiae Scientarum, Hungaricae 2: 1–15. APPLE, M.S. & KOROSTYSHEVSKIY, M.A. 1980. Journal of Theoretical Biology 85: 569–573. ATKINSON, H.J. 1980. In: Nematodes as biological models, Vol. 2. Zuckerman, B. M. (Ed.), pp.116–142. Academic Press, New York, NY. BIRD, A.F. 1974. Annual Review of Phytopathology 12: 69–85 BIRD, D.M. & OPPERMAN, C.H. 1998. Journal of Nematology 30: 299–308. BOFF, M.I.C., VAN TOL, R.H.W.M. & SMITS, P.H. 2002. Biocontrol 47: 67–83 BONGERS, T. 1990. Oecologia 83: 14–19. NEMATODE PHYSIOLOGY: SIGNIFICANT DEVELOPMENTS

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BONGERS, T. & FERRIS, H. 1999. Trends in Evolution and Ecology 14: 224–228. CAMPBELL. 1973. In: Comparative animal physiology. Processer, C.L. (Ed.), pp. 279–316. W. B. Saunders, Philadelphia, PA.. CHEN, J. & FERRIS, H. 1999. Soil Biology and Biochemistry 31: 1265–1279. CHITWOOD, D.J. 2002. Annual Review of Phytopathology 40: 221–249. CHITWOOD, D.J. & SKANTER, A. 2006. Proceedings, 28th International Symposium, European Society of Nematologists. pp. 46–47. COBB, N.A. 1914. Transactions of the American Microscopical Society 33: 92–94. CROLL, N.A. 1970. The behavior of nematodes, their activity, senses and responses. Edward Arnold, London. DAVIS, E.L, HUSSEY, R.S., BAUM, T J., BAKKER, J., SCHOTS, A., ROSSO, M. & ABAD, P. 2000. Annual Review of Phytopathology 38: 365–396. DE CUYPER, C. & VANFLETEREN, J.R. 1982. Comparative Biochemistry and Physiology 73A: 283–289. DROPKIN, V.H. 1980. Introduction to plant nematology. John Wiley and Sons, New York. EVANS, A.A.F. 1998. In: The Physiology and biochemistry of free-living and plant-parasitic nematodes. Perry, R. N. & Wright, D. J. (Ed.), pp.133–154. CABI Publishing, Wallingford, UK. FERRIS, H, VENETTE, R.C., VAN DER MEULEN, H.R. & LAU, S.S. 1998. Plant and Soil 203: 159–171. FERRIS, H, LAU, S.S. & VENETTE, R.C. 1995. Soil Biology and Biochemistry 27: 319–330. FERRIS, H, & BONGERS, T. 2006. Journal of Nematology 38: 3–12. FUCHS, G. 1916. Zoologische Jahrbücher Abteilung für Systematik, Ökologie und Geographie der Tiere 38:109-170. GEMS, D. & RIDDLE, D.L. 1996. Nature 379: 723–725. KIMBLE, J. & WARD, S. 1988. In: The Nematode Caenorhabditis elegans. Wood, W. B. (Ed), pp.191–214. Cold Spring Harbor Laboratory Press. KLEKOWSKI, R.Z., Schiemer, F. & Duncan, A. 1979. Oecologia 44: 119–124. KLEKOWSKI, R.Z., Wasilewska, L. & Paplinska, E. 1974. Nematologica 20: 61–68. KLINGLER, J. 1965. Nematologica 11: 14–18. LEE, D.L. 2002. In: The Biology of nematodes. Lee, D. L. (Ed), pp.369–387. Taylor and Francis, London. LORENZEN, S. & PLATT, H.M. 1994. The Phylogenetic systematics of free-living nematodes. The Ray Society, London. MAUPAS, E. 1899. Archives de Zoologie Expérimentale et Générale 7: 563–628. MELAKEBERHAN, H 1999. Nematology 1: 113–120. MELAKEBERHAN, H. 2006. Nematology 8: 129–137. MELAKEBERHAN, H. & FERRIS, H. 1988. Journal of Nematology 20: 545–554. MELAKEBERHAN, H. & WEBSTER, J.M. 1992. Fundamental and Applied Nematology 15: 179–182. MILJUTIN, D.M., Tchesunov, A.V. & Hope, W.D. 2006. Nematology 8: 1–20. NEEDHAM, T. 1744. Philosophical Transactions of the Royal Society 42: 634–641. NICHOLAS, W.L. 1975. The Biology of free-living nematodes. Clarendon Press, Oxford. OTT, J.A., Rieger, G. & Enderes, F. 1982. Marine Ecology 3: 313–333. PÉREZ, J. 1866. Annales des Sciences Naturelles, Zoologie 6: 152–307. PERRY, R.N. 1996. Annual Review of Phytopathology 34: 181–89

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PERRY, R.N. 2006. XXVIII ESN International Symposium Proceedings. Pp 46. PERRY, R.N. & RIGA, E. 1995. Japanese Journal of Nematology 25: 61–69. PERRY, R.N. & WRIGHT, D.J. 1998. The Physiology and biochemistry of free-living and plant-parasitic nematodes. CABI Publishing, Wallingford, UK. PLINE, M. & DUSENBERY, D.B. 1987. Journal of Chemical Ecology 13: 873–888. PROT, J-C. & VAN GUNDY, S.D. 1980. Journal of Nematology 13: 213–217. REVERSAT, G. 1987. Revue de Nématologie 10: 115–117. RIDDLE, D.L. 1988. In: The Nematode Caenorhabditis elegans. Wood, W.B. (Ed), pp. 393–412. Cold Spring Harbor Laboratory. RIDDLE, D.L. & BIRD, A.F. 1985. Parasitology 91: 185–195. ROBINSON, A.F. 1995. Journal of Nematology 27: 42–50. RODGER, S, BENGOUGH, A.G. GRIFFITHS, B.S., STUBBS, V. & YOUNG, I.M. 2003. Phytopathology 93: 1111–1114. RÜHM, R, DIETSCHE, E. HARLOFF, H.J. LIEB, M. FRANKE, S & AUMANN, J. 2003. Nematology 5: 17–22. SCHIEMER, F. 1982. Oecologia 54: 108–121. SCHNEIDER, A.F. 1866. Monographie der Nematoden. Georg Reimer, Berlin, Germany. VIGLIERCHIO, D.R. 1961. Phytopathology 51: 136–142 WALLACE, H.R. 1973. Nematode ecology and plant disease. Arnold, London. WOMERSLEY, C.Z, WHARTON, D.A. & HIGA, L.M. 1998. In: The physiology and biochemistry of free-living and plant-parasitic nematodes. Perry, R.N. & Wright, D.J. (Ed.), pp.271–302. CABI Publishing, Wallingford, UK. WRIGHT, D.J. 1998. In: The physiology and biochemistry of free-living and plant-parasitic nematodes. Perry, R. N. & Wright, D. J. (Ed.), pp.104–131. CABI Publishing, Wallingford, UK. YEATES, G.W. 1987. Biology and Fertility of Soils 3: 143–146. YEATES, G.W., BONGERS, T., DE GOEDE, R.G.M., FRECKMAN, D.W. & GEORGIEVA, S.S. 1993. Journal of Nematology 25: 315–331. YOUNG, I.M., GRIFFITHS, B S., ROBERTSON, W. M., & MCNICOL, J.W. 1998. European Journal of Soil Science 49: 237–241. ZHENG, L. & FERRIS, H. 1991. Revue de Nématologie 14: 419–426.

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6. MOLECULAR TAXONOMY OF NEMATODES PIERRE ABAD & PHILIPPE CASTAGNONE-SERENO UMR Interactions Plantes Microorganismes et Santé Végétale, INRA/CNRS/Université de Nice Sophia Antipolis, 400, Route des Chappes, 06903 Sophia Antipolis Cedex, France.

Organism taxonomy has been revolutionized by the accessibility of molecular data. Molecular approaches have proved especially useful for sorting out the identification and relationships in those taxa for which morphological data have always presented difficulties. In that respect, nematodes represent a good example since they are remarkably constrained morphologically. The two congeneric sibling species of freeliving nematodes, Caenorhabditis elegans and C. briggsae, well illustrate this fact. Although their discrimination represents a challenge to most trained nematode taxonomists, the recent availability of their complete genome sequence shows that these two species diverged 100 million years ago, i.e., between 5 and 45 (25?) million years before the splitting of the mouse and the human lineages. In addition, these two nematode species have been shown each to bear at least 2000 genes (ca. 10% of the genome) that are not found in the other species.

The “molecularisation” of nematode taxonomy Before these recent advances, the first attempts at molecular taxonomy in the field of nematology occurred more than 30 years ago and the major focus of molecular analyses has been the diagnosis of economically important species and their infraspecific forms in agricultural, medical and veterinary sciences. Among plant parasitic nematodes, root-knot and cyst nematodes have been central to this type of molecular-based research. The molecular biology approach also has been of increasing importance in other economically important taxa, such as Pratylenchus, Xiphinema, Bursaphelenchus and the entomopathogenic nematodes Heterorhabditis and Steinernema. 98

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The “molecularisation” of nematode taxonomy has been successfully developed for use in diagnosis. Thus, any difference in protein or isozyme phenotype, affinity for antibody, DNA polymorphism, DNA probe or sequence data for any gene or region of the genome has been used as a potential diagnostic character. In the same way, studies have used molecular data in phylogenetic analyses, and in those in which it has been applied a phenetic approach has been adopted using distance measures or relationships between species within genera or between genera. One of the first examples of the power of molecular data was given by Antoine Dalmasso in a complete study of allozymes in the Meloidogyne genus. This approach was applied to M. incognita, M. arenaria and M. javanica, a complex of species reproducing by obligatory mitotic parthenogenesis. In terms of identification, these clonal species presented special difficulties which cannot be accommodated by a species definition based upon reproductive isolation. The examination of their isoenzymes revealed their degree of genetic diversity and indicated that they are true species. From that point, enzymatic polymorphism has been widely used on plant parasitic nematodes. In the framework of the International Meloidogyne Project, exhaustive surveys of the main root-knot nematode species were undertaken by Anastasios Triantaphyllou in the late of 1980s based on esterase and malate dehydrogenase phenotypes. In general, however, the most commonly studied enzymatic system is that of esterases. Multilocus studies have been used to investigate nematode dispersal, to trace their origin and for species identification. In principle, several enzymatic systems can be resolved from single individuals, but diverse studies have reported low polymorphism of the resolved loci. Although this approach has important limitations (e.g., the biological material must be kept alive or be frozen until used, and, at a given locus, the technique reveals only a fraction of the actual genetic variation) the alloenzymes represent a useful marker, and the method is still largely in use in many laboratories worldwide, more than 25 years after the original publication. The major focus in molecular taxonomy over the last 20 years has been DNA analysis, and the first data were mainly provided in the early 1980s by the group in John Webster’s laboratory in Canada for both plant-parasitic and entomopathogenic nematodes. At that time, techniques to visualize DNA sequence differences were based on basic approaches such as detection of restriction fragment length polymorphism (RFLP), DNA probes and DNA sequencing. In general, MOLECULAR TAXONOMY OF NEMATODES

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most DNA studies for species and pathotype separation have made comparisons between RFLPs in total DNA of unknown nematode populations against species and pathotype standards. Obtaining the nucleotide sequence has provided access to the ultimate detail of variation in the DNA. Sequencing DNA fragments was quickly developed by techniques that have now become routine and economic. DNA sequences were collated and stored in data libraries (e.g., EMBL and GenBank) and have been universally usable, powerful data. These have also provided search facilities which allow unknown sequences to be identified by similar search engines, such as BLAST. Sequencing was done directly or after cloning into plasmid vectors. Foremost among the factors which have contributed to the rapid increase of molecular data sets in taxonomy is the development of the Polymerase Chain Reaction (PCR). Taxonomists were no longer constrained by the size of the organisms for obtaining a sufficient amount of biological material for analysis. PCR amplifications were conducted upon a single juvenile nematode or an individual egg in a relatively crude assay. The concept of universal primers has reduced the need to initiate each taxonomic project with a time-consuming hunt for primer sequences that will amplify the desired product. These universal amplification primers are oligonucleotide sequences which anneal to genomic regions of such high evolutionary conservation that they serve as amplification primers for a wide taxonomic range of organisms. The random amplified polymorphic DNA (RAPD) technique uses the PCR principle for random amplification of DNA sequences, and does not require any preliminary sequence information of the genome under study. Amplification is performed using a single primer with a very short sequence (8–10 base pairs) under annealing temperature conditions (usually low) that enhance multiple binding at sites scattered throughout the genome. Several DNA fragments are usually amplified and some of these may be present in a proportion of the individuals in a nematode population. Using a large set of primers has the advantage of screening the entire genome. However, the interpretation of RAPD data is sometimes limited by poor repeatability of the results, with the problem aggravated by the small size of nematode stages (larvae) in which the quantity of DNA obtained per individual is reduced, thus preventing accurate assays of DNA concentration. Another limit of these markers is that the RAPD patterns display dominance, preventing identification of 100

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heterozygotes. Nevertheless, the RAPD technique has been often used as the first source of information because results can be generated quickly and easily. A useful approach was developed in the middle of the 1990s based on amplified fragment length polymorphism (AFLP). This technique selects, by PCR amplification, restriction fragments generated from a total digest of genomic DNA. Using this method, sets of restriction fragments can be visualized by PCR without prior knowledge of the genome of the target nematode species. As for other fingerprinting-based methods, such as RAPD, they give access to an almost unlimited number of genetic markers. However, in the AFLP technique, heterozygote genotypes cannot be easily distinguished from other heterozygote genotypes. Significant examples of applications of such methodology were given by the group of Fred Gommers in The Netherlands on the gene pool similarities in potato cyst nematodes and by Philippe Castagnone-Sereno on the virulence and molecular diversity in the parthenogenetic root-knot nematodes.

Most popular markers Different genomic regions have been analyzed depending on the problem examined, in particular those regions that have different rates of evolution and/or different modes of inheritance (maternal vs Mendelian). Rapidly evolving genes have been shown to be useful for comparisons of closely related taxa, and slowly evolving genes for comparisons of distantly related taxa. Two of the most popular markers used in molecular taxonomy have been mitochondrial DNA (mtDNA) and nuclear ribosomal DNA (rDNA). The mtDNA represented a logical choice in molecular nematode taxonomy. This entire circular molecule is present in high copy number and consists of rapidly evolving genes that has allowed large taxonomy application. Several universal primers which bind to highly conserved regions of the mitochondrial genome were demonstrated to amplify DNA from organisms ranging from insects to humans. Ironically, these same primers have not been useful for the amplification of nematode DNA. Nonetheless, several other primer sets have been published that work for numerous nematode taxa. In a survey of mitochondrial genes such as cytochrome oxidase subunits I and II (COI, COII) and the large 16S ribosomal gene, Vivian Blok MOLECULAR TAXONOMY OF NEMATODES

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and collaborators showed extensive variation that distinguished the major species of tropical root-knot nematodes. In addition, mitochondrial genomes have been entirely sequenced in different nematodes species including the plant parasitic nematode genus, Meloidogyne. Considering the estimated rate of ribosomal mitochondrial RNA (16S), it was demonstrated that the ancestral lineages of Caenorhabditis and plant parasitic nematodes diverged during or before the Cambrian period, over 500 million years ago. Nuclear ribosomal DNA still provides one of the most complete tools for a multitude of molecular tasks in nematode taxonomy. The main ribosomal locus in eukaryotic organisms consists of three genes encoding the 18S, 5.8S and 28S subunits of the ribosome. Between these genes are the internal transcribed spacers 1 (ITS1, between the 18S and the 5.8S) and 2 (ITS2, between the 5.8S and the 28S gene). The three genes are reiterated in tandem and between each group are the intergenic spacers (IGS). Virginia and John Ferris showed the usefulness of these markers as taxonomic tools in extensive surveys of cyst-forming nematodes. Early examination by Barry Honda and colleagues of these ribosomal repeats in Meloidogyne was phylogenetically interesting because they demonstrated that a 5S rRNA gene is located in the IGS region between the 18S and the 28S genes. This genomic structure is unusual in higher eukaryotes, and differs from that of Ascaris and Caenorhabditis. The reiteration of these genes in the genome makes their detection considerably easier than for single copy genes. In addition, ribosomal loci exhibit different degrees of conservation along the sequence. rDNA has a general trend of sequence conservation, tending to sequence divergence from 5” to 3” in transcribed regions. With these properties, by studying one single gene, it is possible to cover many taxonomic levels, which has rendered this type of gene one of the most popular in nematode taxonomy.

Phylogeny In addition to the accessibility of molecular data and to techniques that contributed to the rapid increase of molecular data sets, another feature that has stimulated research in nematode phylogeny has been the availability of software packages for computational analysis. Programs for data base searching, sequence editing, data conversions, alignment, and phylogenetic analysis have been obtained for 102

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many computer configurations. Methods for estimating genetic distance as well as several methods for phylogenetic inference, including UPGMA, neighbour-joining, and maximum parsimony, have been shown to be very helpful at resolving nematode phylogenies at different taxonomic levels. ITS2 and COI together have provided a powerful tool for phylogenetic studies of closely related species, and different research groups have characterized the relationships between species in various genera of entomopathogenic nematodes (Heterorhaditis and Steinernema) and of plant parasitic nematodes as diverse as Meloidogyne, Heterodera and Globodera, Pratylenchus, Xiphinema and Bursaphelenchus. The 18S rDNA was useful for phylogenetics at the other end of the nematode taxonomic spectrum. A paper of Mark Blaxter and collaborators in 1998 described, for the first time the phylogeny of the whole Nematoda phylum. However, a potential danger of studying a single gene or region of the genome is that the derived phylogeny may reflect the evolution of the gene rather than the organism. Care is needed also to ensure that only the homologous sequences are compared and that the problems of pseudogenes, gene conversion and duplication are considered. Another problem is that a set of markers appears to be lacking for use at intermediate taxonomic levels, i.e., between the genus and family. To overcome these two limitations, one solution has been to sample a larger gene set; any biases presented by a single gene with a history not reflecting that of the species is hopefully offset by the larger selection of genes that reflect that proper relationship. Candidate nuclear protein-coding genes that have proved to be useful in insect studies, such as elongation factor 1a, glucose-6-phosphate dehydrogenase, phosphenolpyruvate carboxykinase, are now being used for nematode phylogeny studies. In a broader approach, David Bird and collaborators very recently selected a set of orthologous genes initially identified from genome-wide EST analysis to describe the most robust phylogenies for nine nematode species belonging to the order Tylenchida.

Diagnosis and species discrimination The small size of most nematode species of agronomic importance implies that a limited number of morphological characters are available for identification. In addition, identification methods must MOLECULAR TAXONOMY OF NEMATODES

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deliver accurate, rapid, reliable and affordable results. Molecular approaches are becoming more widely applied to meet these demands, in part because of the relative simplicity of their application in the laboratory. In nematode diagnosis, significant progress has been made for major plant parasitic nematode groups. As pointed out by Antoine Dalmasso and subsequent researchers, isozymes provide a means of identifying the four most economically important Meloidogyne species. Species-specific monoclonal antibodies which allowed nematode quantification were developed by Arjen Schots in the late 1980s for the separation of Globodera pallida and G. rostochiensis. Both types of approaches are routinely used by official Plant Protection Services throughout the world. Subsequent attempts to develop molecular diagnostic methods have been based on DNA hybridization techniques. The first studies were based on analysis of RFLP. Although RFLP analyses allowed clear discrimination at both interspecific and intraspecific levels, they could not be used routinely because they were time-consuming and required large amounts of DNA. Species-specific DNA probes were then developed using repetitive DNA, among which are the highly reiterated sequences known as satellite DNA (satDNA). The two authors of this chapter have illustrated its power as a diagnostic marker in the plant parasitic nematode genera Bursaphelenchus and Meloidogyne. In particular, they developed a reliable and rapid yes/no system to discriminate quarantine species such as B. xylophilus and M. chitwoodi from congeneric species. In assays using satDNA as specific probes, an unambiguous separation of species was obtained with the main advantage of avoiding time-consuming DNA extractions. Because of the highly repetitive nature of satellite DNA, this sequence is able to detect one individual in a simple “squash” blot hybridization, even in root tissues. Detailed analysis of the sequences in nematode satDNA allowed the selection of primers that lead to a specific amplification signal in PCR experiments. Beside satDNA, universal primers which bind to the highly conserved regions present in the genes of all taxa, are available (e.g. mtDNA or rDNA). They have been used extensively in PCR technology for diagnostic purposes for most plant parasitic nematodes of agronomic interest.

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Current research and future prospects New molecular approaches that are currently under investigation should enable routine diagnosis. The real-time (quantitative) PCR (qPCR) technique relies on continuous monitoring of amplicon synthesis, and allows a faster detection and quantification of the target DNA, without the need for laborious post-PCR gel electrophoresis. Very recently, rDNA-based qPCR was successfully applied for the detection of the pinewood nematode, B. xylophilus and the cyst nematodes, H. schachtii and G. pallida. The DNA microarray technology provides a promising alternative for high-throughput genotype-based diagnostics. The distinct advantage of this detection approach is that it combines powerful DNA amplification strategies with subsequent hybridization to oligonucleotide probes specific for multiple target sequences. Briefly, hundreds to thousands of 30–50 nt probes for specific targets are arrayed onto a single glass microscope slide, to which fluorescently labelled PCR product or genomic DNA to be tested is then hybridized and detected. Recently, a large research project was initiated in the EU to investigate the feasibility of a microarray-based method for the detection of the quarantine pathogens and pests of potato, including the quarantine nematode species M. chitwoodi. Molecular markers have been successfully used in molecular taxonomy for decades. However, the long-term goal of molecular diagnostics is to develop protocols for the accurate and rapid identification of all nematode species. The need for a speedier system of identifying species arises from the fact that only a small proportion of nematodes have been taxonomically described and that there are many more nematode species to be identified. DNA barcoding is a taxonomic method which uses a short DNA sequence from a particular region of the genome to provide a “barcode” for identifying species. The novelty of the DNA barcoding proposal resides in its enormous scale and proposed standardization. An unknown organism may be identified from its sequence of a target gene. Fast neighbour-joining cluster analysis will link the unknown sequences with some species in the database, usually its closest relatives. Molecular barcode system projects have been developed in nematology mainly for soil and marine nematode identification. The ideal genetic marker for barcode development would combine ease of measurement with a mix of conserved and rapidly evolving segments. In nemaMOLECULAR TAXONOMY OF NEMATODES

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todes, the message emerging from diagnostic research is that the ITS region has sufficient information to identify most nematode species. Nevertheless, in some cases (e.g., mitotically parthenogenetic species of Meloidogyne and other species of recent hybrid origin), ITS would misidentify species. For improved confidence in a successful barcoding system, the mitochondrial COI gene would be one good candidate that would complement ITS as a locus unlinked to the nuclear ribosomal gene region. However, DNA barcoding has met with spirited reactions, some broadly in support but many against. For the latter they see DNA barcoding as a gross oversimplification of the science of taxonomy. While nucleotide sequences are more objective than traditional (i.e., morphological) data in some respects (character choice, character delineation, character state identity), in other respects both are inherently subjective (homology/alignment, divergence metrics). Sequence divergence in standard gene(s) is an extremely crude method for determining species limits, and more appropriate markers that are potentially directly linked to species criteria, such as reproductive isolation, should be used. Therefore, it is worth persisting with the plurality of genetic, anatomical and ethological criteria currently used to identify and test nematode species boundaries.

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7. A HISTORY OF POTATO CYST NEMATODE RESEARCH KEN EVANS Nematode Interactions Unit, Rothamsted Research, Harpenden, Hertfordshire, UK

& DAVID L. TRUDGILL Scottish Crop Research Institute, Invergowrie, Dundee, UK

The beginning “Europe, we have a problem” may not have been exactly how the first person to notice the developing females of potato cyst nematodes (PCN) on a potato crop expressed his feelings, but perhaps it should have been. The year was probably 1881 and the severe damage that cyst nematodes can cause was already known from the manner in which the German sugar beet crop had been devastated by beet cyst nematodes, which were first noted on sugar beet in 1859. Indeed, for quite some time, damage to potatoes was attributed to the beet cyst nematode. However, the story begins earlier than this, from the time at which the potato crop was first brought to Europe from the New World following the Spanish conquests. The first imports of this new crop almost certainly came from Peru and reached Spain in the form of just a few tubers that had survived the long sea voyage and were perhaps left over from ships” stores for the journey. These tubers had probably been handled quite a lot so it is likely that they carried no adhering soil and thus were free of any cysts of PCN. The most likely date for the arrival of potatoes in Europe is 1570 but they were certainly not introduced by either Francis Drake or Walter Raleigh, as believed by some people. The tubers proved viable when they arrived and were bulked up sufficiently for potatoes to be offered for sale in Seville by about 1573. At first, yields were low in Europe because the potatoes from Peru A HISTORY OF POTATO CYST NEMATODE RESEARCH

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were adapted to short-day growing conditions and tuberised poorly in the long days of European summers. When tuber formation eventually began in autumn, there was little time left for tuber bulking before the frosts of winter arrived. Eventually, however, selections were made for lines of potatoes that would initiate tubers under longer day growing conditions. This allowed the potato crop to spread further north in Europe and eventually to become a staple crop throughout the region. Conditions for potato production were found to be ideal in Ireland, with its plentiful rainfall and virtual absence of frosts where the coast is warmed by the Gulf Stream, so that reasonable yields could even be obtained from lines still dependent on short-day conditions for tuberisation. The excellent growth of the crop in Ireland led eventually to a complete dependence on it as a staple foodstuff, encouraged by the confidence that here was a crop that could not be destroyed by fire, a practice used by tyrannical landlords wishing to keep the peasant population on the brink of starvation, and therefore subdued, by burning some of their wheat crops. The situation took a dramatic turn in the early 1840s when weather conditions allowed late blight, Phytophthora infestans, to spread throughout the Irish potato crop. This resulted in two consecutive years (1845/6) of crop failures with much resultant starvation and death of the population. The population of Ireland was further reduced when large numbers of people emigrated to the New World to escape famine. A consequence of this tragedy was the importation of new potato lines from South America in an attempt to identify sources of blight resistance that could be used in potato breeding. It is almost certainly because these tubers carried soil contaminated with cyst nematodes that led to the introduction of PCN into Europe. Cyst nematode infestations initially are cryptic but become obvious when their populations reach densities large enough to cause plant damage and crop loss. Depending on the frequency with which host crops are grown, this often takes about 20 years, so field damage to potatoes was first noticed in the 1870s following introduction of the nematodes in the 1850s. This c. 20-year period is mirrored, for example, in the first record of damage by PCN confirmed on Long Island in 1941 (see Brodie, 1998, pp. 317–331 in Marks & Brodie, Potato Cyst Nematodes, CAB International) following movement of military equipment contaminated with infested soil from Europe to Long Island, at the end of 108

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World War I. Similarly, an infestation appeared in upstate New York in 1967 following the use of contaminated farm machinery from Long Island. The means of spread of cyst nematodes is diverse, and includes the examples given above plus others such as in contaminated guano from Peru (Inagaki & Kegasawa, 1973, Applied Entomology and Zoology 8: 97–102) and birds” feet to the new polders of The Netherlands. The possibility that birds may move cyst nematodes in their guts and deposit them in their droppings was tested by Brodie (1976, Journal of Nematology 8: 318–322), who found that the passage through the gut of some bird species was rapid (so unlikely to allow long distance transfer) and that cyst contents died after exposure to birds” excreta. This also made it unlikely that the guano from Peru was contaminated per se, rather that it was the use of sacks previously used to carry potatoes that contaminated the guano with cysts. Further weight was given to this theory of transport by Franco (1977, PhD thesis, University of London) who traced old lading bills for guano imported into the UK. Most of the imports were made through the ports of Liverpool, Hull and London, and PCN infestations seem to have been centred on these three ports. Interestingly, most of the infestations that developed around London proved to be of Globodera rostochiensis, most of those round Hull proved to be of G. pallida whilst those around Liverpool contained both species. This suggests that perhaps the numbers of cysts introduced to the UK were quite small and that the species passed through genetic bottlenecks. As potatoes gained in popularity, and as improved cultivars were produced by plant breeders, it became increasingly common to ship large quantities of tubers between countries as seed. Inevitably the tubers carried some soil with them, and the soil frequently contained cysts in the days before the importance of PCN was recognised. It is this means of spread that has resulted in the appearance of infestations in so many countries around the world, with Europe acting as a secondary distribution centre, and the species found depends on the species infesting the land in which the seed crop was produced. It has become normal, at least for British people, to blame the stomach upsets they frequently suffer when travelling to warmer countries in the Americas on “Montezuma’s revenge”, and it would perhaps not be unreasonable for Montezuma to wish for revenge A HISTORY OF POTATO CYST NEMATODE RESEARCH

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following the treatment of some of the Mexican people during Cortes’s conquest. However, the Incas of Peru suffered similar treatment by Pizarro’s forces during the slightly later conquest of Peru. Infestations of G. pallida, the predominant species of PCN in Peru (Evans et al., 1975, Nematologica 21: 365–369), have proved particularly difficult to control and to this day threaten the success of the European potato industry, so perhaps might be regarded as Atahualpa’s revenge.

Proper recognition and description of the problem Infestations of PCN have been discovered and reported from more than 65 countries around the world, usually as developing females on small patches of heavily attacked plants to which attention has been drawn. Such patches were quaintly referred to as “nematode nests” by O’Brien and Prentice (1930, The Scottish Journal of Agriculture, 415–433), slightly less quaintly as “lenses” by Wood et al. (1983, New Zealand Journal of Agriculture 11: 271–273) and perhaps most appropriately as “foci” by more recent writers. Following the clarification by Wollenweber (1923, Arb. Forsch. Inst. Kartoff., Berl., No. 7: 1–56) that the cysts he found on potatoes were a species distinct from those found on sugarbeet, and which he described as Heterodera rostochiensis, research was able to begin properly on the characteristics of this species. O’Brien and Prentice, although still confused over the identity of the species, were able to describe in detail the symptoms shown by attacked potato plants (being the first to use the term “feather dusters” for the appearance of heavily attacked plants). They also described the PCN life cycle in detail, the interaction with the fungus Rhizoctonia solani, the depth distribution of infestations, the relationship between disease severity and soil pH, the rate of spread of infestations from an initial focus (about 9 feet or 3 m per year), attempts at chemical control, the use of trap crops, the effects of root “excretions” on hatching, the benefit of crop rotation and the amelioration of crop damage by application of farmyard manure. World War II was probably a factor responsible for PCN coming to the fore as a pest in the UK. The home production of food became of prime importance during the war and, because potatoes have such a relatively high energy production per unit area of land, 110

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the total area of potatoes grown almost tripled during and immediately after this war. The increase in area grown was accompanied by an increase in the frequency of cropping with potatoes and this allowed PCN populations to multiply greatly, to the extent that cropping frequency had to be reduced in certain parts of the country. From data available at the time, Jones (1970, Journal of the Royal Society of Arts 118: 179–199) estimated losses due to PCN in the UK during the late 1940s and early 1950s at about 12% of production. Such an important problem clearly demanded research into ways of reducing these losses. The burgeoning petrochemical industry provided an important lead when it was shown that what was essentially a waste product, dichloropropane-dichloropropene (DD), was an effective soil sterilant and would limit or even almost eliminate the damage caused by plant parasitic nematodes. For the first time, this allowed the damage caused by nematodes to be quantified, and the damage caused by PCN to be directly controlled. At the same time, the potential of various collections of potato germplasm to provide genes for resistance to PCN was realised, and Ellenby (1948, Nature 162: 704) quickly found several important sources of resistance to PCN in the Commonwealth Potato Collection. One of these genes was to become known as the H1 resistance gene and was seized upon by plant breeders for incorporation in new potato cultivars. In the UK, the most important of these was Maris Piper, produced by H. W. Howard at the Plant Breeding Institute (PBI), Cambridge, UK, and first released in 1966. It quickly became widely grown, not only because of its nematode resistance but also because it was liked by consumers, a fact reflected in its current position in the potato sales charts of the UK – still top after more than 40 years. Such confidence did these developments promote that Nollen and Mulder (1969, Proceedings of the 5th British Insecticides and Fungicides Conference, 671–674) presented a scheme using resistant potatoes and soil fumigation to control PCN that would allow potatoes to be grown three times every five years and still lead to the eventual extinction of the nematode. Yet another important development at this time was the intensive research for better nematicides with more specific action than that of a simple soil sterilant. This search led to the release in the late 1960s of two extremely important products in the fight to control nematode damage, namely aldicarb and oxamyl, granular oximecarbamate nematicides. A HISTORY OF POTATO CYST NEMATODE RESEARCH

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Nollen and Mulder realised that their scheme would only work if the nematode infestation was of what was then known as pathotype A, and the scheme quickly became unworkable as pathotypes other than A came to predominate almost wherever H1 resistant cultivars were grown. A further problem was that DD did not always achieve the degree of kill expected, due to soil conditions not being ideal at the time of application, and excessive use of this fumigant quickly led to build-up of undesirable residues in the soil. The oximecarbamate nematicides seemed to overcome this particular problem, although they did have the disadvantage of high mammalian toxicity and being hazardous to handle. They have remained a cornerstone of PCN management programmes to the present day, although aldicarb has been found in groundwater on Long Island and tougher registration requirements in the EU have threatened its future. The availability of PCN-resistant potato cultivars led to the recognition of resistance-breaking pathotypes. Each time that new examples were found they were given code letters. Pathotype A was controlled by the H1 gene and the next type of resistance found was a gene, referred to as H2, discovered by Jack Dunnett (at the Scottish Plant Breeding Station) in the diploid wild species Solanum multidissectum. This led to the identification of pathotype B as one able to break the new resistance. Dutch workers quickly found pathotypes they designated as C and D, and pathotype E was able to overcome all resistance, including H1. The agricultural advisory services of the UK and The Netherlands played important roles in the PCN story in that members of these services worked closely with farmers growing the new PCN-resistant cultivars. Colin Guile reported differences in the colours of the “cysts” of pathotypes A, B and E but Freddie Jones at Rothamsted dismissed these findings when neither he nor one of us (DLT) could detect these differences. Only later did it become clear that Guile was referring to developing females rather than cysts when he published his findings in 1967 (Annals of Applied Biology 60: 411–419), an episode that underlined the importance of precision in the use of scientific terms. His findings were supported by those of Derek Webley, another worker from the National Agricultural Advisory Service (NAAS, later to become the Agricultural Development and Advisory Service, ADAS), who found morphometric differences between the second stage juveniles of the pathotypes (1970, Nematologica 16: 107–112). Further work, using disc 112

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electrophoresis on the so-called pathotypes, by Trudgill and Carpenter (1971, Annals of Applied Biology 69: 35–41) showed that the juveniles had important differences in their protein make-up, a finding taken much further in the two-dimensional electrophoresis studies of Bakker et al. (1992, Fundamental and Applied Nematology 15: 481–490). Overwhelming proof that we were dealing with two reproductively isolated species of PCN rather than one with pathotypes was provided by Parrott (1972, Annals of Applied Biology 71: 271–273), information that confirmed the conviction of Jones et al., (1970, Nature 227: 83–84) that there were two species of PCN. Diana Parrott attempted to make crosses on agar plates between males and virgin females of what she thought were five pathotype A and five E populations. She found she was working with two reproductively isolated groups, one consisting of three of the A populations and the other of seven populations, the five E populations and two of the A. It transpired that the potato clone originally used to identify the pathotype of the populations had extra resistance effective against two of the E populations used. Consequently, these two populations were misclassified as A rather than E. These observations and findings opened the door to a new description of potato cyst nematodes, as two species rather than one. The person assigned to this task was the new boy at Rothamsted, Alan Stone (1974, Nematologica 18: 591–606), who was later to succeed Freddie Jones as head of the Nematology Department at Rothamsted in 1979. The new species (Heterodera rostochiensis and H. pallida) soon became known as G. rostochiensis and G. pallida, when Edda Behrens pointed out the value of the Globodera genus, first suggested as a sub-genus by Skarbilovich in 1959 (Acta Parasitologica Polonica 7: 117–132). Also, the problem of pathotype classification assumed greater importance. Alan Stone, together with colleagues in Europe (John Kort, Hans Ross, Jürgen Rumpenhorst) presented their scheme, in 1976, at a European Society of Nematologists (ESN) meeting in Dublin. It used a range of potato clones with different levels of resistance to classify populations into five pathotypes of G. rostochiensis and three of G. pallida. The classification was not absolute and depended on a threshold multiplication rate of > or < 1 in a standard pot test. The scheme was subsequently recognized as being flawed as it did not take account of the effects of environmental factors on PCN multiplication rates. Also, values apparently related to one pathotype could A HISTORY OF POTATO CYST NEMATODE RESEARCH

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also be generated by mixtures of two other pathotypes within a population. Nevertheless, the scheme was seized on as a means of classifying the virulence of field populations of PCN and is still used by some people to this day. A similar scheme was developed by Canto and de Scurrah (1977, Nematologica 23: 340–349) at the International Potato Center (CIP) in Lima, Peru, initially using the same differential clones and only differing from the European scheme in the code numbers given to the pathotypes. As more differentials became available from the germplasm held at CIP and from breeding exercises, this second scheme eventually differed slightly from the European scheme. Perhaps the most rational scheme is that offered by Nijboer and Parlevliet (1990, Euphytica 49: 39–47), who recognised three pathotypes of G. rostochiensis and only virulence groups within G. pallida.

Better understanding of the pests allows research to become more focused The availability of the pathotype schemes allowed plant breeders to focus their efforts onto exploitation of the resistance found in S. vernei, and some other sources, against the more troublesome G. pallida. However, the resistance was polygenic, making it difficult to breed from, and little was known about the variation in virulence of field populations of G. pallida towards this resistance. A collaborative project between several European countries showed that there were considerable differences in virulence of G. pallida populations and, coincidentally, the Plant Breeding Institute in Cambridge was using a particularly avirulent G. pallida population to screen the progeny of their breeding. Much effort over more than 30 years has gone into breeding for resistance to G. pallida but few commercially acceptable cultivars have been produced, and none has full resistance or the acceptability to make substantial inroads into the commercial market – only 8% of current potato land is planted with G. pallida resistant potato cultivars in the UK. From the mid-1980s, with the production by breeders of cultivars with resistance from different sources, attention began to focus on the range of virulence in PCN and its possible relationship to the original introductions into Europe. It was already evident that there had been more than one introduction of G. rostochiensis into Europe 114

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because all UK populations were avirulent on cultivars with the H1 gene but some populations from The Netherlands were virulent. There appeared to be an even wider range of virulence within G. pallida, some of which was clearly due to repeated fragmentation of the genepool as it spread within Europe. However, comparisons with populations from South America revealed a much greater range of variation in South American populations of G. pallida than in those from Europe, although later studies on the artificial fragmentation of individual European G. pallida populations sometimes revealed as broad a range of virulence as that found in naturally occurring European field populations (Phillips et al., 2002, Nematology 4: 655–666). Studies with mitochondrial DNA (mtDNA), which is exclusively maternally inherited, were done by Vivian Blok and Miles Armstrong at the Scottish Crop Research Institute (SCRI) with the aim of identifying the distinct introductions of PCN into Europe. They discovered that G. pallida has a unique configuration of its mtDNA. In metazoans, the 12 or 13 genes that comprise the mtDNA genome are contained within a single ring. However, in G. pallida, the genes were distributed amongst several small rings (scmtDNAs) ranging from 6.3 to 9.5 kb (Armstrong et al., 2000, Genetics 154: 181–192). The complexity of this genome structure is compounded by variation in the complement of scmtDNAs found in different populations of G. pallida, with some populations lacking particular scmtDNAs while others have more than one variant of the same scmtDNA. Hybridisation studies by Armstrong et al., showed that some mitochondrial genes occurred on more than one ring and sequencing of five of these circles (Gibson et al., 2007, Journal of Molecular Ecology, in press) has revealed that three of these genomes are mosaics, and share long multigenic fragments. These mosaic structures are likely to be the result of intermitochondrial recombination (not thought to occur in the metazoa) and this is supported with evidence for recombination in the repeat region of the scmtDNA (Armstrong et al., 2007, Journal of Molecular Ecology, in press). From the analyses of the different scmtDNAs found in G. pallida populations, there is support for the conclusion that several different introductions of this species have been made into Europe. The mitochondrial genome of G. rostochiensis is similar but with some unique gene structures (V. Blok, pers. comm.).

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The uniqueness of PCN In addition to their mtDNA, PCN are exceptional in several other respects. Molecular studies by groups in the UK, The Netherlands and France, and John Marshall in New Zealand indicated that populations of G. pallida are exceptionally heterogeneous. This is probably necessary as it is a relatively immobile soil pathogen with a narrow range of hosts, so it needs to be able to respond rapidly to selection pressures imposed by those hosts, many of which possess genes for resistance. Populations can become extremely large, sometimes exceeding 100 eggs/g soil (equivalent to c. 3 × 1011 per ha). Such populations are extremely damaging, and Con Ellenby was the first to suggest that the sex of the developing juveniles was influenced by their nutrition, an adaptation that probably helps limit populations and protects the host from being killed. He suggested that when populations are high and the host is damaged, the competition for resources means that poorly nourished juveniles become male and only the well nourished ones become female. DLT produced persuasive data to support this suggestion but it was left to Mugniery and Fayet (1981, Revue de Nématologie 4: 41–45) to produce conclusive evidence. They developed a micro-technique whereby almost 100% of the inoculum survived to become adult and showed that the sex ratio varied with inoculum density, the majority (>90%) of juveniles becoming female with only one nematode per plant and an increasing proportion becoming male as the inoculum density increased. PCN is also exceptional in the changes it induces at its feeding site. The host cells develop into an enlarged, multinucleate syncytium that supplies the developing juvenile with all its food (Jones & Northcote, 1972, Journal of Cell Science 10: 789–809). Nutrients are extracted through a specialized feeding tube secreted from the stylet tip, first recognised in Rotylenchulus reniformis by Rebois (1980, Nematologica 26: 396–405), that acts as a molecular sieve and prevents both the stylet lumen from becoming blocked and the host cells being killed through the loss of vital organelles. Secretions are injected that initiate and maintain syncytium development and, with the development of molecular techniques, these have been the focus of much research. Several studies have reported monoclonal antibodies that recognise secreted proteins but it was not until the late 1990s that the first proteins were identified and genes cloned. 116

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These were the secreted cellulases of both cyst and root-knot nematodes (Smant et al., 1998, Proceedings of the National Academy of Sciences, USA 95: 4906-4911; Rosso et al., 1999, MPMI 12: 585–591). Subsequent work led to the identification of a variety of other enzymes able to degrade plant cell walls, including pectate lyases (Popeijus et al., 2000, Nature 406: 36–37). Remarkably, the genes encoding these cell wall degrading enzymes seem to have been acquired by horizontal gene transfer from bacteria. This process of gene transfer seems to have occurred independently on several occasions within the phylum Nematoda; cell wall degrading enzymes and chorismate mutase are present in root-knot and cyst nematodes, a gene similar to NodL from Rhizobium is present in root-knot nematodes and, perhaps most surprisingly, cellulases that appear to have been acquired horizontally from fungi are present in Bursaphelenchus (Jones et al., 2005, Nematology 7 : 641–646). Other secreted proteins present on the nematode surface that may be important in suppressing or neutralising host defence responses have been identified (Prior et al., 2001, Biochemical Journal 356: 387–394; Robertson et al., 2000, Molecular and Biochemical Parasitology 111: 41–49). More recently, expressed sequence tag analysis of cDNA libraries from infective juveniles or made from excised gland cells has led to the identification of a large number of secreted proteins. As information on the proteins present in nematodes continues to accumulate from genome and EST studies, the challenge for the future lies in deciphering the function of these proteins in the host/parasite interaction. Studies on microarrays of plant genes suggest that several thousand genes may be up- or down-regulated as a result of nematode infection.

The management of PCN populations in crops The growing of resistant (to G. rostochiensis as well as G. pallida) cultivars with a critical eye to performance revealed some interesting features, not least of which was the degree to which they could vary in their ability to tolerate nematode attack. Up to this point, tolerance had been largely considered simply as an expression of resistance, but it was then realised that resistance and tolerance were independently inherited desirable characteristics, with resistance determining the degree to which nematode reproduction is A HISTORY OF POTATO CYST NEMATODE RESEARCH

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possible and tolerance determining the degree to which the crop is damaged for a given level of infestation. This realisation was in no small part due to the work that we (KE and DLT) did on a longterm field trial established by Jones at Woburn Experimental Farm. The treatments had resulted in a series of plots with a range of population densities of G. rostochiensis on which non-resistant Pentland Dell and resistant Maris Piper were grown. There was also the added benefit that G. pallida had been introduced with the seed on one of the Maris Piper plots. We wanted to elucidate the mechanisms behind the expression of PCN damage and found many effects, mostly attributable to the effects on water and major nutrient uptake. Potassium concentrations and, to a lesser extent, those of phosphate plummeted as PCN population densities and crop damage increased. We hypothesised, incorrectly as would be shown later, that reductions in K uptake were central to the impact of PCN on potato growth (experiments with different inputs of N, P and K showed that it was effects on the uptake of N and, sometimes in clay soils, P that were important; the plants were able to take up K in luxury amounts). Perhaps the most important observation to emerge from this experiment, however, was that the resistant cultivar Maris Piper was much less damaged at a given PCN density than the non-resistant Pentland Dell. This sparked the longer term interest that we developed in tolerance, an interest shared by Arne Mulder in The Netherlands. Maris Peer is an early maturing cultivar with no PCN resistance and we both independently (by this time DLT had moved to SCRI) identified it as very intolerant. However, it was not as intolerant as Maris Anchor, even though Maris Anchor has the H1 resistance gene. It emerged in work by KE with Gordon Storey (Storey & Evans, 1987, Plant Pathology 36: 192–200) that Maris Anchor was very susceptible to invasion by the wilt fungus Verticillium dahliae once nematodes had invaded the roots. This was in contrast to another early cultivar, Pentland Javelin, also with the H1 gene, which was very tolerant of PCN attack and, because of basic differences in root structure, did not suffer massive invasion by the fungus. Our work on tolerance showed the importance of environmental effects when studying the relationship between nematode densities, crop yield and PCN multiplication rates. This type of work was also beloved of Wim Seinhorst from The Netherlands, who had presented an early version of his theories at the ESN meeting in 118

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Antibes (1966), the first ESN meeting attended by DLT. In response to a question from DLT, Seinhorst maintained that proportional yield loss was independent of external factors. At a NATO workshop several years later in Martina Franca, Italy (1985), he still nursed this idea, admitting that he had fixed one of the parameters in the equation for his relationship from the results of his own experiments done under carefully controlled conditions. The main features of practical relevance of Seinhorst’s curves are T, the tolerance limit or threshold nematode population density beyond which damage occurs, m, the minimum yield achieved by the crop as nematode density increased, and the constant z, which determines the rate of yield reduction with increasing PCN density. It was the value of z that Seinhorst eventually fixed. Support for Seinhorst’s theory came at the same meeting from a young Ed Caswell, who had data from three sugarbeet fields in California. Ed had measured the preplanting density of beet cyst nematode in 100 small plots across the fields and had measured plot yields at harvest time. Plotting yield against nematode density produced a scatter diagram but, on Howard Ferris’ advice, Ed had taken the means of yield and nematode density in seven or eight error bands and plotted the means against one another. This produced three perfect Seinhorst curves, with slightly different values of T, m and z clearly shown. Since this time, Seinhorst curves have been produced by many workers, most frequently for potatoes by the Italian group that includes Nicola Greco and Mauro Di Vito, among others. Interestingly, however, the value of T is always very low and close to 1 egg per g of soil (Greco & Moreno, 1992, Nematropica 22: 165–173), which is the theoretical lower limit of determination of PCN population densities unless, like Seinhorst, one is working with carefully prepared levels of inoculum in controlled conditions. Seinhorst’s pot studies also yielded an equation for population dynamics. For the mathematically challenged (e.g. DLT!), this equation was even more impenetrable than that for the effect of PCN on yield. The multiplication rate was at a maximum when populations were small (
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directly used to provide advice in the field. Not least, as Seinhorst pointed out, were the problems of accurately quantifying field populations. Michiel Oostenbrink (another outstanding Dutch nematologist) proposed that, for practical purposes, the relationship between PCN population density at planting and crop loss was linear and that the parameter T was so small as to be irrelevant in the field. Eric Brown in the UK also used a linear regression approach to express damage relationships, and the way in which this works so well when comparing the performance of different cultivars was shown by KE with Mike Russell (Evans & Russell, 1990, Annals of Applied Biology 117: 595–610), by taking PCN density and yield measurements from many plants of each cultivar grown in a single plant-plot experimental design. The arrival of more and more powerful desktop computers made possible the development of “expert systems” for managing pests and pathogens in the field. As a theoretical basis was available for quantifying PCN damage and population dynamics it was an attractive option for developing an expert system. However, differences between cultivars in their tolerance of PCN damage, and observations by an ADAS nematologist, Norman French, that damage was more severe on sandy than clay soils, presented further challenges. To obtain data for incorporation in such a system several field trials, coordinated by SCRI, were done on different soil types. The first trials were at sites which had been manipulated to produce a patchwork of plots with a wide range of G. pallida population densities. Each trial tested several cultivars differing in their tolerance and resistance. To minimise variation and sampling errors the sample area in each plot was kept as small as practical, and three independent soil samples were taken, each made up of 40 small (5g) cores. Later, trials in farmers” fields used natural variations in PCN population density to test the model and obtain additional data on the effectiveness of nematicides and PCN decline rates between crops. The results confirmed the influence of environmental factors on the parameters in the Seinhorst equations, and an expert system for PCN management has been developed and is awaiting release. Tom Been and Carrie Schomaker in The Netherlands have also developed an expert system for PCN management based on Seinhorst equations (Been et al., 1995, pp. 305–22 in Haverkort, A.J. & MacKerron, D.K.L., Current issues in Production Ecology, Kluwer Academic Publishers). In addition, they have studied the intensity of 120

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sampling required to detect PCN and to obtain reliable estimates of population densities. This arose from concern to keep the large area of seed potato land in the Dutch polders free from PCN. Early detection is essential as part of a strategy to detect foci of infection and minimise their spread. However, with any system for managing PCN, the intensity of sampling possible is limited by cost, and will almost always be less than that necessary to give the required confidence limits. The growing of cultivars with partial resistance to G. pallida highlighted another problem, one that had been foreseen by Freddie Jones and referred to in his theoretical papers and made a feature of the population dynamics models that he developed with Rob Kempton (1978, pp. 333–361 in Southey, J.F., Plant Nematology, MAFF ADAS Publication GD1, HMSO, London) and Joe Perry (1978, Journal of Applied Ecology 15: 349–371). The problem is that whenever a cultivar with partial resistance is grown, the population of PCN exposed to it is selected for increased levels of virulence towards the particular resistance possessed by that cultivar. Depending on the virulence genes available in the PCN population, the selection can be slow or quite rapid. Sue Turner initiated careful selection work with Alan Stone and has since continued it over several years in Northern Ireland (1990, Annals of Applied Biology 117: 385–397). This work has shown that many populations of G. pallida can be selected to become almost 100% virulent towards cultivars with partial resistance within less than ten generations. More bad news was in the offing when it was found that, in the absence of a host crop, populations of G. pallida often seemed to decline much more slowly than the 32% per annum average found by Jones (1966, Report of Rothamsted Experimental Station for 1965, 301–316) for a large number of G. rostochiensis populations in the UK. Some G. pallida populations seemed to decline at rates as low as 10% per annum. Although no PCN populations have been found to show resistance to oximecarbamate nematicides, further bad news emerged when it was found that G. pallida seemed to be less susceptible than G. rostochiensis to these nematicides. This appeared to be due to G. pallida second stage juveniles reaching a peak of hatching later than G. rostochiensis after planting the potato crop (perhaps 6 weeks as opposed to 3 weeks), at a time when the concentration of nematicide in the soil had declined to relatively inactive levels. With the observation that the soil microflora could be selected by repeated application of oximecarbamates to hasten their A HISTORY OF POTATO CYST NEMATODE RESEARCH

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breakdown, and the discovery that G. pallida juveniles carried more lipid reserves than G. rostochiensis (Robinson et al., 1987, Revue de Nématologie 10: 343–348) and so were better able to resist the action of the chemicals because they were able to survive until the chemicals had broken down to inactive levels (oximecarbamates are more nematostats than nematicides and nematodes starve to death whilst immobilised), the bad news was complete. This meant that resistance was not as effective against G. pallida as against G. rostochiensis (because even the best cultivars were only partially resistant and populations became more virulent each time a particular resistor was grown); rotation was less effective against G. pallida because its decline rate in the absence of potato crops is less than that of G. rostochiensis; and nematicides were less effective against G. pallida. In other words, all of the “traditional” components of integrated management packages for PCN were less effective than they had been when many field populations were of G. rostochiensis (see Evans, 1993, Nematropica 23: 221–231), a consequence of application of those very components – Atahualpa’s revenge indeed. Thus, G. pallida is now predominant in most European field populations of PCN and will continue to become more so. The most traditional method of control is crop rotation and UK rotations for potato crops average 5.7 years (see Minnis et al., 2002, Annals of Applied Biology 140, 187–95), which is far too short to control this species. In Peru, even with the relatively very poor yields that they obtained, which reflects the relatively small amount of root available for PCN reproduction, the Incas insisted that growers follow a 7-year rotation. They did not know the cause but they did know that land developed a “potato sickness” if cropped more frequently with potatoes. New methods for managing PCN populations are, therefore, being investigated at the moment. These include the development of the sophisticated model referred to above to predict population density changes and crop yields, targeted application of nematicides so that they are used more effectively, trap crops to reduce PCN population densities, flooding of land, and biocontrol. Effective ways of mapping the distribution of PCN within fields have been defined and the information used experimentally to direct the application of nematicides to only those parts of the field where populations exceed damaging densities (Evans et al., 2003, Precision Agriculture 4: 149–162). However, accurate information on PCN distribution requires a heavy investment in sampling, and so122

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called “spatial application” of nematicides is probably only practical when it is intended to use both a fumigant and a granular nematicide, with the more expensive fumigant applied in a spatial manner and the granular nematicide applied as a blanket treatment to prevent build-up of troublesome population densities from non-damaging densities such that they would threaten following potato crops. Trap crops have been used with great effect (Halford et al., 1999, Annals of Applied Biology 134: 321–327), and can either utilise potatoes as the trap crop or, more recently and with promising levels of success, a non-host of PCN the weed species Solanum sisymbriifolium (Timmermans, 2005, PhD thesis, University of Wageningen, The Netherlands). In certain potato production areas, such as the fenlands of eastern England, it is possible to build small containment barriers around the large flat fields and flood them by pumping water onto them. This has proved a highly successful way of controlling PCN, with essentially 100% kill after a period of 13 weeks of flooding (Barker, pers. comm.). Unfortunately, only a proportion of the land used for potato production can be treated in this way. Biocontrol of some plant parasitic nematodes (e.g. Meloidogyne spp.), using species of nematophagous fungi, may be a commercial proposition but so far has remained an elusive goal with PCN.

Diagnosis of the PCN species Although G. pallida is becoming the predominant species of PCN wherever management tactics that favour it are deployed, and, of course, where it has actually been introduced, a first step in designing PCN control programmes is the determination of which species is/are present. Following Alan Stone’s description of G. pallida, the information required for identification of the two species using morphological and morphometric observations was available. However, the most useful information, on stylet size and shape and the colour of developing females, had already been provided by Derek Webley and Colin Guile, and formed the basis on which species identity was confirmed for a number of years. The disadvantage was that it required experience and expertise to make the required observations, and the procedure was time-consuming if sufficient individuals were to be examined to estimate with accuracy the proportions of the two species in mixed populations. A HISTORY OF POTATO CYST NEMATODE RESEARCH

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A significant improvement was provided by Fleming and Marks (1983, Annals of Applied Biology 103: 277–281) when they used isoelectric focusing on polyacrylamide gel to separate aqueous extracts of proteins from whole cysts. They showed that the two species of PCN contained specific proteins of similar isoelectric point (at pH 5.7 and 5.9) characteristic for one species or the other. Following staining on the gel, densitometer measurements of the bands allowed the relative proportions of the two species to be estimated, all with no experience or knowledge of nematode morphology and using a method that merely required the following of a simple procedure. The two diagnostic proteins were shown to have similar molecular weights and Robinson et al. (1993, Annals of Applied Biology 123: 337–347) raised monoclonal antibodies to these proteins in an attempt to establish a simple immunoassay for the two species of PCN, an objective completed by Curtis et al. (1998, Annals of Applied Biology 133: 65–79). Unfortunately, this potentially useful assay followed the fate of increasing numbers of promising lines of work in science generally when the funding for the work expired. The utility and convenience of immunoassays also prompted a Dutch group led by Schots et al. (1992, Fundamental and Applied Nematology 15: 55–61) to develop a system based on three antibodies for the determination of the relative proportions of PCN species in species mixtures, but again it was never widely taken up. Perhaps it was inevitable that immunoassays would lose out to DNA-based techniques in the long run, and it now seems likely that real-time PCR procedures will provide the standard assay for PCN species in the future. Andy Barker and Simon Atkins at Rothamsted have designed species-specific primers for such a procedure and Bates et al. (2003, Molecular Plant Pathology 3: 153–161) at the National Institute of Agricultural Botany in Cambridge, UK, have published a working procedure. The ultimate goal in assays would be to determine how a given field population of PCN will react to a given resistant cultivar. But as already mentioned, the resistance in the best G. pallida resistant cultivars is polygenic in nature, with the implication that the virulence that allows PCN populations to overcome the resistance is also polygenic. It may be possible to produce, by controlled matings, PCN test populations that have defined levels of virulence to cultivars with defined levels of resistance, as attempted by Conceiçao et al. (2005, Nematologia Mediterranea 33: 75–85) and in a similar man124

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ner to the avirulent populations of G. rostochiensis produced by Janssen et al. (1990, Revue de Nématologie 13: 265–268). With such material available it may then be possible to design the necessary specific DNA primers.

Hatching factors for PCN No account of research on PCN would be complete without reference to the work that has been done on hatching factors, pioneered by, amongst others, Triffitt (1932, Journal of Helminthology 10: 1812). Few research projects have looked so promising but remained so daunting. The idea of using the PCN hatching factor as a weapon in control programmes dates back at least to 1932, when Triffitt, still under the misapprehension that she was dealing with a potato race of H. schachtii, found that grass root secretions would cause hatching and that a field trial showed that a grass/cereal mixture reduced cyst contents by 23.6% whilst a cereal only treatment affected cyst content only negligibly. Triffitt even began work to identify the active hatching factor, and numerous others have tried since. Some success was obtained with identification of hatching factors for other species of cyst nematodes, notably H. glycines, but the goal remained elusive for many years for PCN. Clarke and Perry (1977, Nematologica 23: 350–368) produced a schematic for the hatching process in PCN that drew upon many observations made in other studies, and revolved around the concept that the hatching factor induces a change in the permeability of the eggshell, probably by displacing internal calcium ions (Atkinson et al., 1980, Annals of Applied Biology 94: 103–109). This allows the disaccharide (trehalose) that is found in the perivitelline fluid surrounding the dormant juvenile to diffuse out of the egg, thereby reducing the osmotic stress on the juvenile. This, in turn, allows the juvenile to become hydrated beyond a critical level and to become active, at which point it cuts open the eggshell and emerges into the cyst interior and, via apertures in the cyst wall, into the soil. This general scheme remains the accepted theory of the succession of events in the PCN hatching process. Devine et al. (1996, Annals of Applied Biology 129: 323–334) showed that several different components of potato root exudates may induce hatching of PCN, and Devine and Jones (2000, Annals of A HISTORY OF POTATO CYST NEMATODE RESEARCH

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Applied Biology 137: 21–29) tested the idea that exogenous application of hatching factors to soil might stimulate PCN hatch sufficiently for it to represent a useful control tactic. One of the best artificial hatching factors found, picrolonic acid, had already been shown to achieve, at best, a 33% decrease in the PCN population, but was unsuitable for routine use due to its binding to soil particles (Whitehead, 1977, Annals of Applied Biology 87: 225–227). Devine and Jones obtained quantities of both potato and tomato root leachates – the potato root leachate from potato plants grown in boxes of gravel and the tomato root leachate from the return feed pipe of a commercial hydroponic tomato production system. They obtained up to 50% reduction in the size of a population of G. rostochiensis, which they attributed to either suicidal hatch or increased in-egg mortality, presumably caused by incomplete hatch stimulation. The goal of identifying a specific PCN hatching factor from potatoes was finally achieved in a Dutch laboratory when a structural formula was produced for an extremely active hatching factor designated solanoeclepin (Schenk et al., 1999, Croatia Chemica Acta 72, 593–606). The extreme activity of this compound meant that only a very small quantity would be required to stimulate sufficient suicidal hatch in the field to achieve a valuable degree of control. However, the problem remains of synthesis and delivery. Perhaps the day will yet come when another crop is engineered to produce PCN hatching factor and thereby greatly reduce the PCN population and make it safe to grow potatoes without resort to nematicides or growing resistant cultivars that are not fully resistant.

The scientists and laboratories that have most influenced PCN research Research work on PCN has covered many different branches of science and reflects the work done in the field of nematology generally. Many laboratories and individuals have contributed over the years to the huge volume of research on PCN and it is impossible to mention all of them, so this account has necessarily been a very personal view that we have given. However, two groups perhaps deserve special mention – the Nematology Department at Rothamsted Experimental Station in Harpenden, UK, and the nematologists from Wageningen, The Netherlands. There were true 126

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leaders of PCN research in both of these centres, most notably Freddie Jones, David Fenwick and Alan Whitehead at Rothamsted, and Wim Seinhorst, Michiel Oostenbrink and Hugo den Ouden from Wageningen. These were the giants on whose shoulders most of us have stood. Their various research interests came together in the modeling work of Jones and Seinhorst, investigations of nematicides by Whitehead and den Ouden, but perhaps most memorably in the Fenwick Can originally designed, in 1940, at Rothamsted but given the “sloping bottom” of Oostenbrink for convenience of use (Fig. 1), and which remains the main method for routine quantitative extraction of PCN from soil. Some of the nematologists who worked at Rothamsted can be seen in Figs. 2, 3 and 4. Despite this substantial and sustained research effort, PCN remains a major constraint to potato production in many countries. There is a continuing need for innovative, high quality research to provide growers with new weapons to tackle this most intractable of pests.

Fig. 1. The iconic Fenwick Can apparatus for recovering cysts from soil. Although not obvious, the model shown features Oostenbrink’s sloping bottom. A HISTORY OF POTATO CYST NEMATODE RESEARCH

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Fig. 2. Basil Goodey and Freddie Jones taking coffee with John Webster in the salubrious coffee room of the Nematology Department at Rothamsted, circa 1964.

Fig. 3. David Fenwick in his favourite pose – with the ladies of the Nematology Department.

Fig. 4. Bertie Winslow and Freddie Jones go back a long way together! (with a little help from Chris Doncaster)

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8. CEREAL CYST NEMATODE COMPLEX ROGER RIVOAL INRA Liason Officer. Formerly, UMR INRA/ENSAR, Biologie des Organismes et des Populations Appliquée à la Protection des Plantes (BiO3P), 35653 Le Rheu, France.

Cereal cyst nematodes (CCN) form a complex of several closely related species which are widely distributed, and found wherever graminaceous plants are cultivated. The main species, Heterodera avenae, was described at the beginning of the 20th century (Wollenweber, 1924). Description of this species was followed by that of H. latipons from the Mediterranean area, H. hordecalis from northern Europe, H. filipjevi from eastern Europe and several others, to total more than 12 species (Wouts, et al., 1995, Nematologica 41: 575–583). Development of research in this important group of nematodes occurred after World War II when European cereal production was increased to satisfy human and animal needs. Research focussed on the main species, H. avenae, which infested more than 50% of the cereal fields of Europe. Oat cultivation, with its high host susceptibility, was certainly responsible for the large increases in the populations of this nematode, previously called the “oat cyst nematode”. This species reduced yields of spring-sown oats and barley in Sweden and Denmark. Even though agriculture and transport mechanisation had reduced oat production (the main nutrient for horses) by the middle of the 20th century, the damaging nematode populations, developed on this cereal, seriously affected the maize crop of northern France and other parts of Europe. Heterodera avenae was demonstrated to be polyphagous on cereals, and was a severe pathogen of wheat in south-eastern Australia and in Asian regions causing “Molya disease” on wheat and barley. Many experiments, in different countries, demonstrated that the degree of damage was determined principally by the size of the initial infestation, as modified by soil and climatic conditions and the crop species and cultivar. Synchrony of H. avenae emergence with cereal sowing time CEREAL CYST NEMATODE COMPLEX

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played a major role in crop damage. In Australia, times of hatching, sowing and rainfall collectively, explained the great losses observed. In the more diverse climate of Europe, a similar synchrony was observed with winter sown crops in southern areas. In northern areas damage was more frequently observed with spring varieties sown when nematode hatch was abundant (Rivoal & Cook, 1993, In: Evans, Trudgill, Webster (Eds), Nematodes in Temperate Agriculture. CAB International, Wallingford, UK. pp. 295–303). Contrasting crop damage with the hatching cycles of the nematode justified the comparative studies mainly between Australian and European populations of H. avenae. Two ecotypes appeared, differing in the induction or suppression of dormancy (diapause) according to temperature conditions. For populations in the Mediterranean climate the diapause was obligate, acting when hot dry conditions prevailed and being suppressed when the temperature fell and soil moisture increased. However, populations from more or less temperate climates had a more facultative diapause from July to the end of winter, and this was suppressed by chilling, ensuring emergence of juveniles when soil temperatures increased in the spring. Reciprocal transfers of northern and southern populations did not alter their basic hatching rythms which resulted certainly from a genetic adaptation to specific climatic conditions (Rivoal, 1986, Revue de Nématologie 9: 405–410). In the 1970s, extensive experiments were done to control H. avenae with chemical nematicides based on the results of experiments using fumigants twenty years earlier against nematode pests and other soil pathogens on a variety of crops. When the delay between nematicide application and juvenile emergence was short, an application of nematicides improved crop production so much that the use of statistical analysis was unnecessary to differentiate the treated plots from the controls! In Australia, engineering low rate distributors of fumigants or systemic nematicides enabled economic control of the nematodes. In several parts of the world, the use of such chemical nematicides was used to demonstrate that these invisible, and frequently unknown pests, the nematodes, were responsible for minimizing crop yields (Brown, 1984, Journal of Nematology 16: 216–222). Fortunately, at the same time, alternative control methods based on plant resistance were being investigated. The initial research was done in northern Europe (Sweden, Denmark) where oat, barley and wheat varieties were found that reduced or inhibited nematode mul130

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tiplication. Quite early, complete resistance was found in the barley cultivars Drost, LP191 and Morocco and in the wheat cultivar Loros. The investigators, being true breeders, demonstrated that the inheritance of resistance was based on a restricted number of genes: Ha1, Ha2 and Ha3 for the three barley cultivars, respectively, and Ccn1 for the wheat, Loros. This research, and the results achieved, was of great importance in nematology as a prerequisite for successful crop production. Intensive screening for resistance was further developed in several countries and resulted in the identification of additional sources of genetic resistance in the various cultivated cereals and related wild species. Several of these genes were introduced into the breeding programmes which produced commercial resistant varieties of oats, barley and wheat, particularly in northern Europe and in Australia. Significant progress was achieved, using molecular technology, to identify markers for various types of CCN plant resistance and for developing marker-assisted selection (MAS) to pyramid resistance genes to H. avenae in new cultivars in Australia and western Europe (Nicol et al., 2003, Nematology Monographs and Perspectives 2, 1–19). Screening for resistance sources using a wide range of populations of H. avenae, and also of H. filipjevi, H. hordecalis and H. latipons, showed variation in resistance efficiency depending upon the species of nematode and the population tested. Particularly within H. avenae, the virulence of populations, determined by their ability to overcome resistance genes, enabled the differentiation of pathotypes. These pathotypes have been recognised using the International Test Assortment of barley, oat and wheat cultivars, with their respective resistance genes, as developed by Sigurd Andersen in Denmark. Seeds of the differential hosts were further distributed in different European and Asian countries to enable a comparison of the virulence spectrum among the different nematode populations tested. Heterodera avenae populations were divided into three pathotype groups based upon the reactions of barley cultivars with the particular resistance genes (Rha1, Rha2, Rha3), each pathotype group being further divided by reactions of other differentials which led to double integer codes (Andersen & Andersen, 1982, EPPO Bulletin 12, 379–386). This nomenclature became difficult to use because it was based on a simple descriptive approach of incompletely understood interactions. A more flexible scheme, based upon the same differential reactions but using a decaCEREAL CYST NEMATODE COMPLEX

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nary notation of differentials, was further proposed in order to give unambiguous labels to each pathotype. Virulence phenotypes were labelled by using the sum of the number of susceptible differentials for each of the cereal species. Both of these differentiation schemes ensured the existence of seven to eleven virulence phenotypes resulting from previous extensive selection pressure or the recent use of particular resistance genes, as in Scandinavia (Cook & Rivoal, 1998, In: Sharma (Ed.), The cyst nematodes. Chapman & Hall, London, UK. pp. 322–352). The identification of such a large number of pathotypes raised the question as to the true identity of the species involved. The pathotypes were difficult to distinguish morphologically despite the use of improved optic and electron microscopy, coupled with automatic image analysis processing which enhanced the accuracy of observations and measurements of cyst and juvenile features. Controlled matings between pathotypes of H. avenae confirmed that they belonged to the same species, and testing of the F2 and F3 progenies of the pathotypes on barley cultivars confirmed that this species was more complex than previously considered. In the 1980s, and more recently, biochemical and molecular techniques based on the analysis of proteins and DNA polymorphisms have enabled the reliable identification of most species involved in the CCN complex. These analyses demonstrated, in particular, that several populations of the “Gotland race” (Sweden) were in fact western European isolates of H. filipjevi (R. Holgado and others). Until that time, molecular technology had failed to distinguish pathotypes of CCN and markers for virulence traits, but rather had established the new taxon, Heterodera australis, even though no morphological features of the cysts and juveniles differentiated this new species from H. avenae sensu stricto! (Subbotin et al., 2002, Russian Journal of Nematology 10: 139–148). Procedures for extracting nematodes and preparing them for identification have not evolved very much over the years. Population dynamic studies were based on standard sampling (soil cores) and extraction procedures. The Kort elutriator was specifically adapted for cyst extraction from wet soils and was demonstrated to be more efficient than the old Fenwick can. Centrifugation with sugar or MgSO4 solutions improved the extraction of cysts or eggs from soil for studies on population dynamics and resistance. Significant improvements have been made in the use of resistance/virulence 132

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tests. Originally, soils were imported from infested areas and distributed in pots and containers where individual plants were sown to evaluate their capacity for hosting the populations tested. This procedure was replaced by the production of cysts that were to be used as inoculum being isolated by a plastic net so as to be separated from their progenies. Finally, the increase in knowledge on breaking diapause allowed us to obtain juveniles in the numbers needed to perform miniaturized tests in tubes filled with sand/kaolin medium or in Petri dishes with agar. The Petri dish technique was better adapted for controlled matings between single females and males. Miniaturized tests in Petri dishes were adopted to establish that populations of H. avenae differed in the capacity of the juveniles to produce females (part of the fitness component) which was important for designing virulence/resistance investigations and for the management of nematode densities (Rivoal et al., 2001, Nematology 3, 581–592). In cereal production from low-value crops, strategies for controlling CCN population densities relied on the adoption of integrated control measures. In several countries in western Europe, manipulation of densities by growing crops with different host capacity in field microplots allowed the determination of damage thresholds for different cereals and for the population changes during their culture. In the 1980s, in Australia, a bioassay (SIRONEM) was developed to indicate the potential for damage to occur, and that helped in advising on control options using nematicides, resistant cultivars or agronomic practices (Brown, 1987, In: Brown, R.H. & Kerry, B.R. (Eds) Principles and Practice of Nematode Control in Crops. Academic Press, London, UK. pp.351–387). In long-term experiments, monocultures of host cereals led to an unexpected decrease of H. avenae densities due to biological antagonists (fungi). However, there was a marked contrast in the results between those which developed in suppressive soils and those from the dry land production areas. In 1991, a project entitled “Sustainable management of H. avenae” was submitted to the European Union in the proposal “Competitiveness of Agriculture and Management of Agriculture Resources 1989–1993”. The project, involving scientists from France, UK, Spain and Germany was not approved, even though well appreciated. The EU was already producing too much small grain cereal, and to be successful such an application would have had to have been more ingenious to propose a research programme that decreased crop yield! Nevertheless, long term experiments were initiated on the effects of CEREAL CYST NEMATODE COMPLEX

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resistance on nematode densities and recolonization by susceptible varieties, based on a CCN population genetics approach for the first time. (Lasserre et al., 1996, Theoretical and Applied Genetics 93: 1–8). In contrast to the economic importance of cereals in the world and the wide distribution of the CCN complex there are, unfortunately very few scientists that have been involved in this topic. Meetings of CCN scientists were relatively scarce but warmhearted. First contacts began in the 1970s, at the Eucarpia breeder meetings with the exchange of preliminary virulence results for H. avenae. The complexity of pathotyping in these populations led to the organization of an EPPO Colloquium on “Cereal Cyst Nematodes” at Rennes (France) in June, 1982. More than 40 scientists, from different parts of the world attended this meeting which ended around a famous St John’s fire, well served with a bottle of fine Breton apple brandy! Results of virulence tests were further examined and discussed every two years at the beginning of each ESN Symposium, until 1998. In addition to exchanging materials, bilateral visits and hosting of Ph.D., Fellows in different countries of Europe (e.g., UK, Germany, Belgium, France), there were overtures to nematologists from developing countries that were made through training courses entitled “Soil borne pathogens of cereals or wheat” and organized by CIMMYT (Julie Nicol ) in Turkey (2003) and China (2005). Evidence from these notes and landmarks should be used to demonstrate that the Cereal Cyst Nematode Complex is acknowledged as a global economic problem especially on wheat and barley production systems, both in the past and the present. Global warming could enhance dramatically the noxiousness of these pathogens in both dry land and rainfed production of cereals as well as in the intensive production systems in western Europe. Remote sensing techniques based on thermal-infra red measurements have demonstrated already that H. avenae populations are a major factor increasing plant water stress in infested areas (Nicolas et al., 1991, Revue de Nématologie 14, 285–290). Although substantial progress has been made, it is clear that additional studies are needed to evaluate the economic importance of CCN in developing countries (e.g., those in north Africa, eastern and western Asia) and also in developed countries (e.g., western and eastern Europe, USA) which face both greater climatic constraints and reduced fertilizer utilization. A major research challenge is the concern over the genetic diversity of these species and populations and their phylogenetic relationships. 134

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Fig 2. Mating between male and female of Heterodera avenae produced on wheat cultivated in Petri dish.

Fig. 1. Attack of Heterodera avenae on Triticum durum in southern France.

Fig. 5. Below: Scientists who attended the EPPO Colloquium on “Cereal Cyst Nematodes”. June, 13–26, 1982, Rennes, France.

Fig 3. Above: Kort elutriator for extracting cyst nematodes from wet soil.

Fig 4. Left: Resistance or virulence tests for cereal cyst nematodes in miniaturized conditions.

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Researchers must identify and confirm sustainable management solutions, and these require a deeper understanding of the population dynamics of H. avenae and other species in the complex. Active research on resistance sources associated with their molecular characterization is necessary for a more rapid integration into cereal cultivars. A new and important challenge is offered to both young and old nematologists involved in traditional or more advanced scientific skills, originating from developed and developing countries. As happened in the earlier years with the previous CCN research group, they should join in efforts to create a critical mass of scientific capacity to deliver sustainable solutions in applied and theoretical situations. I thank Roger Cook for his critical reading of the manuscript.

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9. THE SCIENCE AND ART OF SOYBEAN CYST NEMATODE RESEARCH TERRY L. NIBLACK Department of Crop Sciences, University of Illinois Urbana-Champaign

& DON P. SCHMITT Nematology, University of Hawaii, USA

Fig. 1. Soybeans from seed to harvest: A) seed and vegetative growth; B) flowers; C) pods; D) harvesting operations.

Introduction In a single century, the global soybean production area has increased from about one million ha (the first official record in the USA in 1924 was 1.8 million ha) to somewhat more than 80 million ha at the beginning of the 21st century, yielding ca. 200 x 109 kg seed (Fig. 1). Among the numerous factors that can limit soybean yield, Heterodera glycines, the soybean cyst nematode (SCN), is one of the most important according to research data and testimonials (Fig. 2). THE SCIENCE AND ART OF SOYBEAN CYST NEMATODE RESEARCH

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Much has been learned about the species since its description by Minoru Ichinohe over 50 years ago, but a surprising number of things we “know” about the nematode reside in the realm of opinion and folklore. (The facts, on the other hand, are well-reviewed in the 2004 book Biology and Management of the Soybean Cyst Nematode, Second Edition, edited by D.P. Schmitt, J.A. Wrather, and R.D. Riggs). Even more remarkable in view of its known economic impact is that this fascinating animal receives limited attention from soybean farmers, the research community and funding agencies. Out of necessity, the focus of information in this article is largely presented from a North-America-centric point of view because the SCN literature is primarily from the USA.

Fig. 2. Soybean cyst nematode: A) females on soybean roots; B) maturing female; C) cysts and females with gelatinous matrices.

We know that SCN is present in most countries where soybean is grown, but we do not know the details of the struggles that nematologists in most other countries are having with SCN research (except through some personal communication). For example, the species was described by a Japanese scientist and a couple of papers were published by Japanese researchers in the 1970s on ultrastructure and ecology, but little other information has been published thereafter from that country. From Brazil, a major producer of soybean, few publications can be found postdating the one that described the detection of the nematode in that country (in 1993) even though SCN is an economically important soybean pathogen in Brazil.

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Ancient history, distribution, and economic importance The story of SCN as a plant parasite began long before the domestication of soybean ca. 6,000 years ago. Numerous genes for resistance in soybean and its wild relatives and for virulence in the nematode, are known or posited. (We define virulence simply as the ability to reproduce on a resistant host, in accordance with R.S. Hussey [University of Georgia, Athens, Georgia] and G.J.W. Janssen [Syngenta Seeds AB, Sweden] in a description of screening for resistance in the 2002 book Plant Resistance to Parasitic Nematodes (edited by J.L. Starr, R. Cook, and J. Bridge.) There is general agreement that the ancient origin of SCN is in Asia. Greg Noel (Fig. 3) has suggested that SCN was unintentionally brought to the West in the late 19th century by agronomists who imported soil from Asia for studies of soybean nodulation, the source of which was then unknown.

Fig. 3. Gregory R. Noel (left), nematologist, and Richard L. Bernard, soybean breeder.

The distribution of SCN has followed the distribution of soybean production as it has expanded, and now includes the Middle East (Iran), as of 1999, and at least one area in Europe (Italy), as of 2001. In the United States alone, economic losses due to SCN are near one billion US dollars annually. It is somewhat surprising, therefore, that in North America, only a relatively small group of nematologists and plant pathologists work on the problem. Part of the reason for this is historical, and part is lack of demand on the part of soybean producers. Unfortunately for our ability to reduce THE SCIENCE AND ART OF SOYBEAN CYST NEMATODE RESEARCH

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soybean losses, SCN appears to have adapted so well to life in the midwestern US, where most North American soybean is grown, that it does not usually cause visible symptoms (such as stunting and chlorosis) even while reducing yields by 5 to 30% (Figs. 4–5). As crop management decisions are based strongly on commodity prices and sometimes aesthetics, with limited attention given to yield limiting factors, SCN is often ignored.

Fig. 4. Visible symptoms of infection by SCN: left, chlorosis; right, stunting.

Fig. 5. Symptomless SCN-resistant and susceptible soybean infected with yield-limiting population densities of SCN: left, Missouri; right, Iowa.

Based primarily on subjective assessments (e.g., windshield surveys), annual losses due to SCN in the US range from about 0.5% to 13%. Some damage function data exist from microplot and field research that demonstrate the potential yield suppression by SCN alone. However, since most data for yield loss (such as those illustrated in the graphs below) are not research-based, it is difficult to determine whether losses are increasing, decreasing, or remaining stable over time. Year-to-year assessments are heavily influenced by environmental conditions. In spite of these reservations, there is no doubt that loss is occurring directly and through disease complexes. 140

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Actually, the greatest losses are most likely occurring through multiple interactions of SCN with several other pests including insects, fungi and weeds (Fig. 6).

Fig. 6. Average estimated loss in soybean yields attributed to SCN: A) southern USA; B) USA overall. (Note that soybean production in the southern states has declined to a fraction of its former extent, whereas total soybean production in the US has increased).

The disease The disease caused by SCN has been classified under a number of names such as “moon night” (a Japanese appellation) and “fireburned seedlings” (Chinese). These two names reflect the symptoms associated with severe infestations: oval patches of stunted, chlorotic THE SCIENCE AND ART OF SOYBEAN CYST NEMATODE RESEARCH

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plants in the field (see Fig. 4). The earliest documentation for the disease was 1881, but it has certainly been a problem for many centuries. Greg Noel reported that an Asian post-doctoral fellow working with him described an ancient Chinese character for soybean as a glyph that appeared to have the outlines of H. glycines females incorporated in it (Fig. 7). It is difficult to date this glyph, but one can speculate from other evidence that SCN was causing problems at least 2,500 years ago. Referring to Book 26 of The Annals of Lü Buwei compiled in China in 239 BC, rules were outlined for controlling “three robbers”, one of which was “the land stealing the crops.” The prescription for this “robber” was “overworked soils [need] fallowing.” Since the diseased soybeans in these fields were described by the Chinese farmers as “fire-burned seedlings,” the cause could have been SCN. Fig. 7. Ancient Chinese character for soybean (courtesy of Zonglin Liu and Greg Noel).

As if any were needed, further evidence that nematodes are often overlooked as a primary cause of disease can be had from the testimony of Nash N. Winstead (Fig. 8). Upon graduation from the University of Wisconsin, he was employed by North Carolina State University (NCSU) and stationed at Castle Hayne, NC near Wilmington. He noted that fertility research had been underway for at least 8 years to solve a soybean “leaf yellowing” problem in the area. Winstead, having just completed his doctoral program and, recalling lessons taught by J.C. Walker and other plant pathologists at the University of Wisconsin, felt that there must be a root disease. Sure enough, he found cysts on the roots, which he sent to Hedwig Hirschmann at NCSU to confirm. This was the beginning of much activity by regulatory agencies to determine the distribution of SCN. A quarantine was actually instituted and maintained until the early 70s, when it became clear that regulatory efforts to contain the distribution of SCN within the US had failed. SCN was found in Canada in the late 1990s. 142

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Fig. 8. Nash N. Winstead

Fig. 9. Minoru Ichinohe

Fig. 10. Lawrence Miller

Species and infraspecific variation In 1952, Minoru Ichinohe (Fig. 9) described the species, Heterodera glycines, in Japan. It is puzzling that it took so long to describe H. glycines, especially when we consider that Heterodera schachtii was described about 100 years earlier. Heterodera glycines and H. schachtii are very similar in morphology, but were thought to be genetically isolated by host range (at least). They may not be as genetically isolated as we think. In the 1970s, Lawrence Miller from the Virginia Polytechnic Institute & State University (VPI) in Blacksburg, Virginia, (Fig. 10) reported on a series of unconfirmed experiments in which he made viable crosses between H. glycines and H. schachtii. His results have recently been verified by Alison Colgrove, a postdoc working with Terry Niblack at the University of Illinois, Urbana-Champaign, Illinois, with phenotypic as well as genotypic tests. In addition, such crosses are occurring naturally in sugar beet – cabbage – soybean rotations in Michigan, monitored by George Bird of Michigan State University. These results are new at the time of writing (March 2006) and have not been subjected to the scrutiny of peer review as yet, but they will perhaps muddy the waters regarding the species concept in Heterodera. SCN also exhibits physiological variation that impacts research and management alike. This understatement introduces one of the most controversial and colorful chapters in the history of SCN research: the problem of infraspecific variation for virulence on soybean (Fig. 11). In the 1960s, even before the release of the first SCN-resistant soybean cultivar, John Ross, a USDA-ARS scientist, THE SCIENCE AND ART OF SOYBEAN CYST NEMATODE RESEARCH

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reported on “physiological strains” that differed among states with known SCN infestations, according to differences in virulence on known resistant soybean lines. In the 1970s, A.C. Triantaphyllou of NCSU, demonFig. 11. Earliest known photograph illustrating the effects of strated SCN adaptatwo different isolates of SCN on the same soybean cultivar tion to resistance (Control CK; Arkansas ARK; North Carolina NC) within a few genera(courtesy of R. D. Riggs). tions. In the 1980s and 90s, Terry Niblack confirmed that an SCN population adapted to a “new” host in about 6 generations. For example, some populations adapted to lima bean and tomato, previously considered nonhosts of SCN; each took six generations to develop into a viable, sustainable population. Similarly, it took about six generations for a population to adapt to the soybean plant introduction (PI) 437.654, originally reported as resistant to “all known races” of SCN. In Missouri and Illinois, field SCN populations are able to parasitize (at some level) all known sources of resistance to SCN. Clearly, the relevant genes for virulence are already present in many SCN populations because adaptation occurs too quickly to be explained by mutation. As indicated in the previous paragraph, physiological variation among SCN populations was reported by C.A. Brim and John P. Ross in the 1960s before the release of the first resistant soybean cultivar (Pickett). Ross and subsequent researchers observed that certain SCN populations could parasitize the three known sources of resistance at the time, the soybean plant introductions (PI) Peking, 88788, and 90763 (the PI designation refers to its classification in the USDA Soybean Germplasm Collection, curated by Randall Nelson), and so it was essential that a framework be devised to describe these populations so that changes in adaptation to resistance could be monitored more easily. The group that first addressed this issue met in 1969, and consisted of Morgan Golden (USDA-ARS, Fig. 12), J.M. Epps (USDA-ARS), R.D. Riggs (University of Arkansas, Fig. 13), L.A. Duclos (University of Missouri), J.A. Fox (VPI), and 144

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Fig. 12. A. Morgan Golden

Fig. 13. Robert D. Riggs

Fig. 14. Donald P. Schmitt

R.L. Bernard (USDA-ARS, Fig. 3). From this group, the eminent nematologist Robert Riggs devoted a portion of his 48-year career to studying physiological variability in SCN. Also from this group, the soybean breeder, R.L. Bernard, was later responsible (together with nematologist, G.R. Noel) for developing the germplasm that is the basis for more than 90% of the SCN-resistant soybean cultivars now used in the midwestern US. The group solved the problem of terminology by describing the variation in virulence among four SCN populations in terms of what became known as the “race scheme.” In the years immediately following publication of the 4-race scheme, SCN populations were found and described that did not fit the description — a situation that was not remedied until 1988, when Riggs and D.P. Schmitt (then at NCSU, Fig. 14) expanded the race scheme to its logical extent: 16 races.

A digression into classification of SCN resistance An interesting and unforeseen consequence of the initial development and publication of the race scheme was that breeders began to use it to classify resistance in soybean cultivars rather than virulence in nematode populations – a very important distinction. In the race scheme, the decision whether to label an SCN population as virulent or not depended on its ability to develop into adult females on a resistant line relative to its ability to develop on a susceptible stanTHE SCIENCE AND ART OF SOYBEAN CYST NEMATODE RESEARCH

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dard host; the cutoff value was 10%. Soybean breeders adopted the same 10% rule to identify soybean cultivars as resistant. There were two major problems with this as they usually used only one isolate of SCN as representative of an entire race, for example, a cultivar showing resistance to one isolate of race 3 was pronounced “resistant to race 3,” a generalization that was not warranted (Fig. 15); and those cultivars that expressed partial resistance were being ignored.

Fig. 15. Illustration of the distribution of resistance to race 3 in soybean cultivars labeled as “resistant to race 3.“

This issue and others were discussed in the soybean breeders” board meetings (held annually in St. Louis), which led to a decision to hold a special meeting to address the problem of categorizing resistance. At the special meeting, documented by nematologist Don Schmitt and soybean breeder Grover Shannon (then of Delta Pine Land, Inc.) in voluminous notes, the consensus of opinion was summarized and developed into a manuscript to be published in Crop Science. Despite the general agreement at the meeting, some breeders rescinded their support for publication of the scheme that was developed unless their personal opinions were included in the manuscript. Thus the manuscript began to change form but, fortunately, one of the breeders who had attended the meeting was the final editor of the paper. He indicated that the original agreement should be published and so it was! The consensus stood: there would be four levels of resistance based on levels of reproduction of SCN on 146

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the test line compared to a susceptible standard: if < 10%, the line was resistant; 10 to 30%, moderately resistant; 31 to 60%, moderately susceptible; and >60%, susceptible. Due to some concern about the interpretation of “moderately susceptible,” the term was later changed to “slightly resistant”. In Illinois, the SCN screening program, which determines the levels of resistance in 400 to 600 soybean cultivars to five different SCN isolates each year (conducted by T.L. Niblack, G.R. Noel, and J. Bond [Southern Illinois University]), a different scale is used: 0 to 9%, highly resistant; 10 to 24%, resistant; 25 to 39%, moderately resistant; 40 to 59%, low resistance; and > 60%, no effective resistance. This change was implemented for three reasons, at least one of which is not scientifically justifiable. First, most of the SCN-resistant cultivars in the lower Maturity Groups (4 and below, which are grown mostly north of 34° north latitude) are derived from PI 88788. One of the characteristics of this source of resistance is that it does not produce a necrotic reaction, but reduces SCN populations by acting on developmental stages after the second stage juvenile. Sometimes, many females are allowed to develop, but their fecundity is so limited that (in the field) the ultimate effect is a reduction in the SCN population. Without resorting to counting eggs, labelling as “resistant” only those PI88788-derived cultivars that allow only 10% or less female development would be to undervalue a large number of usefully resistant cultivars. Second, over the 18year period that data were collected, the 10–25–40–60% thresholds define statistically “natural” categories. Finally, and perhaps indefensibly, the word “susceptible” is not used to describe a cultivar released by a commercial company as “resistant.”

Back to the subject at hand: variation for virulence in SCN Shortly after the expansion of the race scheme by Riggs and Schmitt, a new, highly-resistant soybean cultivar, Hartwig, was released, developed by Sam Anand, University of Missouri, and named after the most legendary, confident, and tenacious soybean breeder of the latter half of the 20th century, Edgar E. Hartwig (Fig. 16), USDA-ARS, who influenced SCN research in the southeastern US in various ways for many years. Resistance in the cultivar Hartwig was derived from PI 437654 THE SCIENCE AND ART OF SOYBEAN CYST NEMATODE RESEARCH

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Fig. 16. Edgar E. Hartwig

Fig. 17. T. L. Niblack

and “Forrest”. Soon thereafter, three more lines were released with SCN resistance derived from PI 209332, PI 89772 and 548316, respectively. Since the SCN race scheme did not include these PIs, there was no framework ready to describe SCN populations virulent on them. The obvious solution, to add them to the race scheme, would have increased the possible number of races to 256, an untenable number for any practical use. Furthermore, the race system was not suitable for use in genetic studies (for various reasons discussed elsewhere in reviews and research papers). To address these problems, a group of nematologists, geneticists, and soybean breeders convened, in 2001, to discuss replacing the race scheme with another virulence phenotyping framework. The group included T.L. Niblack (Fig. 17), P. Arelli (USDA-ARS), G.R. Noel (Fig. 3), C.H. Opperman (NCSU), J.H. Orf (University of Minnesota), D.P. Schmitt (Fig. 14; then of University of Hawaii), J.G. Shannon (University of Missouri), G.L. Tylka (Iowa State University), and R.D. Riggs (Fig. 13), the latter being the only remaining active scientist from the group which had developed the original race scheme. The solution of this group was called the HG Type Test (a name that only a committee could love), published in 2002. The framework included an easily-adapted list of all the sources of SCN-resistance known to have been used in breeding programs. This system could be adapted for use in different countries, and has been adapted for use in making cultivar recommendations in 148

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Illinois by including only the three sources of resistance available to Illinois soybean growers. The HG Type Test has been adopted by nematologists and plant pathologists, but has met with resistance and even hostility from a few public and private soybean breeders who find themselves unable to co-opt it for use in their breeding programs. The recommendation by the HG Type committee was for those who release SCN-resistant germplasm to simply identify the source of resistance, but until now many private companies have been unwilling or unable to do so. It will be interesting to see how this is resolved. The next permutation of virulence phenotyping for SCN is likely to be accompanied by genotype tests, which are not currently available. Those working on the genetic basis of virulence include labs headed by K.N. Lambert (University of Illinois), D.McK. Bird, and C.H. Opperman (NCSU). The first genetic map of SCN was published in 2005, and genomic analysis of the nematode is in its infancy. SCN serves as a model organism for investigation of plant parasitism by nematodes, exemplified by labs headed by R. S. Hussey, E.L. Davis (NCSU), and T.J. Baum (Iowa State University). Challenges will continue because of the complex genetics of SCN. One of the many tasks is to determine how SCN isolates maintain their diversity after 100 to 300 generations of inbreeding. “Random mating” doesn’t really occur; matings in soil are usually between full- or half-siblings. Single-cyst-descent inbreeding should result in SCN populations being fixed for most loci, and yet this is not what we observe. The imagination is the only limit to unravelling the genetics and behavior of this pathogen.

Management Farmers tend to perpetuate their habits and those of their ancestors in crop management. To survive, though, they must make sufficient profit to sustain the farming operation with an adequate return to management to support the family. Therein lies the challenge for pest control advisors, whether from the private or public sector. The most frequently used rotation recommendation that is considered to be fundamental to SCN management is a 3- or 4-year rotation including one resistant soybean cultivar and one susceptible cultivar, as follows: Year 1, resistant soybean cultivar; Year 2, THE SCIENCE AND ART OF SOYBEAN CYST NEMATODE RESEARCH

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corn (maize); Year 3, susceptible soybean; Year 4, corn. Ask anyone involved in making SCN recommendations, and this is what they will tell you. One would think that such a recommendation, one that affects so many soybean growers, would have been based on well-documented field studies. Instead, it was developed in a discussion over a pitcher of beer (sources wish to remain anonymous) as a best guess based on assumptions about mortality rates and “race shifts.” To be fair, field studies were done in North Carolina and elsewhere, but in general, research to determine the rates of mortality and “race shifts” have not given clear and definitive results. Rotation to nonhosts clearly reduces SCN population densities, but what happens to the genetic structure of the population? The point here is not that the value of rotation should be questioned, but that certain aspects of the long-term effects of nonhosts and resistant cultivars on SCN populations and crop profitability still need to be elucidated. SCN populations have shown evidence of marked adaptations to life in the Soybean Belt, i.e. the Corn Belt states, where annual alternation between maize and soybean became the standard in the latter part of the 20th century. Those adaptations, such as an apparent shift to earlier induction of dormancy and an increase in overwinter survival rates, interfered with the portability of management recommendations from one region of the US to another, not to mention from the US to other countries. Changes in the disease caused by the nematode have also occurred. For example, as mentioned earlier in this paper, in many of today’s intensive soybean production areas, SCN causes few or no visible disease symptoms. Soybean growers, admonished for years to look for stunted and chlorotic plants as evidence of the presence of SCN, are now hearing that symptoms are no longer characteristic of SCN infection. This can create frustration for both growers and those who craft recommendations for SCN management. There is purely anecdotal evidence that the parasites and predators of SCN are catching up, geographically, with the nematode. From conversations with nematologists and plant pathologists who were around in the Soybean Belt when SCN was first spreading into the area, it appears that fewer nematodes were required to cause significant yield loss several decades ago than are required today. Damage thresholds established in the 70s and 80s are no longer applicable today. 150

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Concluding comments “Moon night” type symptoms helped us recognize the organism ultimately classified as soybean cyst nematode. Today, management is compromised because the symptoms are not obvious. Nematologists and those scientists working in nematological roles have to envy weed scientists. Growers will use extreme means to control weeds, even the species that compete very little with the soybean crop. Many of these same growers will simply ignore SCN because the neighbors will not notice anything striking or unusual. This fact points to an issue of education that should be addressed. Extension is being down-sized (eliminated in some states). Private practitioners are rarely educated in nematology and they are few in number. Will a serious crisis be necessary to get the attention of the public and private sector? What we really need are soybean cultivars genetically engineered to break out in pink polka-dots when infected by SCN (not really kidding). Among the generally underappreciated plant-pathogenic nematodes that cause significant economic injury to important crop plants, SCN ranks as one of the least appreciated. Dedicated to solving the puzzles that SCN has set us, many fine researchers contributed ideas, data, and opinions; not all of the researchers or their opinions have been mentioned in this paper, of course. Our editor tells us we must stop somewhere!

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10. NEMATODES /VIRUSES /PLANTS: “A 32-YEAR LOVE AFFAIR” DEREK J. F. BROWN College of Tourism, Bansko, Bulgaria

It was not possible to write an authoritative and dispassionate historical account of a subject that one was intimately involved in for 32 years! My “love affair” with virus-vector nematodes, their associated viruses, and the many plant species affected by both nematode and virus is professionally referred to as my scientific research career. Such reference provides no insight into the passion, emotion, commitment, excitement, frustration, intellectual and physical challenges, and involvement with colleagues and students worldwide, many of whom became personal friends. Consequently, this is an unashamedly personal “historical account of plant virus transmission by soil nematodes”. I take this opportunity to thank everyone – nematodes, viruses, plants and the numerous colleagues and students – and dedicate this chapter to Professor Charles E. Taylor and Professor Franco Lamberti (now, sadly, both deceased) who individually and collectively were my constant and supportive mentors.

The “love affair” ended in April 2002, but it began in May 1970 with the words ringing in my ears “much of the work is repetitive, it is mainly laboratory and glasshouse based, and there are few opportunities for travel”. Thus began my career as a plant nematologist/plant virologist, and how misleading would this initial guideline prove to be. However, my research area, virus-vector nematodes and the viruses they transmit, had much earlier beginnings.

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The early beginning: soil-borne plant diseases vs. aerial-borne plant diseases. In the period 1882 to 1900, several reports demonstrated and confirmed that some plant diseases were soil-borne. For example, healthy grapevines became diseased when planted in soil taken from old vineyards in which diseased grapevines were growing. It would be almost another 50 years before the disease was described as grapevine fanleaf virus (GFLV), and a further 30 years before the soil-borne vector of the virus, the nematode, Xiphinema index would be identified. In 1886, tobacco plants were shown to become infected in a similar manner with a mosaic disease that, in 1943, was identified as being caused by tobacco rattle virus (TRV). Whereas the concept of soil-transmission of plant diseases was largely ignored until the late 1940s, aerial vector transmission became the vogue in the first half of the 20th century. Quite simply it was much easier to see, on the crops, insects that had the potential to be the disease carrying agent than to believe that some mysterious organism present in the soil could transmit a crop disease. The impediment to research that this misconception had is probably best exemplified in the raspberry crop in the UK. In 1922, in eastern Scotland a devastating disease was recorded that apparently occurred spontaneously in plantations of cv. Baumforth’s Seedling raspberry. The disease agent was never identified, and disappeared when cv. Lloyd George was planted. Eventually, cv. Lloyd George became infected with a mosaic disease and was replaced by cv. Norfolk Giant. In 1941, the original lethal disease in cv. Baumforth’s Seedling recurred in cv. Lloyd George, and the disease was named “leaf curl“ because of its resemblance to an aphid-borne North American disease of the same name. Despite comprehensive research over the next twelve years an aerial vector for the disease was never identified. In 1956, the disease now known to be caused by raspberry ringspot virus (RRSV) was demonstrated to be soil-borne. During the next couple of years several other viruses, including tomato black ring nepovirus (TBRV) and arabis mosaic nepovirus (ArMV) causing diseases in crops in the UK, were shown to be soil-borne. Thus, during the 1950s, soil-borne viruses had been identified as being more common and of more economic importance than had previously been realized. With the development of new methods to reliably recover viruses from infected NEMATODES /VIRUSES /PLANTS: “A 32-YEAR LOVE AFFAIR”

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plants, to transfer them between herbaceous test plants, and to identify them by their serological properties, research on soil-borne viruses intensified independently in the UK, continental Europe and the USA.

Soil nematodes: could they really be responsible for spreading soil-borne diseases? Initially the wrong nematode types were investigated as potential plant disease transmitting agents, thus resulting in early studies failing to establish any nematodes as vectors. In 1912, in southern England the occurrence of the hop cyst nematode, Heterodera humuli, was correlated with the spread of “nettlehead“ disease in hops. A decade later the nematode was exonerated as being the vector, and it was not until fifty years later that the disease was identified as being caused by ArMV and was being transmitted by the nematode, X. diversicaudatum. During the 1940s, in the USA wheat mosaic-infected soil was treated with calcium cyanide, carbon disulphide, chloropicrin and methyl bromide and these treatments prevented transmission of the disease. A decade later experiments failed to associate nematodes with transmission of the virus. Several nematode species in the USA were meticulously tested as potential vectors of lettuce big vein virus, but with negative results. Subsequently, the virus was shown to be transmitted by the chytrid fungus, Olpidium brassicae. Meloidogyne spp. were similarly tested as vectors of tobacco mosaic and cucumber mosaic viruses as were Helicotylenchus nanus and Pratylenchus spp. as vectors of carnation mottle virus, but again with negative results. Despite several studies testing soil nematodes as potential vectors of various soil-borne diseases and plant virus diseases, the investigations all proved fruitless. By the early 1950s, it was concluded that conducting research to associate nematodes with soil-borne diseases was a pointless exercise. However, there were still a few scientists who doggedly continued to try to prove that a soil nematode could vector a soil-borne disease.

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Grapevines – Prohibition – Breakthrough: the American story Grapevines were first cultivated around the Black and Caspian Seas, and grapevine fanleaf nepovirus (GFLV) and its nematode vector, Xiphinema index, probably originated from and co-evolved in this area. The Phoenicians brought grapevine rootstocks to Greece and around 600BC to Marseille, France from where they were distributed, almost certainly with GFLV and X. index, to Italy and, subsequently, to Spain and Portugal. Grapevines were transported to the New World from Spain in the 17th century and by the end of the 17th century to Alto California (California, USA). Here the “Mission“ grapevine, which is highly susceptible to GFLV, eventually formed the basis of what became an extensive wine-producing industry. It has been speculated that GFLV and X. index were introduced into California by Colonel Agoston Haraszthy who imported rooted grapevines from his native Hungary in 1851. Even more likely, the virus and vector were imported in 1861 when he imported 100,000 grapevines of 1400 cultivars collected from many wine producing areas in Europe, where by this time both virus and vector were widespread. It also has been suggested that GFLV and X. index were first introduced into the United States in 1900, when imported rootstocks became widely used after the spread of Phylloxera. The evidence for this is that GFLV was not present in over 100 clones of grapevine cultivars that were still growing at the site of a trial that had been established in California in 1890 and abandoned in 1903. A third scenario is that in the 1920s, the USA Volstead Act (alcohol prohibition) resulted in most commercial wine-production vineyards in California being destroyed. Thirteen years later when the Act was rescinded there was massive, rapid replanting of vineyards. This would obviously have provided an excellent opportunity for the introduction into California of virus and vector and their widespread distribution. GFLV was discovered in California in 1948, and thereafter a series of investigations were established in 1954, to identify its soil-borne nature. Firstly, healthy grapevines were planted in containers holding: i) soil from the rhizosphere of GFLV infected grapevines, at sites where the disease was spreading; ii) steam-sterilized vineyard soil and; iii) sterile soil. The following spring 62 of 70 vines in the untreated soil were infected with GFLV, but the virus was not present in grapevines in NEMATODES /VIRUSES /PLANTS: “A 32-YEAR LOVE AFFAIR”

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either of the other two treatments. Thus, GFLV had been shown to be soil-borne. The next tests were to prove crucial, as they would finally demonstrate that a soil nematode was the vector of GFLV. Soil collected from the rhizosphere of GFLV infected vines was wet screened as 500g samples through a sieve with 75 µm diameter apertures. The debris collected on the sieve, after thorough washing, was poured over the roots of healthy grapevines growing in pots of sterile soil. Xiphinema index and Criconemoides xenoplax were present in most, but not all, of the samples. The following year GFLV infection had developed in 20 of 35 grapevines. In the next set of tests hand-picked groups of nematodes of both species, from GFLV infected soil, were placed on the roots of healthy grapevines growing in sterilized soil. Eventually, 1 of 12 plants with C. xenoplax and 5 of 12 plants with X. index developed symptoms of GFLV infection. The single infection associated with C. xenoplax was subsequently assumed to have been caused by contamination, as subsequent experiments with this nematode species proved negative. Finally, further experiments confirmed X. index as the vector of GFLV.

The real beginning and its aftermath The publication in the USA in 1958 by Hewitt, Raski and Goheen confirming that X. index was the vector of GFLV stimulated the search for nematode vectors of other soil-borne viruses occurring in the USA and Europe. This was the real beginning of virus-vector research and here is presented a brief chronology of scientific landmarks that have marked this area of science (adapted from Taylor, C.E. & Brown, D.J.F. 1997. Nematode Vectors of Plant Viruses. CAB International, Wallingford, England). 1958 – Hewitt et al. identify X. index as the natural vector of GFLV in vineyards in California, USA. 1959 – Jha & Posnette and Harrison & Cadman report X. diversicaudatum as the natural vector of ArMV in Europe. 1960 – Sol et al. report that tobacco rattle tobravirus (TRV) is transmitted by Paratrichodorus sp. (=Trichodorus pachydermus) – In Scotland the first international symposium is held on virus-vector nematodes and their associated viruses. 156

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1961 – Harrison et al. are the first to report a Longidorus sp. as a vector of a nepovirus. – Harrison reports a Trichodorus sp. as a vector of TRV. 1962 – Hoof reports the transmission of pea early-browning tobravirus (PEBV) by a Paratrichodorus sp. 1964 – Harrison reports the association of serologically distinguishable strains of nepoviruses with specific longidorid species as their vectors. – Taylor & Raski report that viruses in the vector are not retained through the moult or through the egg. 1966 – Hoof identifies differences in the ability of L. elongatus populations to transmit TBRV. 1968 – Hoof reports differences in the ability of trichodorid populations to transmit TRV isolates and concluded that transmission occurs only when the virus isolate “suits“ the nematode population (specificity). 1969 – Taylor & Robertson identify “virus-like” particles, based on the particle morphology, at specific sites within the feeding apparatus of Longidorus. 1970 – Taylor & Robertson identify “virus-like” particles, based on the particle morphology, at specific sites within the feeding apparatus of Xiphinema and of Paratrichodorus. – McGuire et al. identify “virus-like” particles, based on the particle morphology, at specific sites within the feeding apparatus of X. americanum. 1971 – Hoof reports differences in the ability of Xiphinema populations to transmit nepoviruses. 1972 – Yagita & Komuro report the transmission of mulberry ringspot nepovirus (MRSV) by L. martini in Japan; the first record of a new vector and virus association reported outside Europe and North America. 1973 – Salomao reports the transmission of pepper ringspot tobravirus (PRV) by P. minor (= P. christiei) in Brazil, South America. 1974 – Harrison et al., establish that RNA-2 of the bipartite genome of RRSV, which codes for the virus coat protein, is involved in the recognition between the virus and its vector. – Siddiqi establishes Trichodorus and Paratrichodorus as two distinct genera.

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– A NATO Advanced Study Institute “Nematode Vectors of Plant Viruses“ is held in Italy. 1975 – Yagita publishes evidence that L. martini, the vector of MRSV, has only three and not the usual four juvenile development stages. – “Nematode Vectors of Plant Viruses” by F. Lamberti, C.E. Taylor and J.W. Seinhorst (Eds), published by Plenum Press, London and New York. 1977 – Heath et al., publish a nematological atlas for Britain containing maps of the distribution of Longidorus, Paratrichodorus, Trichodorus and Xiphinema species. 1978 – Trudgill & Brown report the infrequent transmission of RRSV by L. macrosoma to be associated with an apparent lack of release of virus particles from the site of retention within the vector. – McNamara identifies sources of potential contamination in virus transmission experiments which could account for several reports of apparent non-specific associations between vectors and viruses. 1979 – Lamberti & Bleve-Zacheo reappraise members of the X. americanum-group, which results in uncertainty in the identification of X. americanum transmitting North American nepoviruses in all previous reports. 1981 – Trudgill et al. report differences in the efficiency of transmission of nepoviruses by longidorid vectors. 1983 – Trudgill et al., propose a set of criteria for assessing reports of longidorid nematode transmission of nepoviruses. 1984 – Hoy et al. report the differential transmission of strains of tomato ringspot nepovirus (ToRSV) by X. californicum, a member of the X. americanum-group. 1985 – Brown demonstrates differences between populations of X. diversicaudatum in their ability to transmit strains of strawberry latent ringspot nepovirus (SLRSV). 1986 – Brown demonstrates differences between populations of X. diversicaudatum in their ability to transmit strains of ArMV and shows that the vector’s ability to transmit virus is inherited. – Robertson & Henry associate retention of virus particles with the layer of carbohydrate lining the oesophagus in Xiphinema and Paratrichodorus. 158

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1989 – Brown et al. establish that differences in the frequency of transmission of isolates of TBRV by a population of L. attenuatus do not correspond with the serological groupings of the isolates. – Brown et al. establish a set of criteria for assessing reports of transmission of tobraviruses by trichodorid nematodes. 1990 – Ploeg et al. confirm specificity of transmission between trichodorid species and serologically distinguishable strains of TRV. 1992 – Ploeg et al. show that the genetic determinants of vector transmissibility are associated with TRV RNA-2. – Halbrendt & Brown report that several North American populations of the X. americanum-group nematodes have only three, and not the usual four, juvenile stages. – Vrain et al. report that molecular taxonomy methods support the establishment of several morpho-species in the X. americanum-group from North America. – “Dorylaimida. Free-living, Predaceous and Plant-parasitic Nematodes” by M.S. Jairajpuri and W. Ahmad, published by Oxford and IBH Publishing Co., New Delhi, India; includes the Longidoridae and Trichodoridae. 1993 – Ploeg et al. report that virus coat protein mediated resistance, which has been shown to be effective with several viruses transmitted by insect vectors, is not an effective control strategy for TRV when the virus is transmitted by the nematode. – “Aphelenchida, Longidoridae and Trichodoridae: Their Systematics and Bionomics” by D. Hunt, published by CAB International, Wallingford, England. 1994 – Brown et al. report that several X. americanum-group species can each transmit three distinct North American nepoviruses, that differences occur between populations of X. americanum sensu stricto in their ability to transmit North American nepoviruses and that the specific associations between these nematode species and the North American nepoviruses is different from that which occurs between European virus-vector species and their nepoviruses. – Jones et al. report Paralongidorus maximus as the vector of an atypical isolate of RRSV in vineyards in Germany.

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1995 – MacFarlane et al. report that the transmission of tobraviruses by nematodes is not determined exclusively by the virus coat protein. – Mayo et al. suggest that flexible peptides on the C-terminus of the coat protein of nematode-transmitted viruses, especially tobraviruses, may be responsible for virus and vector recognition. – Ramel et al. report the first unequivocal evidence of a nepovirus, a strain of ArMV transmitted by X. diversicaudatum, causing a disease in a graminaceous plant, barley. – Robbins et al. report that several Longidorus spp., including L. martini (see 1975), have only three and not the usual four juvenile stages. – “The family Trichodoridae; Stubby Root and Virus Vector Nematodes” by W. Decraemer, published by Kluwer Academic Publishers, Dordrecht, The Netherlands. 1996 – Brown et al. identify the site of retention of TRV in Trichodorus. – MacFarlane et al. confirm that each of the four Open Reading Frames (ORFs) contained in PEBV RNA-2, and that flexible peptides on the C-terminal of the coat protein of the virus, are involved in determining vector transmission of the virus. – Kreiah et al. report that SLRSV coat protein mediated resistance is an effective control strategy when the virus is transmitted by its vector. 1997 – “Nematode Vectors of Plant Viruses” by C.E. Taylor and D.J.F. Brown, published by CAB International, Wallingford, England. 1998 – Weischer & Brown define terminology for specificity, exclusivity and complimentarity of nematode transmission of viruses. – Wang & Gergerich using immunoflourescent techniques provide the first unequivocal evidence that “virus like” particles at specific sites within the feeding apparatus of Xiphinema americanum senso lato nematodes, as reported by McGuire et al., in 1970, are indeed nepovirus particles. 2000– Karanastasi et al. develop advanced serological techniques involving immunogold labelling to provide the first unequivocal evidence that “virus like” particles at specific sites within the feeding apparatus of Paratrichodorus, as reported by Taylor & Robertson in 1970, are indeed tobravirus particles. 160

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2001 – Karanastasi et al. report that differences in the body cuticle of Paratrichodorus species clearly separate them from Trichodorus species, and that similar differences reveal two separate groups of Trichodorus species. Chen et al. develop a method for separating specific nematode species from species mixtures using antiserum and lectin-coated magnetized beads. – Vassilakos et al. identify that the tobravirus 2b protein acts in trans to facilitate vector transmission. 2003 – Karanastasi et al. identify specific retention of tobravirus particles in the feeding apparatus of trichodorids, including nonvector species in which the virus is retained at sites from where it can not be transmitted to plants. – Chen et al., develop a magnetic capture system for recovery of specific X. americanum nematodes from mixtures of soil nematodes. – Wang et al., develop a multiplex polymerase chain reaction using ribosomal genes for identifying single individuals of several Xiphinema species including X. index. 2004 – Boutsika et al., develop a molecular diagnostic method for identifying trichodorid virus-vector species and their associated tobacco rattle virus. With research on nematode transmission of viruses developing rapidly after the seminal paper by Hewitt et al., in 1958, it was considered pertinent to arrange an international forum to exchange information between the various researchers. A symposium was held in July 1960 at the Scottish Horticultural Research Institute. Most notably, Dutch researchers attending the conference received a telegram from their co-workers in Wageningen, The Netherlands, confirming that Trichodorus (Paratrichodorus) pachydermus was the natural vector of TRV. Evidence that L. elongatus transmitted the Scottish strain of tomato black ring virus (TBRV) was also discussed. Thus the three groups of vector species viz. Longidorus, Trichodorus (Paratrichodorus) and Xiphinema had been identified. Interestingly, the conclusion of the delegates was that nematode-transmitted viruses were localized problems of insignificant economic importance in comparison with the soil-borne wheat and oat mosaic virus diseases occurring over extensive areas in the USA. Thankfully, this conclusion was ignored!

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The Golden Years In 1970, I joined the then small Zoology Department at the Scottish Horticultural Research Institute with Charles Taylor as Head of Department. Four years later I was fortunate enough to attend what was to be for me the most important meeting of scientists involved in virus vector research. Franco Lamberti, Charles Taylor and Wim Seinhorst were the joint organisers of the NATO Advanced Study Institute at Riva dei Tessali, Italy. Eighteen lecturers and 48 delegates from numerous countries throughout the world reviewed and debated all aspects of the biology of virus-vector nematodes. Two weeks of unadulterated bliss was spent discovering what was known about the nematodes and viruses and being able to hone one’s research ambitions and ideas. By then much was already known about the nature of the association between virus and vector, and about virus acquisition, retention and transmission by vectors. The presentations on virus transmission, taxonomy, morphology, feeding behaviour, ecology, geographical distribution and control could only serve to stimulate my enquiring mind. The contacts made whilst attending this meeting would feature greatly in my career, enabling me to work around the world with some of the very best contemporary nematologists. Importantly, friendships were established that were to endure throughout one’s career, and the memories – the unfortunate scientist who walked through a glass door, luckily not causing himself any injury; or the colleague who alarmed Teresa Lamberti when she looked out of her bedroom window in the early morning to be confronted by him a few meters away with binoculars dangling, but we all knew that he was a very enthusiastic amateur ornithologist!?! By the 1980s, virus-vector nematodes were an established component of many nematology teaching programmes. In 1984, the European Society of Nematologists established a Virus-Vector Workshop as part of each of their biennial meetings, including the Second and Third International Nematology Congresses. Most personally gratifying were the numerous specialist training workshops in which I was privileged to be invited to participate and sometimes organize – University of Coimbra, Portugal; University of Cordoba, Argentina, Zhejiang University, China etc. To give a 3 hour lecture starting at 06.00 attended by what seemed to be hundreds of enthu162

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siastic Chinese students is unforgettable, and I was still answering questions whilst entering the taxi to speed to the airport. Another significant event was the arrival of my first student, who came for 3 months and eventually was to stay with me for 5 years. The excitement felt when witnessing my first student achieve his Ph.D., never diminished when my other students achieved the same distinction. Each one was and remains precious, and it is most gratifying to me that each has gone on to do even greater things than I could ever have hoped to achieve.

PERSONALITIES Numerous outstanding individuals have been, and many remain, involved in the various research components that collectively are referred to as virus-vector research. Taxonomy and systematics Early taxonomy of the virus-vector nematodes was most ably served by David Hooper at Rothamsted. Stalwarts of virus-vector taxonomy have been Michel Luc, France, the late Juan Heyns, South Africa, Piet Loof, Netherlands, August Coomans, Belgium, Wilfrieda Decraemer, Belgium, Dieter Sturhan, Germany, Franco Lamberti, Italy, Robert Robbins, USA Luiz Ferraz, Brazil and Jingwu Zheng, China. Each, undoubtedly, was amongst the most productive and influential scientists serving the taxonomy of the Longidoridae and Trichodoridae. Luc producing a definitive taxonomic guide of Xiphinema species, Decraemer a taxonomic guide to the Trichodoridae and Robbins a guide to the Longidorus species. Coomans, Luc and Heyns produced a remarkable compilation on Xiphinema systematics, and Lamberti was unflinching in devoting many years to attempting to clarify the X. americanum complex of species whilst all other taxonomists studiously avoided what must be the most difficult group of all. Meanwhile, Charles Taylor and Walter Robertson provided insight into aspects of the ultrastructure of longidorids. From the late 1990s to the present, major advances have been made in the molecular taxonomy and systematics of longidorid and trichodorid nematodes by Maurice Moens and colleagues in Belgium, and by Roy Neilson, Vivian Blok, Mark Phillips, John Jones and colleagues in Scotland.

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Biology Many scientists worldwide have contributed to the biology of virus-vector nematodes. Amazing visual insights into virus-vector biology were provided by the series of films showing details of trichodorids feeding on plant roots produced by Urs Wyss, Germany, and similar studies were done by Eirini Karanastasi, Greece and with Xiphinema and Longidorus spp. by David Trudgill and Walter Robertson, UK. Mariella Coiro and the author demonstrated the number of progeny that a single Longidorus and Xiphinema species can produce, and also the time taken for a single egg to be produced. The discovery by John Halbrendt, USA and the author that some Xiphinema species have only three and not four juvenile stages was highly controversial. A comment by one of the referees, a highly respected systematist in the USA, was that the authors would be best advised to find new careers as the Nematoda had four and never three juvenile stages! The research was eventually published, but not accepted by several European based virus-vector taxonomists, until they themselves identified species that clearly only had three and not four juvenile stages. This was followed by the revelation that also several Longidorus species had only three juvenile stages. Several years earlier a Japanese scientist published irrefutable evidence that this phenomenon occurred, but his work had been ignored. Perhaps the debate continues! Ecology As with the biology of virus-vector nematodes many scientists worldwide made significant contributions also to our understanding of their ecology. An important observation made by David McNamara was that X. diversicaudatum stored in plant-free, sterilized soil quickly became translucent and died within a few weeks, whereas those stored in untreated, plant-free soil appeared healthy and survived for at least 22 weeks. This observation has never been explained, although it was speculated that soil microorganisms might be responsible. John Halbrendt and the author raised populations from single females taken from naturally occurring populations of X. americanum sensu lato and revealed that little morphological variation occurred between specimens within a raised population, but that several morphological variants (species?) occurred in natural soil populations. On the other hand, 164

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Charles Taylor and the author showed that the occurrence and distribution of virus and associated vector nematodes at field sites changed very little over 25 to 30 years. Distribution The first comprehensive, systematic survey of soil nematodes was made for the Longidoridae in the British Isles and Italy as part of a co-operative project between the Istituto di Nematologia Agraria, Bari, Italy and the Scottish Horticultural Research Institute. This was developed into a European Plant Parasitic Nematode Survey (EPPNS) that eventually produced detailed distribution maps on the occurrence of many plant parasitic nematode species throughout Europe. The distribution patterns provided the impetus for numerous research studies on the biotic and abiotic reasons for the location of various species. Also, the distribution patterns, combined with morphological data provided new insights into the systematics and possible origins of the species. The original survey was subsequently complemented by additional surveys in the former USSR, the Mediterranean region, North America, and Latin America. Virus and vector associations The information in the late 1950s that X. index transmitted GFLV stimulated many researchers worldwide to seek further virus-vector associations, and during the next 15 to 20 years many new associations were reported. A research team in the former East Germany was amongst the most prolific, although several of the virus-vector associations appeared to be anomalies. With the more recent development of new, stringent testing procedures, not least being the introduction of experimental procedures in which individual nematodes are tested for their ability to transmit virus, a set of criteria for assessing reports of virus transmission by nematodes was developed by David Trudgill, David McNamara and the author. Applying these criteria to all published reports of virus transmission by nematodes they concluded that approximately two thirds of the reports failed to fulfill the criteria. Charles Taylor and Walter Robertson in the UK and Jim McGuire and colleagues in the USA identified the sites of virus retention in each of the vector genera in the late 1960s and early 1970s. Regrettably, to this day the specific nature of the association and particularly the mechanism of release of the virus from these sites remains a subject of conjecture. NEMATODES /VIRUSES /PLANTS: “A 32-YEAR LOVE AFFAIR”

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Throughout the 1960s, 70s and 80s virologists made significant advances in determining the genetic structure of nematode-transmitted viruses, eventually revealing that vector transmission, and specificity of transmission, was encoded in the RNA-2 segment of the nematode-transmitted virus genomes. The most exciting discoveries were made during the later 1990s early 2000s by several Ph.D., students e.g., Ton Ploeg, Nikon Vassilakos, Evangelis Vellios, Eirini Karanastasi, Konstantina Boutsaki, Rodanthi Holeva, Quing Chen, and Cleber Furlanetto at the Scottish Crop Research Institute. Individually and collectively in research teams, these researchers have revealed much of what we now know about the specificity of virus transmission by nematodes, the genetic determinants of virus and vector recognition and the distribution of viruses in plant roots. SWANSONG For the individual farmer the occurrence of a nematode-transmitted virus disease in a crop is of paramount importance. For example, in the UK, when “pick your own” raspberry and strawberry crops were being advocated as the potential financial saving of small farms, the occurrence of a virus transmitted disease in a crop situated in a “suitable” field, at least in part, resulted in the collapse of these family farm enterprises. Similar disease outbreaks around the world in fruit orchards, potato fields, and vineyards result in alternative crops having to be grown. Such crops, especially replacement crops in orchards and vineyards, very often have lower market returns resulting in an insidious, slow decline of the profitability of the farm enterprise, eventually leading to their financial collapse. Interestingly, the Golden Anniversary, 50 years, of the European Society of Nematologists coincided, within a couple of years, with the publication of the original report of a soil nematode being the natural vector of a plant virus. Today the Society and this research area have each fully matured. Unquestionably, during the last half century virus-vector nematodes, and in Europe potato cyst nematodes, have been among the foremost stimuli in plant nematology, presenting a plethora of research opportunities to the nematology community. With virus-vector nematodes most of the challenges have been successfully met and nematode taxonomy, systematics, biology, ecology, interactions, and control, have benefited greatly from the impetus throughout the general area of research. New methods, especially those continually being generated in molecular 166

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biology, provide exciting research opportunities for the global nematology community. Many intellectual and practical questions involving virus-vector nematodes, and their transmission of viruses, remain to be answered. These intriguing challenges are available to the current generation of nematologists who, if they accept these opportunities, may find that they also have embarked on a 32-year “love affair”. BIOGRAPHIC NOTE: – Derek Brown took early retirement from the Scottish Crop Research Institute in 2002 and relocated to the international, mountain ski resort of Bansko, Bulgaria where he, his wife June, and friends Vlada and Lyubomir Penev, operate a self-catering family hotel (www.penbro.com). Derek was appointed Deputy Rector of the College of Tourism in Bansko, where he holds a personal Chair in Rural, Village and Eco Tourism. He can be contacted at: [email protected].

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11. HORTICULTURAL HAZARDS: IN AND OUT OF HOT-WATER BATHS AND OTHER TRANSIENT TECHNOLOGIES SIMON R. GOWEN School of Agriculture, Policy and Development, The University of Reading, Earley Gate, Reading, UK

& PHILIP A. ROBERTS Department of Nematology, University of California, Riverside, California, USA

Introduction The 1950s and 1960s were important decades for the burgeoning of interest in nematodes and the opportunities for their management. Many nematologists were recruited to university and research station positions, a number of important meetings were held in the US, Caribbean and Central America and we saw the formation of SON in 1961 and ONTA in 1967. In 1968, a Caribbean Symposium on Nematodes of Tropical Crops was held at the University of the West Indies (UWI), Trinidad. This was jointly organised by UWI, the Commonwealth Development Corporation (which had interests in sugar cane, coconuts and bananas), the Commonwealth Institute of Helminthology (now part of CABI Bioscience), and FAO. The meeting brought together nearly 50 scientists including many of the leading nematologists from the USA, and several from the UK and other parts of Europe. The objectives of this meeting were to stimulate greater research effort in the management of nematodes of tropical crops and to promote teaching and training programmes in plant nematology. One of us (SRG) was to be a direct beneficiary; “I first met Nigel Hague at this meeting and it was he who encouraged me to do a higher degree in plant nematology (Fig. 1). Also present were Fred Jones and David Hooper, and it was through them that I received some specialist training at Rothamsted Experimental Station in England prior to 168

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undertaking a series of contracts in Jamaica, St Lucia and Ecuador for the UK Government’s Overseas Development Administration.”

Fig. 1. Nigel Hague and Simon Gowen at Reading in June, 2006.

The other of us (PAR) also benefited from specialist training at Rothamsted with Fred Jones, David Hooper, Alan Stone and colleagues, from where a career in nematology research and extension ensued in California. Using two contrasting examples, burrowing nematode on banana and stem and bulb nematode on garlic, we recount some of our experiences and insights on work to develop nematode management strategies and tactics for horticultural crops. Although the examples are as different as bananas and garlic, the underlying themes, experiences, and outcomes are remarkably similar, and we suspect they are much like other nematode-plant problems and their solutions in horticulture.

The burrowing nematode (Radopholus similis) and the banana The centenary of the description of the burrowing nematode, Radopholus similis [Tylenchus similis], by Cobb in 1893 was overlooked by nematological societies, a sad omission! Cobb’s material HORTICULTURAL HAZARDS

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was collected from some banana plants growing in gardens adjacent to Government House in Suva, Fiji. The same population was there when Al Taylor visited, in 1967.

Fig. 2. Uprooted banana plant and lesioned root illustrating the damage caused principally by the burrowing nematode, Radopholus similis.

Radopholus similis is thought to be indigenous to the western Pacific, and its pan-tropical distribution is probably a result of the movement of nematode-infested banana suckers from that region during this past millennium. Nematode infestations of bananas in the New World were recorded as early as 1910, by which time this fruit had become established as an export commodity. A Jamaica Department of Agriculture report mentions that a dreaded banana disease, thought to be caused by bacteria, was suspected to be due to “eel worm at the root”. The problem had also been reported in French West Africa before World War II. Not until the 1950s, did the disease known as “blackhead toppling” begin to be recognized as the major banana production constraint (Fig. 2). Two unrelated issues had contributed to this. 1. When the banana trade began in the latter part of the 19th century the industry was based upon one variety “Gros Michel” (Musa AAA). Unfortunately, this variety, although popular with 170

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consumers was highly susceptible to Panama wilt caused by the fungus, Fusarium oxysporum f. sp. cubense. This was devastating for plantation owners and smallholders. With no effective treatments to combat the disease the “industry” changed to the “Cavendish” varieties which were immune to the pathogen. What was not known at the time was that the Cavendish varieties (also Musa AAA) had less tolerance to R. similis than did Gros Michel. This change of variety was done over a relatively short period of time and neither propagation nurseries nor quarantine officials were aware that field-produced suckers were likely to be infested with nematodes. Thus the nematode problem became more wideFig. 3. A pit dug around a banana plant showing spread upon the adoption the distribution of roots and highlighting the technical problem from a nematode management of Cavendish as the perspective. export variety. The connection between the arrival of the nematode problem and the increase in the cultivation of Cavendish varieties has rarely been recognized.. 2. The period after World War II saw bananas return to the international export trade and the drive towards their more intensive production. As the losses from blackhead toppling became more serious greater attention was given to the cause, creating many careers in practical field nematology. Contemporaneous with this was the ascendancy of the agricultural chemical industry. The Shell Chemical Company and the Dow Chemical Company had developed soil fumigants that had been shown to be very efficient in controlling nematodes. One product, DBCP, HORTICULTURAL HAZARDS

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first described by C. W. McBeth and G. B. Bergeson in 1955, was non-phytotoxic and could be applied to established banana plants. DBCP became the standard field treatment wherever bananas were grown for export. The liquid formulation was applied at six points around each plant with special hand-operated injectors, not an easy or particularly pleasant task. At last a partial solution to this hitherto undiagnosed and poorly understood problem was available (Fig 3). In the Caribbean and Central America, the United Fruit Company and Standard Fruit Company and the Jamaica Banana Board (in association with the major chemical companies) led the research on banana nematodes. The research activities moved from the descriptive and taxonomic to the investigative and practical. For a long time these organisations had employed plant pathologists, and the textbooks Banana Diseases Including Plantain and Abaca by C.W. Wardlaw and Banana Plantain and Abaca Diseases by R.H. Stover were standard references for all banana researchers and provided useful descriptions of the nematode problems. Several nematologists, including A. Vilardebo, M. Luc and R. Guérout from France, J. Edmunds in the Windward Islands, D.I. Edwards in Central America, and P. Maas in Surinam were assisted by a handful of chemical company representatives in the development of fumigants and the newly discovered non-fumigants for the banana industry. It was recognized that treating established bananas in the field was not the only solution and that much of the problem was to do with the infection on the planting material. The blackhead-toppling disease was described by R. Leach (also a plant pathologist) in Nature 181: 204–205 (1958). At this time C.A. Loos and S.B. Loos were working for the banana companies, and in a series of papers highlighted the problem of blackhead disease and how it might be managed (1960, Phytopathology 50: 383–386; 1961, Plant Disease Reporter 45: 457–461). In Australia, C.D. Blake and R.C. Colbran were also developing and promoting different “seed” treatments for the banana farmers in New South Wales and Queensland. All of these scientists had concluded that longer-lasting control could only be achieved with treatments based on the concept of “clean seed”. The options were as follows: – Cut away the dark brown necrosis with a knife or machete and discard suckers with the severest necrosis (Fig. 4). 172

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Fig. 4. Nematode infested banana corms, formally the only method for propagation and the reason for the widespread distribution of Radopholus similis.

– Heat-treat banana suckers in hot water baths (a method first recommended by A. Mallemire in West Africa in 1939). – Dip suckers in a nematicide (DBCP) suspension. – Establish disease-free nurseries. These recommendations were sensible but tedious and quite difficult to manage and implement. Hot-water treatment was practiced on some banana estates but never became universally adopted. The logistics of the treatment were daunting. Each sucker weighs 12 kg and about 2,000 suckers are required to plant 1ha. The equipment needed for treating such volumes of plant tissue had to be robust and efficient to heat water tanks and maintain them at a temperature of 50 °C. The treatment was effective but did not eliminate all nematodes in the corm tissue and nematode population densities increased to damaging levels after a few crop cycles. Dipping in nematicide or coating suckers with mud impregnated with nematicide (a technique advocated by T. Mateille, P. Topart and P. Quénéhervé in the Ivory Coast) were also recommended. Eventually, regulations concerning availability or use of the emulsifiable concentrate formulations of these toxic products were tightened and the practice was discontinued. No matter how successful the treatments of planting material were they did not resolve the problem of re-planting “clean seed” in land that was already infested with R. similis. Although DBCP treatment was adopted by many banana growers and gave good nematode control with economic benefits, it had to be properly applied, which HORTICULTURAL HAZARDS

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required good field supervision. Thus, when the granular formulations of non-fumigant organophosphates and oxime carbamates became available, many growers preferred them because of their relative ease of application. The compounds cadusafos, carbofuran, ethoprophos, phenamiphos, oxamyl, and in some countries aldicarb, were used widely as granules sprinkled at 2–3 g a.i. around banana plants two or three times a year (Fogain & Gowen, S., 1997, Nematropica 27: 27–32). With some changes in their registration and formulation, such compounds continue to be used on commercial plantations to this day. However, registration of DBCP was withdrawn in 1977 following the reports of its carcinogenic properties and fumigants have ceased to be used for treating banana fields since that time. A.C. Tarjan (1961, Nematologica 6: 170–175) found that R. similis would disappear from soil if deprived of a host for 6–12 months. The question of other hosts, including weeds, was addressed by D.I. Edwards & E J. Wehunt (1971, Plant Disease Reporter 55: 415–418), by J. O’Bannon, and by those working with the R. similis population that was peculiar to the citrus growing region of central Florida. However, for dedicated banana farmers, the concept of leaving banana land free of bananas was not popular and such fallowing was rarely practiced. In addition, the task of removing all vestiges of corm and root from a field is time-consuming and the probability of leaving foci of nematodes in a field was always high. It is interesting to reflect that at this time most of the detailed studies on R. similis were done by the nematologists from Florida. Thus R. similis, the cause of spreading decline of citrus received much publicity and notoriety, occupying significant sections in nematology textbooks such as Victor Dropkin’s Introduction to Plant Nematology and Eli Cohn’s chapter in Economic Nematology. As a result, many surveys were conducted in countries wherever bananas and citrus were grown together. Radopholus similis was invariably found in the bananas but never in citrus roots. The R. similis on citrus in Florida, known for a while as R. citrophilus, remains an enigma (see Duncan, 2005, In: Luc, M., Sikora, R.A. & Bridge, J.(eds), Plant Parasitic Nematodes in Subtropical and Tropical Agriculture. 2nd edition, CABI, Wallingford, UK). By the 1970s, the opportunity to plant bananas in new land not hitherto used for bananas, and thus free of R. similis, was uncommon. The introduction of commercial bananas to Belize was one such example. Unfortunately, the companies developing this new area 174

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chose to use suckers sent from a neighbouring country with the recommendation to heat-treat before planting. If time had been spent in preparing a proper disease-free nursery the R. similis problem in Belize might have been avoided or delayed for many years – an example of managers failing to heed the advice of the scientists!

Fig. 5. Contemporary technique for production of banana plants by tissue culture. Careful attention is required when tissue-cultured plants are being hardened-off prior to planting.

The development of the Belize banana industry came just too soon for another technology that revolutionised banana propagation, and has made the different corm treatments, including hot water treatment, obsolete. Micro-propagation or tissue culture of banana meristems on defined media in sterile conditions was first demonstrated in Taiwan in the early 1970s. This has now become the standard technique for mass-producing banana plants and enables the movement of material free of major pests and diseases (some viruses excepted). Even in non-exporting countries such as Uganda, the use of tissue-cultured plants is becoming an accepted practice with some plant production businesses dedicated to this technology (Fig. 5). One-half of the problem with nematodes on bananas has been solved by micropropogation. There remains the task of controlling nematodes on the growing crop. In 2006, the only treatments used by commercial producers are those with the non-volatile nematiHORTICULTURAL HAZARDS

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cides. From a sustainability point of view this situation will become less acceptable as environmental and human health considerations become higher on the political agenda. In addition, commercial producers regularly spray their crops with fungicides to control the leaf spot disease Mycosphaerella fijiensis, an aggressive pathogen that has also spread throughout the banana producing regions. Molecular biology might provide an answer; H. Atkinson, at the University of Leeds, has demonstrated the possibility of the transgenic approach (Atkinson et al., 2004, Transgenic Research 13:135–142). If the popular cultivar Cavendish were to be modified by transformation with cystatin-forming constructs, some of the arguments used by objectors to genetic modification would not be relevant since Cavendish is sterile. However, current public opinion, at least in northern Europe, is resolutely against any form of genetic modification. In the future, some hard choices will have to be made on this issue. A frequently asked question is “are there any banana varieties with resistance and or tolerance to R. similis?” The short answer is no, but that is not quite true. Between 1970–72 I (SRG) worked with a banana breeding scheme in Jamaica where K. Shepherd had developed several disease-resistant tetraploid cultivars which also showed good tolerance to R. similis in the field, but were not resistant, and for different reasons were not considered suitable for the export banana trade (Gowen, 1979, Nematropica 9: 79–91). The nematologists with the banana breeding group at the United Fruit Company laboratory in Honduras (E.J. Wehunt, D.I. Edwards and J. Pinochet) also evaluated the material in their collection and found R. similis resistance in some of the diploid breeding lines (Pinochet & Rowe, 1979, Nematropica 9: 76–78). The most promising of these, a cultivar named “Pisang Jari Buaya” was eventually used as a parent in the breeding programme directed by Phil Rowe. By this time, the laboratory had been given to the Honduran Government and became the Fundación Hondurena de Investigación Agricola (FHIA). Some tetraploid hybrid varieties that Phil Rowe produced are now being field-tested, and in some countries in the tropics are being grown for local markets. Unfortunately, as with the new varieties from the Jamaican programme, the banana marketing companies do not consider the fruit of these nematode- and disease-tolerant varieties to be as good as that of the (nematode-susceptible) Cavendish varieties. 176

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Nematodes, particularly R. similis but also Helicotylenchus multicinctus, Pratylenchus coffeae and Meloidogyne spp. continue to be key pests wherever bananas are grown in the tropics (Gowen., et al., 2005, In: Luc, M., Sikora, R.A. & Bridge, J. (eds), Plant Parasitic Nematodes in Subtropical and Tropical Agriculture. 2nd edition, pp 611–643, CABI, Wallingford, UK). In the future, the system of intensive production of export fruit maintained with the use of nematicides might well have to change if consumers and regulatory authorities conclude that the practice of continuous re-application of nematicides to banana plantations is unacceptable. Not all banana varieties are as susceptible to nematodes as the Cavendish clones. Also, the new nematode-tolerant varieties have been bred for resistance to diseases and, if managed carefully, should not require fungicide treatment. The export banana industry has changed its preferred variety once before; there should be no reason why this cannot happen again, and if it means growing a diversity of cultivars so much the better as no industry should be dependent upon one genotype!

The stem and bulb nematode (Ditylenchus dipsaci) and garlic One usually associates garlic in hot water with those wonderful saliva-inducing aromas in the kitchen as soups and other savoury dishes are prepared, and not with factory-scale nematode killing on garlic “seed-cloves” being prepared for large-scale commercial plantings. Hence, it is little wonder that researchers and staff would come out of their labs and offices to investigate the source of pungent odors when the garlic hot water dip treatments were being made at the University of California (UC) Kearney Field Station each autumn during the 1980s. I (PAR) had arrived as a postdoctoral fellow at the UC Riverside campus from Rothamsted Experimental Station where my practical nematode experience was with the round cyst nematodes (Globodera spp.). Shortly thereafter, I was appointed as a Nematology Extension Specialist for California annual field and vegetable crops. At the time that my appointment was announced I was at a meeting of California nematologists that was being held at Riverside, and among the attendees was Dr. Bert Lear, a senior proHORTICULTURAL HAZARDS

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fessor at UC Davis (Fig. 6), and the Godfather of the hot water treatment for disinfecting garlic cloves containing Ditylenchus dipsaci. Dr. Lear announced that all the calls he had been receiving about how to control this nematode on garlic would now be directed to me, since I had instantly become the expert on control of all nematodes in crops grown annually. “Oh really”, I replied in trepidation about the honor that Bert was bestowing upon me. Of course, I knew absolutely nothing about garlic or stem and bulb nematode (apart from a lecture or two on this nematode from Howard Atkinson at the University of Leeds), and even less about the hotwater treatment regimes. Bert was true to his word; within a few weeks I was fielding calls about stem and bulb nematode on garlic, and as a result spent the next dozen or so years researching various approaches to controlling the problem.

Fig. 6. Dr. Bert Lear, University of California, Davis: a pioneer of hot-water treatment for garlic clove treatment, shown early in his career and later in life.

The stem and bulb nematode has been known on garlic in Europe since 1877; in The Nethelands it was referred to as kroefziekte and in France as maladie vermiculaire de l’oignon (Johnson & Roberts, 1994, In: Compendium of Onion and Garlic Diseases. H.F. Swartz & S.K. Mohan, (eds), American Phytopathological Society, APS Press, St. Paul, USA). In the United States, the disease was first recognized on onions from Canastota, New York, and is now found in many states including California. The nematode is a persistent but unpredictable problem in horticultural bulb crops, including onions and garlic, 178

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tulip and narcissus. Typical symptoms (Fig. 7) on infected onion and garlic plants include erratic stands, stunting, looping and bending of leaves below the soil surface, spikkel formation (swelling), and extensive longitudinal splitting of cotyledons and leaves (Newhall, 1943, Phytopathology 33: 61–69; Roberts, 2006, In: Compendium of Onion and Garlic Diseases. 2nd edition. H.F. Swartz and S.K. Mohan, (eds), American Phytopathological Society, APS Press, St. Paul, USA.). Leaves are short and thickened and frequently exhibit brown or yellowish spots and bloat (stem swelling). Infected seedlings Fig. 7. Close-up of garlic plants showing become twisted, enlarged, and symptoms of seed-borne infection by the stem deformed and, in severely and bulb nematode, Ditylenchus dipsaci, in a infested fields, die (Fig. 8). As California field. the season progresses, the foliage collapses and a softening of the bulb begins at the neck and gradually proceeds downward. Scales become soft and light gray. Symptoms

Fig. 8. View of garlic field symptom distribution caused by infection with the stem and bulb nematode, Ditylenchus dipsaci. A. Symptoms resulting from seed-borne infestation showing uniform distribution (left) compared with non-infested planting block (right); B. Patchy distribution resulting from planting non-infested cloves in a field with a focus of infestation from a previous planting. HORTICULTURAL HAZARDS

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occurring in infested garlic seed lots are often not apparent until mid-season when bulbs often become desiccated, shrunken, and low in weight. Infected bulbs often decay at the base due to the presence of secondary invaders such as bacteria, fungi, maggots, thrips larvae, bulb mites, and saprophagous nematodes (Fig. 9).

Fig. 9. Close-up of young garlic bulbs showing symptoms of decay at the base resulting from seed-borne infection by the stem and bulb nematode, Ditylenchus dipsaci.

Although D. dipsaci has a large number of host races, it can be managed effectively as a soil-borne problem by crop rotation. The onion-garlic race of D. dipsaci attacks onions, garlic, leeks, chives, shasta pea, parsley, celery, mints, lettuce, hairy nightshade, and salsify. Rotating garlic or onions with 4 years of non-host crops between plantings in infested fields will ensure that the subsequent Allium crop is not infested. Elimination of volunteer onions or garlic and host weeds ensures host-free rotations. The nematode can be spread in infested soil, debris from bulb processing and storage houses, and in other materials and on equipment. Hot water formaldehyde dip treatments were first developed for nematode and fungus disinfection of narcissus bulbs in Europe and the USA (Anonymous, 1967, Hot-water treatment of plant material. Bulletin 201. London: Her Majesty’s Stationary Office; Chitwood & Blanton, 1941, Journal of the Washington Academy of Science 31: 296–308; Hawker, 1944, Annals of Applied Biology 31: 31–33). Lear and Johnson adapted hot-water treatment for garlic and reported, in two papers in the 1960s, the protocols that they had developed and the improvements made by various temperature and exposure time treatments, together with the use of chemical additives to boost treatment efficacy (Lear & Johnson, 1962, Plant Disease Reporter 46: 635–639; Johnson & Lear, 1965, Plant Disease 180

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Reporter 49: 898–899). As with any heat treatment of live plant material, the key to successful hot-water treatment is to expose the nematode to a thermal death regime, but not to injure the “germ” potential of the planting stock. For D. dipsaci on garlic, exposure to 49° C for 20 minutes was enough to kill the nematode and not injure the garlic. However, so close are the thermal injury thresholds for plant and nematode that the hot-water treatment alone was not 100 % effective in eliminating the nematode infestation. Lear and Johnson experimented with additives to the hot-water dips and found that formaldehyde as an additive was effective in obtaining complete nematode kill. The standard commercial treatment for managing D. dipsaci on garlic was developed based on the results of the Lear and Johnson experiments. They had found with garlic cloves that temperature-time combinations of greater than 49° C for 16 minutes and 51.5° C for 4 minutes are lethal to D. dipsaci. They also found that garlic cloves can tolerate 50° C for 20 minutes and 49° C for as long as 25 minutes. To optimize this regimen, 49° C for 20 minutes that included 0.75% aqueous formaldehyde was found to give the best control. Furthermore, a pre-soak dip in water for 30 minutes at 38° C was found to activate dormant nematodes and optimize nematode eradication from seed-cloves. The excellent adaptation of D. dipsaci to survive extreme drying via its anhydrobiotic capacity and clumping behavior, especially in the fourth juvenile stage, makes the dehydrated nematode highly resistant to chemical toxicity, and thus emphasises the value of a warm pre-soak to hydrate and activate the nematode before the hot dip treatment. An additional beneficial component was to plunge the hot-dipped cloves into a cooling tank for 10 minutes at 18° C, to ensure protection of the garlic tissue from heat damage. Based on these studies and additional industrybased modifications for large scale dipping the following regimen was used for many years by the garlic industry: Seed cloves are separated from bulbs using rubber rollers (a process called cracking). The cloves are then placed in a warming dip of water for 30 minutes at 38° C followed by immersion in 0.74% aqueous formaldehyde for 20 minutes at 49° C, then 10 minutes in 0.06% benomyl at 18° C and air dried. The benomyl in the cooling stage is used for surface sterilization to minimise fungal contamination. The California garlic industry, which produces the majority of the US processed garlic (used for garlic flavoring of food HORTICULTURAL HAZARDS

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as garlic salt, garlic powder, or minced garlic) utilized this hot-water treatment of seed-cloves on a routine basis during the 1960s to the 1990s, when formaldehyde was banned from use due to its carcinogenic properties and hence risk to worker health. During this period, two extensive research efforts were undertaken by one of us (PAR) with support from the garlic industry (at that time organized under the banner “American Dehydrated Onion and Garlic Association”, or ADOGA). During the 1980s, considerable interest in nematology was focused on the development and implementation of nematode control programs using plant and soil treatments with non-fumigant nematicides of both organophosphate and carbamate chemical classes. The chemical companies pressed hard with these products as potential replacements for the soil fumigants such as EDB and DBCP that were banned because of human health and environmental contamination concerns. While numerous nematologists worldwide investigated the potential of aldicarb (Temik), phenamiphos (Nemacur), ethroprop (Mocap), carbofuran (Furadan), and oxamyl (Vydate) treatments to control most of the important nematode problems on a wide range of agronomic and horticultural crops, in California, we examined their potential for control of D. dipsaci on garlic. Several other researchers working with bulb crops in Europe, such as Nigel Hague and Alan Whitehead in the UK (1979, Plant Pathology 28: 86–90; 1979, Annals of Applied Biology 93: 213–220) and William Haglund and Harold Jensen in the USA (1983, Journal of Nematology 15: 92–96; 1983, Plant Disease 67: 43–44), had shown that several of these nematicides were effective in controlling soil-borne nematode problems on bulb crops. However, we were interested in determining whether applications at planting time could successfully control “seed-borne” infestations of D. dipsaci on garlic, as an alternative to the hot-water formaldehyde dip treatments. In the US, federal and state agencies were already scrutinizing industrial uses of formaldehyde and the commercial-scale dip treatments were complex and difficult to maintain. We summarized results from a series of nine field experiments in which granular formulations of the non-fumigants were applied at planting directly onto garlic seed cloves in the seed furrow to assess efficacy for control of D. dipsaci infested cloves (Roberts & Greathead, 1986, Journal of Nematology 18: 66–73). The treatments were compared with the standard hot-water formaldehyde dip and to non-treated controls. As with many results reported 182

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in these types of non-fumigant nematicide comparisons, we found a mixed performance of the compounds. Aldicarb and phenamiphos at 2.52 and 5.04 kg a.i./ha, but not at lower rates, were highly effective and recommended as alternatives to the hot-water/formaldehyde dip. The other nematicides were either phytotoxic (carbofuran and ethoprop) or failed to provide adequate control (fensulfothion, oxamyl). As an outcome of this applied research, phenamiphos (Nemacur 15G) was subsequently registered for this use on garlic by Bayer Corporation in the mid-1980s, while Union Carbide Co., producer of aldicarb (Temik) at that time, determined that the market potential for registration on garlic was too small. Nemacur was not used much by the garlic growers and did not replace the use of the clovedip treatment. Many nematologists could recount similar stories about researching the potential of these non-fumigant nematicides for various nematodes on various crops, and although some important commercial treatments were developed and used, we are seeing today the demise of these compounds due to human and environmental health issues. Most such products have disappeared and/or their registered use on crops has been reduced. At the time of writing, a 2006 nematology meeting in California was informed that Temik use on cotton remains the only primary use of non-fumigant nematicides on California crops, and that use is targeted for early season insect control. A second research effort was undertaken, beginning in the late 1980s and early 1990s, in which we assessed options for modifying the hot-water formaldehyde dip treatment. During this time formaldehyde was banned from use in this and other industrial-scale processes. The garlic growers were deeply concerned about maintaining clean planting stock. We conducted a series of twelve field experiments in which various temperature and time regimes were compared using water without additives and with sodium hypochlorite or abamectin as additives (Fig. 10) (Roberts & Matthews, 1995, Journal of Nematology 27: 448–456). We re-affirmed that the differential between thermal tolerances of D. dipsaci and garlic cloves was too small to allow any effective disinfection regime based on hot-water alone. However, we discovered that abamectin at 10–20 ppm in the cool dip following a water hotdip of 49° C for 20 minutes was very effective, as was sodium hypochlorite at 1.1–3.3% aqueous solution as the 20-minute hot dip. These regimes were recommended to the HORTICULTURAL HAZARDS

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Fig. 10. Experimental hot-water treatment for Ditylenchus dipsaci disinfection of garlic cloves. A. Hot-water treatment tanks, equipped with thermostat and stirrer. B. Sack of garlic cloves being loaded into tank; C. Sacks of treated cloves air-drying before planting.

industry as viable alternatives to the formaldehyde dip treatments. As far as we know, these alternatives were never adopted in any broad scale for garlic industry use. Instead, the industry then moved toward a system of producing nematode-free seed-clove stock. Production of nematode-free, seed garlic cloves is achieved by meristem tip culture, followed by greenhouse, screenhouse, and isolated production in non-infested soil coupled with hot-water treatment of cloves. Once nematode-free seed garlic cloves are available, an effective regimen combines periodic testing for nematodes (California has an official state seed certification process for nematode-free seed-clove garlic) and planting for seed increase on fields that have not been planted to host Allium crops for at least 5 years. Adapting well-known techniques to specific situations for nematode management in horticultural crops is a recurring theme. An example related to the garlic nematode problem is the use of hot-water treatment to disinfect strawberry of foliar nematode, Aphelenchoides fragariae. Hot-water treatment of dormant strawberry crowns to be used for planting has been available for about 70 years (Christie & Crossman, 1935, Proceedings of the Helminthological Society of Washington 2: 98–103; Hodson, 1934, Journal of the Ministry of Agriculture 40: 1153–1161), but a survey of the literature revealed a large variation in recommended exposure periods and temperatures and differences in sensitivity among A. fragariae populations (Qui et al., 1993, Suppl. Journal of Nematology 25(4S): 795–799). Becky Westerdahl and colleagues at U.C. Davis pursued interest from the California strawberry industry to refine the hotwater treatment to manage this problem for California growers. They assessed various time and temperature regimes on five common strawberry cultivars, and determined minimum-maximum 184

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exposure periods that killed A. fragariae without reducing subsequent plant growth and flowering to be 20–30 minutes at 44.4° C, 10–15 minutes at 46.1° C, or 8–10 minutes at 47.7° C (Qui et al., 1993, Suppl. Journal of Nematology 25(4S): 795–799). This work was completed sixteen years ago. Becky Westerdahl has just informed us that the California strawberry growers, who have not used the new information and refined procedures developed for nematode management, had just called her and said they are now ready, in 2006, to implement and utilize the technology. So one can never know when a research investment will pay off, only that new knowledge gained and recorded always retains potential value for future application. We certainly live with nematodes as a chronic, persistent problem in horticulture that will always require management approaches. What did we learn from these experiences? Hindsight suggests that we should have developed the nematode-free, seed-clove stock program for garlic in the first instance, instead of the time-consuming and expensive applied research effort over two decades on chemical and water-bath treatments for nematode eradication or control. The same could be said for the foliar nematode problem in strawberry. We worked with non-fumigant nematicides because they were readily available, a “hot commodity” in applied nematology research, and with plenty of funding and “in-kind” support from the chemical companies. We worked with the hot-dip treatments because the industry was equipped to utilize the technology, because with D. dipsaci hot-water without additives “nearly did the job” and effective adjustments were feasible, as our results proved. But how quickly society’s perspective has changed over what is safe or not as a process in the workplace or as an application in the food production chain. Sodium hypochlorite (common bleach) is found and used in almost every household. Nevertheless, the garlic industry became concerned about using large volumes of heated, dilute bleach for seed clove treatment. Compared with an effective, nematode free, seed production program, we would have to agree. At first glance, it all seems to have been a waste of time and effort. However, as nematologists, we learned in detail (as did the garlic industry) about the stem and bulb nematode problem in garlic, about the infection process, the biology of the nematode, the role played by secondary infections by other organisms of nematode infected plant tissues, the efficacy of different nematicide treatments, and how to utilize nematicides. We also learned how to HORTICULTURAL HAZARDS

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grow a high quality crop of garlic, and developed a deep appreciation for garlic in our lives as a food additive. Who knows when the knowledge gained from such studies will find relevance and application to this or related problems under different circumstances in the future, as demonstrated by the strawberry growers?

Summary Whether the nematode problem is on leaves, stems, bulbs, corms or roots, and the affected crop is narcissus, bananas, garlic or strawberries, nematologists are needed to help the grower find a workable solution. In a world in which pest management strategies need to be based upon a more conceptual understanding of pests and their complex relationships with plant hosts and other organisms, the horticultural industry requires specialist practitioners with greater field expertise. Unfortunately, today we are training fewer nematologists and applied researchers: as more research effort is devoted to cell and molecular biology. Whilst the new technologies have advanced our understanding of species diagnosis and some elegant solutions to seemingly difficult or intractable problems have been proposed there is a great danger in forsaking the practical and applied skills. We hope that any molecular solutions will be as effective as hot-water solutions and not any more transient.

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12. THE SPREAD OF NEMATOLOGY TO DEVELOPING COUNTRIES: A CASE STUDY MICHEL LUC Formerly, Muséum National d’Histoire Naturelle, Paris, France

Until approximately 1950, plant nematology had been, almost exclusively, a subject of interest to the developed countries of Europe and America, and had received very little attention in tropical countries (with some notable exceptions: Jobert, Göldi, Meloidogyne, coffee, Brazil; Nowell, Bursaphelenchus (Rhadinaphelenchus) cocophilus, coconut, Caribbean area; Linford & Oliviera, Rotylenchulus reniformis, pineapple, Hawaii; Butler, Ditylenchus angustus, rice, Bangladesh). A possible reason for this may have been that the other plant pests of the tropics (particularly insects and fungi) were so dramatic in their effects compared to nematodes, which rarely produce specific, above-ground symptoms, that nematodes were just not noticed! However, as soon as some far-sighted people began to explore the possibility of nematode damage in the tropics, the real importance of nematodes there began to be revealed. These pioneering studies were usually made in countries that had previously been colonies, and were made by scientists from the former colonial powers. The following account by Michel Luc illustrates how the first scientists were, rather haphazardly, charged with the task of exploring tropical nematology. As in Luc’s case, the scientists in question were often not originally specialists in nematology, but needed to be re-trained in the subject, or even learn the subject in the field!

The birth of nematology in the Ivory Coast (Côte d’Ivoire) I had been working for two years as a plant pathologist at the main research station of ORSTOM (Office de la Recherche THE SPREAD OF NEMATOLOGY TO DEVELOPING COUNTRIES

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Scientifique et Technique Outre-Mer), in Adiopodoumé, near Abidjan, Ivory Coast, when, in May 1953, the Director of ORSTOM (Raoul Combes, a plant physiologist and Professor at the Sorbonne, Paris) asked me to change my career direction and to become a plant nematologist. That decision stemmed from the development of banana cultivation in the Ivory Coast and French Guinea for which nematodes were suspected to be an important limiting factor. After having received some “international” training in the UK (Rothamsted, J.B. Goodey), Belgium (Ghent, L.A.P. de Coninck), The Netherlands (Wageningen, M. Oostenbrink and J.W. Seinhorst), France (Lyon: V. Nigon; Versailles, M. Ritter) I returned to the Ivory Coast in early 1955 with a simple mission defined by the Director: “Go to Adiopodoumé, establish a lab of Nematology and decide, on the spot, what needs to be done.” So my research career in nematology was opened in a quasi-virgin land (only nine references on plant parasitic nematodes for all of West Africa including the ex Belgian Congo!). During the initial stage, I established a list of the materials (primarily, good quality microscopes), chemicals etc. that were considered necessary as the basic equipment of a nematology lab. I also obtained corresponding pro-forma invoices for these items. When, however, I requested permission to buy all that material, the answer from the Director of ORSTOM was very simple: “ORSTOM has no money for that. Your activity will be relevant to agronomy, so the money has to come from SARA”. An explanation is needed here: as the financial support for the Adiopodoumé station by its administration, ORSTOM, was not sufficient, the Director of the station, Professor G. Mangenot (also an eminent botanist at the Sorbonne), obtained from the governor of the Ivory Coast (a French colony at that time) permission to establish a national administrative group called SARA (Section Autonome de Recherche Agricole) managed directly by Professor Mangenot and receiving a comfortable amount of money from the colony’s funds. So I met Professor Mangenot at the Sorbonne (he spent 6 months of every year in Paris, and 6 months in the Ivory Coast) and presented him my list of materials. His reply was: “I am awfully sorry, but nobody has informed me that you must become a nematologist, so I have no funds at all for you”. I was so upset at the thought of having to return to the Ivory Coast to create a new lab in a new subject without a penny that I became severely ill (with jaundice, for the 188

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first and only time). When recovered, I flew to the Ivory Coast in a very pessimistic frame of mind. Fortunately, my plant pathologist colleagues helped me and I could begin the first surveys. The results were very exciting as a great number of the records were of new species (but this is outside the present subject area). Professor Mangenot had also, in the meantime, returned to the Ivory Coast and was facing an important problem. He had discovered a new plant in the primary forest of the Ivory Coast, a small Moraceae of the genus Dorstenia. However, in the greenhouses of the station, all the plants became stunted, the leaves became yellow, the stems dried and the plants died. All plant pathologists, entomologists, plant physiologists, soil scientists, agronomists etc. were mobilized to examine the problem but they did not detect a probable cause, and the plants continued to die (fortunately, after having produced seeds in some cases). So, I took my turn at examining the problem. I stained the root system and – miracle! – I could observe small nematodes fixed on the roots as well as kinds of small black galls containing females, eggs, juveniles and males of the same nematode. I identified it as a second species of Tylenchulus (it is now an Ivotylenchulus species). Professor Mangenot had some doubts about the effect of “such a small animal”, but I demonstrated to him, by using surface sterilised seed placed in autoclaved soil, plus re-infestation on some lots (the usual nematological methodology), that the nematode was, in fact, the cause of the observed stunting. Some time later Professor Mangenot said to me: “If my memory does not fail me, you presented me in Paris a list of materials you need in order to develop your lab. Please resubmit the list to me and I’ll see what I can do. I have some funds that could, perhaps be used for that purpose”. I gave him the list and a few days later he said to me: “Well, finally I have funds enough to accept all the items on your list”. Some months later I received many of the requested materials, most importantly an up-to-date Zeiss microscope. After that unexpected good fortune I had no greater supporter than Professor Mangenot and I had no, or few, problems in buying materials, recruiting local and French assistants, or building glasshouses and a new laboratory. He also facilitated the recruitment of new nematologists, and provided funds to travel for missions outside the Ivory Coast and to attend various meetings. The description (Luc, 1957, Nematologica 2:329-334) of THE SPREAD OF NEMATOLOGY TO DEVELOPING COUNTRIES

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Tylenchulus mangenoti (= Ivotylenchulus mangenoti) – that was my thanks to the Director – should have been my first nematological publication. Actually it was the second one as I had to wait for the description of the host plant! Thanks are due to that “small animal” for effectively provoking the development of plant nematology in West Africa.

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13. CONTRIBUTIONS BY LATIN AMERICAN NEMATOLOGISTS TO THE STUDY OF NEMATODE PLANT DISORDERS AND RELATED IMPACT ON CROP PRODUCTION R.H. MANZANILLA-LÓPEZ1, P. QUÉNÉHERVÉ2, J.A. BRITO3, R. GIBLIN-DAVIS4, J. FRANCO5, J. ROMÁN6 AND R.N. INSERRA7 1 Plant Nematode Interactions Unit, Rothamsted Research, Harpenden, Herts, UK; 2IRD, Laboratoire de Nématologie Tropicale, PRAM, BP 8006, 97259 Fort-de-France, Martinique, France; 3,7Florida Department of Agriculture and Consumer Services, Nematology Section, Gainesville, Florida,USA; 4 University of Florida/IFAS,FLREC, 2305 College Avenue, Davie, Florida, USA; 5 Fundación para la Promoción e Investigación de Productos Andinos, IBTA-CIP-COTESU, Casilla 4285, Cochabamba, Bolivia; 6 Agricultural Experiment Station, Crop Protection Department, P.O. Box 21360, Rio Piedras, Puerto Rico.

A brief history of plant nematology in Latin America and the Caribbean Islands The majority of Latin American and Caribbean countries can be generally characterized as having tropical climates. However, the topography of these countries creates a great variety of climatic condition that result in the growth of tropical, sub-tropical and temperate crops that are favored in most areas by an abundance of rainfall. These different climates favor also the development and establishment of a great diversity of nematode pests that have different temperature and plant host requirements. The major nematological problems afflicting the agriculture of these countries are caused by both indigenous nematode species on native plant crops (such as cultivated Solanaceae) and by exotic species that were introduced with new crops. Exotic nematode pests pose serious problems to Latin American agriculture. These pests include the root-knot nematode, Meloidogyne ethiopica, which may have arrived during the slave trade between Africa and the New World, and the burrowing CONTRIBUTIONS BY LATIN AMERICAN NEMATOLOGISTS

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Fig. 1. G. Steiner

Fig. 2. A. Ayala

Fig. 3. J. Romàn

Fig. 4. L. G. E. Lordello

Fig. 5. From left to right, back row: P. Lax, J. Franco, M. Doucet, G. Cap, E. Chaves. Front row, C. Gallardo, E. Lorenzo and R. H. Manzanilla-López.

nematode, Radopholus similis, introduced with mixed banana genomes from the Far East by early Spanish and Portuguese immigrants. The recent introduction of the soybean cyst nematode, Heterodera glycines, into Argentina and Brazil illustrates the continuing importance of efforts to prevent the importation of exotic species into Latin America. The exchange of crops and their pests between the Americas and Europe caused also the inadvertent movement of native nematode plant pests from Latin America into Europe where some, such as the potato cyst nematodes, have become established as major pests. As a consequence of these introductions, many exotic nematode species from Latin America were described and studied in European countries long before they were reported and studied in their areas of geographic origin. 192

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In the past, a lack of advanced agricultural research in Latin America resulted in an inadequate knowledge of nematological plant problems. The first report of a plant parasitic nematode from Latin America may have been that of C. Jobert, in 1878 in France, of a root-knot nematode infecting coffee in Brazil. Later, root-knot nematodes were reported on coffee in Mexico by G. Gándara (1906) and on coffee and sugarcane in Puerto Rico by G.L. Fawcett (1915) and J. Matz (1925) (Román, 1978). The re-description of R. similis by N. A. Cobb, in 1915, used a nematode population from Jamaica. Nematological research in Latin America received a strong impetus in the second half of the twentieth century from scientific and financial assistance by European and North American countries. One of the founding fathers of plant nematology in the United States, G. Steiner (Fig. 1), promoted nematological studies in Puerto Rico where he worked and cooperated with L. F. Martorell in training the first young nematologists from Puerto Rico, A. Ayala and J. Román (Figs 2, 3). L. G. E. Lordello (Fig. 4), the founder of plant nematology in Brazil, also was trained by Steiner. Investigations into nematode plant pests were conducted in Peru in the early 1960s by A. Martin and continued by J. Franco (see Fig. 5) and G. Gomez under the guidance of nematologists, such as J. Sasser and W. Mai from the United States. Soon after, nematological studies were initiated by M. Costilla (Fig. 6) in Argentina, J. Franco in Bolivia, A. Valenzuela and E. Dagnino in Chile, R. Barriga in Colombia, M. Jiménez in Costa Rica, F. Pineda in Cuba, L. Gullón in Dominican Republic, J. Escobar (Fig. 7) in Ecuador, L. Abrego (Fig. 8) in El Salvador, A. Kermarrec in Guadeloupe and Martinique, C. Sosa-Moss (Fig. 9) in Mexico, R. Tarté (see Fig. 10) in Panama, A. Martin and C. Bazán in Peru, J. Edmunds (Fig. 11) in St. Lucia, and F. Dao (Fig. 12) in Venezuela amongst others. Many of these pioneer nematologists faced not only the inevitable scientific challenges, but also a language barrier that isolated them from much of the main stream of nematological research. In most Latin American countries, nematology laboratories were developed from scratch by self-taught nematologists who bought and translated books (in some countries the only library of nematology is the one accumulated by the nematology teacher) and trained new staff members. Several phytopathologists, such as R. H. Stover, involved in management of banana diseases at the United Fruit Company and CONTRIBUTIONS BY LATIN AMERICAN NEMATOLOGISTS

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Fig. 6. M. Costilla

Fig. 7. J. Escobar

Fig. 8. L. Abrego (left)

Fig. 9. C. Sosa-Moss

Fig. 10. Participants in the Planning Conference: International Potato Center headquarters in Lima, Peru, 1978

its subsidiaries, promoted nematological studies in Central American countries, especially in Costa Rica, Honduras and Panama. A growing awareness of nematological problems on crops and of the dramatic yield increases obtained by the application of chemical nematicides were major forces in the development of nematological projects in Latin America. Chemical companies found new markets for their nematicides. Private and government funds became available to Latin American scientists for nematological training in universities and in nematology research institutes in Europe and North America. Scientific societies were established to promote nematological studies in Latin America. The Organization of Nematologists of Tropical America (ONTA) was founded by Ayala and Román, in 1967, with the initial aim of fostering cooperation between nematologists in Latin America and those in the United States. This organi194

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Fig. 11. From left to right: A. Ayala, J. Edmunds and J. Román

Fig. 12. F. Dao and J. A. Meredith

Fig. 14. J.C. Magunacelaya

Fig. 15. J. Esquivel

Fig. 13. E. Aballay

zation has received great support from nematologists in the United States and Europe. The contribution provided by A.C. Tarjan and R. Rodríguez-Kábana in promoting ONTA’s scientific activity and cohesiveness played a pivotal role in enabling this emerging Society to obtain international scientific recognition. The Brazilian Society of Nematologists (SBN) was founded by Lordello, in 1974, to promote nematological research in Brazil. Under Lordello’s leadership, the number of nematologists in Brazil increased dramatically and SBN became the most important and largest nematological society in South America. Other nematological societies were established later in Mexico and Peru. A cooperative agreement between the Venezuelan and Dutch governments provided funding and experts to teach plant nematology in Venezuela in the 1970s. Many nematologists from Latin America attended these international courses and received training in Venezuela. This initiative led to the estabCONTRIBUTIONS BY LATIN AMERICAN NEMATOLOGISTS

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Fig. 16. From left: F. Franco, K. Evans, I. Cid del Prado (fourth in row) and J. Cristóbal (sixth in row) with growers.

Fig. 17. From left to right, back row: T. Powers, A. Ciancio, R. N. Inserra, R. H. Manzanilla-López, S. Hockland. Middle row, M. Rodríguez, M. Mundo-Ocampo, J. Rowe, N. Marbán-Mendoza. Front row, B. Tello and Z. Handoo.

Fig. 18. M. Canto-Sáenz

Fig. 19. R. Crozzoli

lishment of excellent nematological facilities in Maracay and enabled the Central University of Venezuela to offer graduate level courses in nematology. Nematologists such as M. Doucet (see Fig. 5) and S. del Toro (Argentina), E. Aballay and J.C. Magunacelaya (Figs 13, 14) (Chile), A.R. Monteiro and S. Ferraz (Brazil), J. Escobar and Carmen Triviño (Ecuador), R. López-Chavez and J. Esquivel (Fig. 15) (Costa Rica), F. de la Jara, I. Cid del Prado (see Fig. 16) and N. Marbán-Mendoza (see Fig. 17) (Mexico), M. Canto-Sáenz (Fig. 18) (Peru), J. Román (Puerto Rico) J.A. Meredith (Fig. 12), who was succeeded by R. Crozzoli (Fig. 19) (Venezuela) and many others played an important role in nematological training and research. The history of plant nematology in Mexico is typical of the development of the science in other Latin American countries. The 196

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establishment and development of plant nematology in Mexico has been affected by the political and economic events faced by that country from 1906 until today (Montes-Belmont, 2000). C. SosaMoss was the promoter of plant nematology in Mexico, and his teaching and research have received international recognition. The teaching programs and research were favored by international cooperation with European and North American universities and by the oil boom that started in the late 1970s. A funding shortfall for nematological research occurred in the 1990s, but interest in nematological studies has renewed as a result of international trade agreements that have affected the phytosanitary regulations and Mexican agropolicies in recent years. Graduate and postgraduate courses in plant nematology have been offered at the University of Chapingo and Postgraduates” College since 1967. Similarly, nematological teaching activities were promoted at the National Agricultural School, La Molina, in Peru and eventually in other universities in countries throughout the region. The increase in production and marketing of crops such as soybean, vegetables, citrus, grapes and other fruit crops that has occurred in recent years in Latin America has favored nematological research and teaching programs in the region. The remainder of this chapter highlights aspects of research on some of the most important phytonematological problems in Latin America and of the contributions by the scientists involved in these studies.

Nematode problems on bananas in Latin America and Caribbean Islands Latin America and the Caribbean Islands supply about 80% of the world banana trade. Most exports are based on the triploid dessert bananas, mostly of the Cavendish subgroup. These are all minor variants of one genotype, and there is no other major fruit or vegetable that depends solely on one variety. In the 1960s, the triploid dessert bananas completely replaced the cultivar, Gros Michel, which was extremely susceptible to fusarial wilt. However, the replacement Cavendish variety is more susceptible to the burrowing nematode, R. similis. Research on the effects of nematodes on Musa spp. production in Latin America and the Caribbean began as long CONTRIBUTIONS BY LATIN AMERICAN NEMATOLOGISTS

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ago as 1870 when bananas were first cultivated for export to North America (Champion, 1968). Today, there is a marked decrease in nematological research in tropical American and Caribbean Musa producing countries due to the reduction in the number of nematologists, the shortage of funds for research and the lack of encouragement to train new nematologists. Ironically, these recent shortages in nematological training and research are happening in a period of great need due to the drastic changes in the nematode-banana management system caused by increased concern for environmental quality (product, soil, water) and human health related to the use of chemical nematicides, as well as to the withdrawal from use of non-fumigant nematicides and the absence of effective alternatives (e.g., biological control). 1. Main nematode species Ashby (1915), in Jamaica, was the first to describe burrowing nematode symptoms in banana rhizomes as “Black-head disease of bananas”. The burrowing nematode in Jamaica, initially identified as Tylenchus similis by N. A. Cobb and later reclassified as Radopholus similis, was subsequently found, in 1939, in the French West Indies and in other banana growing areas of Central America and the Caribbean. Other nematode pests of banana, such as Pratylenchus coffeae, Helicotylenchus multicinctus and Meloidogyne spp. are of less economic importance. However, there is increasing occurrence in Brazil and French West Indies of Meloidogyne spp. parasitizing banana vitroplants soon after planting in areas where competition with the burrowing nematode is low. 2. Biology, damage and economics In a series of leading publications on the life cycle and histology, Clive Loos described the root symptoms and pathology of R. similis in banana roots. Subsequently, the difference in pathogenicity among R. similis isolates was extensively studied in Central America and the Caribbean. This research was aimed at explaining the discrepancies observed in R. similis damage worldwide in terms of yield loss, plantation longevity and nematode management efficacy. Until recently, it was thought that R. similis had two races, one of which was non-pathogenic to citrus (R. similis) and another, considered to be a sibling species (R. citrophilus), pathogenic to both citrus and banana. Recent research, however, does not support the validity of this “sibling” species. 198

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The interaction between the burrowing nematode and the fungus Fusarium oxysporum cf cubense was important in the expression of Panama disease, and had devastating effects on the cultivar Gros Michel in banana plantations in Central America and the Caribbean. Research on this interaction led to the replacement of Gros Michel with cultivars from the Cavendish subgroup, which are resistant to Fusarium, but susceptible to R. similis. The role of plant hosts other than bananas in spreading and maintaining nematode populations in new plantations was investigated extensively for regulatory reasons and as a prerequisite for nematode management. The importance of the burrowing nematode as a widespread cause of banana losses was first reported by Leach (1958) in Jamaica. Since then, crop losses have been estimated in the different producing countries on the basis of yield improvement after nematicide treatment. These yield responses varied greatly from 15 to 275%. Such differences are due to several factors including soil type, plant physiology and climate. Damage can vary from a hidden lengthening of the vegetative period to the most obvious symptom of attack by R. similis – toppling over of the entire banana plant. Tropical storms and hurricanes are especially prevalent in the Caribbean and in Central America and result in much greater numbers of uprooted plants when compared with other banana producing areas of the world. At present, the percentage of necrotic roots combined with nematode enumeration is the basis of most banana nematode monitoring in Latin America. 3. Management measures The golden era of nematicides (1960–1990) The implementation of good phytosanitation practices and the use of clean propagative material in non-infested land have been recommended since the 1960s. Peeling and steam/hot-water treatments of rhizomes has been emphasized as the method to sanitize infected banana rhizomes. However, applications of fumigant nematicides such as 1,2-dibromo-3-chloropropane (DBCP) as dip treatments to sanitize infected rhizomes or as injection treatments in the soil around the infected banana plants became a common management practice for nematodes in banana plantations. Non-fumigant, mostly systemic, nematicides (organophosphates and carbamates), were also successful as post-planting treatments. CONTRIBUTIONS BY LATIN AMERICAN NEMATOLOGISTS

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The application of non-fumigant nematicides still remains the most widely used control method in Latin America with granular or liquid nematicides applied through a sure-fill system and hand-held applicators to ensure safe applications. In the past, these treatments were mostly applied on a calendar basis, but are now done after monitoring by banana plant uprooting and/or the enumeration of nematodes in the roots. Threshold levels vary from place to place (from 4,000 to 6,000 nematodes per 100g of banana roots in some plantations in Costa Rica to only 1,000 per 100g of roots in Martinique) reflecting regional differences in R. similis pathogenicity and also cultivation practices (see Gowen et al., 2005). Resistant cultivars Development of resistant cultivars has been a research priority in Latin America and the Caribbean with many scientists involved. Unfortunately, resistance to the burrowing nematode in many banana cultivars is difficult to incorporate without non-desirable traits, thereby resulting in practical difficulties in breeding programs. Alternatives to chemical control During the last decade, several factors have influenced changes in management of banana nematodes e.g., loss of important non-fumigant nematicides, absence of effective alternatives (biological control) to nematicides and increased concern relating to nematicides, for environmental quality (product, soil, water) and human health. The repercussion of these changes was even more acute in the replant crop systems of the Caribbean (due to a lack of clean propagative material and clean land) than in the large plantations of Latin America where bananas are grown continuously without replanting. As a consequence, research on alternatives to chemical treatments has been more intense in the Caribbean. The efforts have concentrated on replant practices with vitroplants on cleaned soils. This concept was known for a long time, but its application was feasible only after disease-free vitroplants from meristem culture became available. The suppression of nematode populations when replanting old banana plantations poses difficult challenges. Many cultural practices were attempted in the Caribbean in order to free the soil from R. similis. They include bare or weeded fallow and rotations with Pangola grass, Sudan grass, or with crops such as sugarcane and pineapple. However, some of these cultural practices 200

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were not implemented because of the high cost of planting and maintaining the rotation crop along with an inability to develop markets for the rotation crops. Some improvement in decreasing nematode populations was achieved by chemical destruction of infected banana plant rhizome and root tissue in the soil. The implementation of this practice not only extended the longevity of the banana plantations in the French Antilles, but also greatly decreased (by 63 % from 1996 to 2004) the application of nematicides (see Gowen et al., 2005). 4. Future prospects During the past 50 years, many (and perhaps the most important) advances in our understanding of banana nematodes and their management have been obtained through research in the laboratories of the United Fruit Co. in La Lima, Honduras and in the research plots of the Banana Board of Jamaica at Bodles. However, due to the emigration of the nematologists, shortage of support funds for basic research and failure to train new nematologists, the focus of research has moved from centers in one country to another. Presently, most of the research is now done in Costa Rica and the French Antilles. The golden era of nematicides is definitely behind us. Development of banana varieties that are not only resistant to black Sigatoka, but also to the burrowing and lesions nematodes, is the next major step. 5. Contributors to the study of nematode banana disorders Clive Loos conducted his studies on R. similis on banana while working at the United Fruit Research Laboratories, La Lima, Honduras. Loos, together with his wife Sarah, also worked with the United Fruit Company in Panama and with the Banana Board in Kingston, Jamaica. Loos’s studies emphasized the importance of using clean banana rhizomes to avoid the dissemination of the nematode. Unfortunately, these preventative approaches to exclude the nematode from banana plantations were too late or poorly implemented at the time, and the nematode was widely disseminated with infected banana rhizomes throughout the major banana producing areas of Central America. Jesse Román conducted research on R. similis on banana in Puerto Rico for many years. After obtaining a Ph.D. in nematology under the supervision of Hirschmann at North Carolina State University, he worked as a nematologist at the Agricultural CONTRIBUTIONS BY LATIN AMERICAN NEMATOLOGISTS

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Experiment Station in San Juan, Puerto Rico. Román studied R. similis pathotypes of cooking banana (plantain) and the banana plantations of Puerto Rico, with emphasis on the genetic characterization of the banana race of this nematode. The results of his studies, in 1985, indicated that both R. similis citrus and banana races have five chromosomes rather than five or four. His data were debated among nematologists because they did not support the elevation of the citrus race to species level. Today his findings are congruent with the results of molecular and mating studies indicating that both the citrus and the banana race are the same species. Many young nematologists from the United States initiated their career in the late 1960s and early 1970s working on R. similis pathotypes and chemical management in banana plantations of the United Fruit Company in Honduras, Costa Rica and Panama. W. G. Wehunt (Fig. 20) and D. I. Edwards found a great variability in the host preferences of banana race populations. Wehunt became a research nematologist at the USDA Experiment Station in Byron, Georgia and later in Arkansas. Q. Holdeman studied the host range and races of the burrowing nematode in both Central America and in California where he was senior nematologist at the California Department of Food and Agriculture in Sacramento. R. A. Dunn, Extension Nematologist at the University of Florida, Gainesville, started his career in Costa Rica working on nematode pests of banana. The English nematologist, S.R. Gowen, worked for WINBAN on banana nematodes in both the Windward Islands and in Ecuador, in the 1970s. He supervised many local students working on nematode control and resistance in bananas, and has published extensively on his observations. G. Pinochet also worked for many years at the United Fruit Company, Division of Tropical Research in Panama on nematode resistance and control before retiring in Spain. There has been a very significant French contribution in Latin America to the study of banana disorders caused by nematodes. A. Vilardebó, who was located at IRFA (CIRAD) in Montpellier, France, conducted biological and management studies in the French West Indies since the early 1970s. Today, his studies are continued by P. Quénéhervé (Fig. 21) who aims to implement non-chemical management approaches for banana production in the French West Indies, and is currently working on sources of resistance to banana nematodes. This French nematologist acquired experience in tropical Nematology at IRD (ex ORSTOM) in West Africa and moved 202

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Fig. 20. E. J. Wehunt

Fig. 21. P. Quénéhervé

Fig. 22. D. W. Fenwick

Fig. 23. R. Griffith

Fig. 23. R. Griffith

Fig. 25. E. A. Goeldi

from West Africa (Ivory Coast) to Martinique in the early 1990s. During the last decade, biological and management studies on banana nematodes have been conducted in Costa Rica at the National Banana Corporation (CORBANA) and Chiquita Brands by nematologists, such as M. Araya and G. Fallas, amongst others, who specialize in banana diseases.

Coconut palm disorder caused by the red ring nematode Biology, parasitism and damage The red ring disease of coconut has been reported only in tropical Latin America and the Lesser Antilles. This disorder, which also affects other palms such as African oil and date palms, is caused by CONTRIBUTIONS BY LATIN AMERICAN NEMATOLOGISTS

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the red ring nematode, Bursaphelenchus (= Rhadinaphelenchus) cocophilus and usually involves an insect vector, the palm weevil Rhynchophorus palmarum. The palm weevil transports and deposits B. cocophilus juveniles into feeding wounds it causes in the palm leaf axils. Subsequently, the nematodes colonize palm stem tissues causing the decline of infected palms. Premature yellowing and senescence of leaves and a distinct band of orangish lesions, appearing as a red ring in cross sections of the parenchymal stem tissues, are characteristic symptoms of this disease in coconut palms (Griffith et al., 2005). Nematode feeding activity causes serious vascular damage to coconut and oil palms, which are stunted, unproductive and eventually killed by the infection. It is not clear if the association of the red ring disease and their causal agents originated on native oil palms in the neotropics and moved onto the coconut palms introduced from South-east Asia and oil palms introduced from Africa, or developed as a disease complex in the neotropics around the turn of the century. Direct damage induced by the nematode and associated weevil results in annual crops losses of 10–15% for the coconut and oil palm industry in the neotropics. This disorder has also an aesthetic impact by seriously affecting the landscape industry. The red ring nematode and the palm weevil are regulated by many countries to protect their coconut industry (South-east Asia) or their landscape palms (USA) from these pests. Management Phytosanitary measures aimed at reducing weevil populations and other sources of nematode infection are the best method to reduce the incidence of red ring disease. Early removal of nematode infected palms followed by herbicide treatment of the stem is a common practice in affected coconut plantations. Applications of insecticides are necessary if weevils are present in the culled palms. The use of systemic chemical nematicides can effectively cure palms infected by B. cocophilus as these chemicals suppress nematode population levels and thereby assist palm recovery. New management approaches based on mass-trapping weevils with sugarcane and a synthetic aggregation pheromone are very effective in reducing the number of weevil vectors. Updated information on red ring disease of coconut and oil palms are provided by a recent review of this nematode palm disorder by Griffith et al. (2005). 204

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Latin American contributors to the study of the red ring disease Early studies on this palm disease were conducted by non-Latin American Nematologists such as N. A. Cobb and D. W. Fenwick (Fig. 22). However, C. Salazar, a Latin American phytopathologist reported this disease in Venezuela in 1934. Fenwick was a British nematologist working at the Field Station of the London School of Hygiene and Tropical Medicine (1938) and later at Rothamsted Experimental Station (1945–1958) in England. He subsequently changed his research interests to tropical nematology in Trinidad and Tobago, West Indies and became Director of Red Ring Research, at Trinidad and Tobago Coconut Research Limited in Trinidad. Fenwick’s traditional plant parasitic nematology background led him to suggest that the red ring disease of palms was a soil borne disorder. According to Fenwick, experimental root colonization supported his view. However, he also emphasized the role of the weevil in vectoring and spreading the nematode and the disease. This had originally been suggested by Cobb and Nowell in 1919. After Fenwick’s retirement, these studies were continued by several scientists in Trinidad, including G. Blair and R. Griffith. Their work did not support Fenwick’s theory and Griffith provided convincing evidence that the disease was mainly vectored by the weevil rather than it being soil borne. R. Griffith (Fig. 23) was educated in Trinidad where he graduated from the Imperial College of Tropical Agriculture in Trinidad (1960). He later expanded his scientific knowledge and training by obtaining an MS degree from the University of Wisconsin and a Ph.D. from the World University Round Table. His studies on coconut diseases made him an international expert on palm diseases and led to his appointment as Director of Red Ring Research, Coconut Research, Ministry of Food Production, in Centeno, Trinidad. Griffith promoted studies on red ring disease through cooperative research with scientists from other countries. The contributions of K. Gerber to the studies of weevil vectors of the red ring nematode are noteworthy. Gerber (Fig. 24), a nematologist from Austria, spent almost 2 years in Trinidad and other Latin American countries working on this subject. She adapted very well to the Caribbean life style, but experienced health problems induced by tropical diseases such as, dengue fever. She is now enjoying her retirement in Austria. In the 1990s, C. Chinchilla made important contributions to our understanding of the chemical ecology of R. palmarum in Costa CONTRIBUTIONS BY LATIN AMERICAN NEMATOLOGISTS

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Rican African oil palm plantations that have led to methodologies for mass trapping of the red ring nematode vector that can be implemented in concert with aggressive phytosanitation. Nematode coffee disorders Lesion and root-knot nematodes are the most common nematode pests of coffee and occur in almost all coffee growing areas in Latin America. Their damaging effects are influenced by environmental conditions. Sandy soils are more conducive to root-knot nematode infections than heavy soils, which are tolerated by lesion nematodes. Lesion nematodes Pratylenchus brachyurus, P. coffeae and P. gutierrezi are the most common lesion nematodes infecting coffee in Latin America. Other undescribed damaging species closely related to P. gutierrezi also occur. Symptoms, damage and management Coffee roots infected by lesion nematodes are yellowish or brown in color and decay rapidly. The infected plants are chlorotic and stunted. These symptoms may result in coffee seedling mortality in nurseries and in tree decline followed by premature death. Poor bean quality is another adverse effect of lesion nematode infections. The use of non-fumigant nematicides is effective in suppressing lesion nematode populations and the incidence of plant mortality. However, non-chemical management strategies, including the use of coffee resistant rootstocks (Coffea canefora cv. Robusta) are the best management approaches for long lasting protection of coffee plantations from lesion nematodes (see Campos and Villain, 2005). Root-knot nematodes The early studies on root-knot nematode pests of coffee were initiated by European nematologists. As mentioned in section 1, Jobert found, for the first time, these pests infecting coffee in Brazil. A few years later (1887), E. A. Goeldi (1859–1917) (Fig. 25), a Swiss zoologist and naturalist, who was working as a visiting scientist in the National Museum in Rio de Janeiro, Brazil confirmed the observations published by Jobert. The results of his investigation allowed him to correlate the root-knot nematode with the serious coffee decline that was occurring in plantations in the Province of Rio de 206

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Janeiro. In his report, published in 1892 in the National Museum Archives (Arquivos do Museu Nacional) volume 8, Goeldi emphasized the potential menace of this organism to the Brazilian coffee industry. Goeldi studied the morphology of the root-knot nematode populations infecting coffee, which he described as a new species, Meloidogyne exigua. This species, the coffee root-knot nematode, became the most widely spread root-knot nematode in the coffee plantations of Latin America because it was probably distributed with nematode infected transplants. Of the 17 species of root-knot nematode found infecting coffee worldwide, nine have been reported in Latin America: M. arabicida; M. arenaria; M. coffeicola; M. exigua; M. hapla; M. incognita; M. inornata; M. javanica; and M. paranaensis. The root symptoms observed in infected coffee roots vary with the species. Root swellings (galls) are consistent symptoms induced by M. exigua infections, but inconspicuous galls, peeling, cracking of the root cortex and destruction of the feeder roots are commonly observed in roots infected by M. arabicida, M. coffeicola, M. incognita and M. paranaensis. These four species are considered to be the most damaging ones on coffee in Latin America. They debilitate and kill coffee trees and have a devastating impact on coffee plantations. Yield losses are very severe (20% or more) with high mortality of planting material in nematode infested nurseries. The indirect financial losses caused by nematode damage to the coffee industry is discussed by Campos and Villain (2005). The management of root-knot nematodes on coffee relies on: i) phytosanitation and production of certified propagative plant material to avoid the spread of these pests in non-infested land, ii) chemical approaches and iii) non-chemical approaches such as resistant varieties and rootstocks, rotation and soil amendments with organic matter. Coffea arabica lines resistant to root-knot nematodes have been successfully selected in Brazil and provide a very useful tool for managing these pests (Campos and Villain, 2005). Contributions of Latin American nematologists to the study of nematode pests of coffee L.G.E. Lordello (1926–2002) was the “Father of Brazilian Nematology”. He initiated his research on nematode pests of coffee and described a new species of root-knot nematode, Meloidogyne coffeicola, which was found causing severe damage in coffee plantaCONTRIBUTIONS BY LATIN AMERICAN NEMATOLOGISTS

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tions in the states of Paraná and São Paulo. Lordello also described severe coffee decline caused by M. incognita in coffee plantations in São Paulo, Espirito Santo and Paraná State, and provided evidence of the damage caused by the lesion nematode, P. brachyurus, to coffee. These early studies elucidated the important role of nematodes in the decline of coffee plantations and fostered research projects that were carried out by several Latin American nematologists. The pioneering work that Lordello conducted on nematode pests of coffee reflects his biological knowledge and background. He was educated in Brazil where he obtained a Ph.D. in zoology from the University of São Paulo, Escola Superior de Agricultura “Luiz de Queiroz“ (ESALQ), Piracicaba, São Paulo, where he was already conducting research and teaching as professor in the Department of Zoology. In spite of his reluctance to travel, he went to the United States to be trained by Steiner. In the early 1960s, Lordello established the first course in nematology in Brazil, which he taught at both undergraduate and graduate level. Lordello worked extensively on nematodes of agricultural relevance, including many projects on root-knot nematodes on coffee. Lordello was also a well respected taxonomist describing approximately 50 new taxa. A. Jaehn (Fig. 26), who was trained by Lordello, dedicated the majority of his short scientific career to developing methods for management of root-knot nematode on coffee in the state of São Paulo, which is one of the largest coffee producing states in Brazil. Jaehn became well known for his work on cover crops, organic matter and pesticides as a means to manage root-knot nematode in coffee nurseries and plantations. Jaehn died prematurely from an incurable disease. The molecular and morphological characterization of root-knot nematode pests of coffee in Latin America has played an important role in elucidating the taxonomic status of root-knot nematode populations occurring on coffee. This work has been conducted almost exclusively by R. M. D. G. Carneiro (Fig. 27), another of Lordello’s students. Her studies, concerning the genetic variability of root-knot nematode species on coffee, led to the description of two new root-knot nematode species infecting coffee, M. izalcoensis found originally on coffee in El Salvador and M. paranaensis in Paraná, Brazil. This latter species had originally been referred to as M. incognita biotype IAPAR by R. Gomes Carneiro, another Brazilian nematologist involved in the management of root-knot nematodes on coffee. 208

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Currently, M. paranaensis is the most pathogenic root-knot nematode in coffee plantations in Brazil, the second most important being M. incognita. Additional morphological work on root-knot nematodes using SEM has been Fig. 26. A. Jaehn Fig. 27. R. M. D. G. Carneiro conducted by J. M. dos Santos (Fig. 28). He teaches courses at the graduate level, and is involved in management studies of root-knot nematodes in coffee plantations including biological, chemical, organic and cultural approaches. Important inforFig. 28. J. M. dos Santos Fig. 29. V. P. Campos mation on the effect of biological control agents such as Pasteuria penetrans, Arthrobotrys spp., Paecilomyces lilacinus, and Verticillium chlamydosporium (= Pochonia chlamydosporia), and cultural and chemical methods to manage root-knot nematodes on coffee have been provided by V. P. Campos (Fig. 29). The search for coffee rootstocks resistant to root-knot nematodes has been successful in Latin America. This cooperative work between nematologists and coffee breeders (W. Gonçalves and L. C. Fazuoli) in Brazil resulted in the selection of the rootstock, Apoatã IAC 2258 (C. canephora), which is resistant to M. exigua, M. incognita (some races) and M. paranaensis. Other resistant rootstocks were selected in Central America, such as the rootstock Nemaya that is resistant to M. exigua and M. incognita. The Mexican nematologist, N. Marbán-Mendoza, was involved in these selection studies that were planned by the nematologist, F. Anzueto. The early contribuCONTRIBUTIONS BY LATIN AMERICAN NEMATOLOGISTS

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tions (1974) of L. Abrego on the biology and management of coffee lesion nematodes in El Salvador are now continued by the French nematologist, L. Villain, who clarified the species composition of coffee lesion nematodes and their role in inducing coffee decline in Central America.

Potato disorders caused by nematodes in Latin America The potato crop and potato cyst nematodes Potato (Solanum tuberosum) is a very important crop in Latin America not only because of the area of land dedicated to its cultivation, but also because of its nutritional properties. It is native to the Andean regions of South America where the Incas cultivated it some 2000 years ago. Some wild potato varieties are native to North America (USA and Mexico) and Central America (SIAP, 2002). The potato tuber was called “Popotl” by the Nahoas of Mexico and it is now known as “papa”. It was introduced to Europe by the Spaniards. From 1600–1845, for example, potatoes were the staple food in Ireland and Irish migrants took it to the USA in 1719. Many countries of the old world adopted the crop as a staple food because of its high yield and nutritional value. Potato production has increased in Latin America at an average annual rate of 2.2% for the last three decades. Cultivation has expanded annually in Ecuador (3.0%), Peru (2.0%) and Brazil (1.0%). However, increased area of cultivation does not necessarily equate to increased yield, especially in areas where growth is poor and yield is low. Latin America is the only region of developing countries with a commercial deficit (307, 000 tons) in potatoes. This deficit is partly explained by the importation of seed potatoes to meet regional requirements. However, a significant improvement in productivity could increase competitiveness in the fresh potato market. At present, potato production tends to be concentrated in areas of higher productivity where surplus tubers can be sold. Andean countries such as Bolivia and Peru have traditionally produced potatoes for local consumption, but production is becoming increasingly linked to market forces and commercialization. Annual potato production in the Andean region is estimated as 7.8 million tons from an area of 640,000 ha. 210

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Commercial production in Mexico occurs all year around in the highlands (2000–3000 m above sea level), but especially in spring and summer. The cultivated area is approximately 67,000 ha with a yield of 1,635,000 metric tons (SIAP, 2002) and an average yield of 20.5 tons/ha. Production located in irrigated valleys (high input agriculture) occupies 17,000 ha, while that in rainfed valleys where subsistence farming is practiced occupies 50,000 ha. In Latin America, highland potato cultivation is by slash and burn of forests, the consequences of these practices include loss of diversity and genetic erosion, expansion of the agricultural frontier, low yield (4 tons/ha in Bolivia vs 20 on irrigated fields), and land being disqualified for seed potato production. In Latin American countries, including Mexico, major limitations to crop production include limited use of certified seed of genetic and phytosanitary quality, the many pests and diseases, and poor practices in production processes. Globodera rostochiensis and Meloidogyne spp. are among the most important phytopathological problems for the Mexican potato industry whereas G. rostochiensis, G. pallida and Nacobbus aberrans sensu lato are the major limiting factors for the Andean region of South America. Two major international bodies have been created to address and solve these problems: CIP (International Potato Center, in Peru) fosters scientific research on edible tuber crops and their pathogens and pests in the Andes; whilst PRECODEPA (Programa Regional Cooperativo de Papa) aims to improve production and technology for the potato industry in member countries (Central America, Caribbean and Mexico). Potato cyst nematodes Most Latin American countries are located between latitude 30N and 60S. Although considered tropical, subtropical highlands and mountain ranges provide these countries with the conditions to grow temperate crops such as potato. Latin America is the center of origin of major crops, including potato (Solanum spp.), tomato (Lycopersicon spp.), chilli pepper (Capsicum spp.) and maize (Zea mays). It is likely that a combination of geography, climate and vegetation have allowed many species of cyst nematode to exist and thrive at levels that can cause damage to crops (Sosa-Moss, 1987). The potato cyst nematodes are believed to have co-evolved with potatoes in the Andean region of Peru and/or Bolivia. Brucher CONTRIBUTIONS BY LATIN AMERICAN NEMATOLOGISTS

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(1960), however, suggested that they originated in the mountains of North Argentina, where the pest occurs, apparently naturally, in inaccessible places (CIP, 1978). Globodera rostochiensis and G. pallida are the most common cyst nematodes in Latin American potato growing areas. Records for G. rostochiensis began in 1952 in Peru. In 1971, Sosa-Moss identified G. rostochiensis in Guanajuato, Mexico (Iverson, 1972). Ironically, these introductions to Latin America probably originated from The Netherlands rather than from the Andean regions of South America. Globodera rostochiensis and G. pallida occur in potato growing areas in the Andean region from Venezuela to Chile. They occur also in Costa Rica and Panama (Central America). A historical account of the records of Globodera can be found in Sosa-Moss (1987). Along with G. rostochiensis on potatoes, species such as G. solanacearum, and G. virginiae coexist with other native populations of Globodera that are similar to G. rostochiensis and are found on native potato species and on other Solanaceae in Mexico. Many of these native nematode species remain undescribed, including Heterodera mexicana (= G. mexicana), a species inquirenda reported by Campos Vela in 1967. Native Globodera species have specific host ranges and are of great economic and regulatory importance to Mexican agriculture as they are potentially as damaging as the golden cyst nematode (R1A pathoype), which causes an average of 50% crop loss in heavily infested fields. In spite of the presence of many Globodera species, however, G. pallida has not yet been found in Mexico. Potato cyst nematode infection adversely affects plant growth, production and tuber quality in all potato growing areas of Latin America where these nematodes occur. Economic losses caused to the potato industry in Bolivia by G. rostochiensis and G. pallida average US $ 13 million annually (Franco et al., 1998/1999). Studies on the geographical distribution, species identification, life cycle and pathogenicity of these potato pests are routinely conducted in South America. Molecular identification of these nematodes is now used for regulatory purposes. Control strategies in Mexico include the use of nematicides, crop rotation e.g. with barley (Hordeum vulgare), broad bean (Vicia faba), butter bean (Phaseolus lunatus), corn (Zea mays), spring and hairy vetch (V. sativa and V. villosa), together with the use of improved potato varieties (e.g. Alpha, Atlantis, Atzimba). However, 212

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these varieties are not nematode resistant. Potential use of native resistant or tolerant potatoes in order to develop commercial resistant varieties has been suggested (Sosa-Moss, 1987). Evaluation of genetic material provided by the International Potato Center, rotation with non-host crops, organic amendments and trap crops are all management approaches adopted in South America. False root-knot nematode or potato rosary nematode The false-root knot or rosary nematode, Nacobbus aberrans, is a serious pest of potato and other crops in the Andean region of South America. The results of molecular studies of nematode populations from different geographical regions of Latin America support the hypothesis that N. aberrans s.l. is a species complex (Anthoine and Mugniéry, 2005; Reid et al., 2003). Populations of this pest infecting potato occur in Mexico, but their distribution is limited. In Mexico, this species is a common and important pest of other crops, including chilli pepper, tomato, and beans. Nacobbus bolivianus is found in Bolivia and potato pathotypes of N. aberrans s.l. have a wide distribution in the Andean region, including Argentina, Bolivia, Chile, Ecuador and Peru. Many different aspects of nematode biology, parasitism and integrated crop management have been studied (at least in a preliminary fashion) in Latin America (Manzanilla-López et al., 2002). In the Andean region this nematode is the main limiting factor to potato production. The motile stages are able to infect potato tubers and are disseminated with infected potato seed. The lack of certified seed in many Andean countries has resulted in dissemination of this pest throughout the majority of the potato growing areas of South America. Quality and yield of potato tubers are seriously affected and the value of crop losses in South America averages US $ 53 million annually. These figures are the results of long-term field studies conducted in the high elevation potato growing areas of Bolivia. No resistant potato varieties have been bred, although native, cultivated potatoes (Gendarme) are partially resistant to certain Bolivian populations of Nacobbus (known as rosary potato nematode). Development of varieties resistant or tolerant to the false root-knot nematode is a major objective of management studies in South America since a chemical approach is not feasible in the subsistence potato growing areas of the Andes. Crop rotations are difficult to implement because of the wide host range of this pest. CONTRIBUTIONS BY LATIN AMERICAN NEMATOLOGISTS

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Implementation of sanitation practices for the production of certified seed potatoes free of Nacobbus is another aspect of management research being undertaken. Several control strategies including thermo-therapy, solarization, cultural management, burning of root debris, manipulation of sowing dates, organic amendments (compost), green and animal manures (e.g. chicken manure 7–10 t/ha), crop rotation, trap crops, integrated crop management and, more recently, biological control have been used. More details can be found in Brodie et al. (1993) and Ortuño et al. (2005). Despite the acknowledged importance of Nacobbus in economic nematology, it remains a Latin American problem, as it is only in that region that it causes significant yield loss in staple crops such as potato (up to 80%). Other affected crops include tomato (50-90%), and beans (35%), the former being of high export value. Despite the progress achieved so far in understanding this nematode’s biology, parasitic strategies, ecology and taxonomical problems, an effective management program is needed and, hopefully with international collaboration, this will be achieved. Latin American contributors to the study of potato disorders caused by nematodes Many Latin American nematologists have been involved in studies concerning potato cyst nematodes. C. Sosa-Moss discovered and identified the golden nematode in Mexico. He promoted cooperative studies concerning the cyst forming nematodes parasitizing solanaceous plants in Mexico with the participation of M. Luc, L. Miller, D. Mugniéry, R. Mulvey and A. Stone. His academic accomplishments left an indelible imprint on the Autonomous University of Chapingo and the Postgraduates” College. Important studies on potato cyst nematodes were started in Peru in the La Molina Experimental Station, by A. Martin in 1964. These studies were continued by J. Franco, M. Scurrah (see Fig. 10) and M. Canto-Sáenz who elucidated the biology of these nematodes, and selected potato varieties resistant to these pests, at the International Potato Center, Lima. These three Peruvian scientists obtained their Ph.D. in American and British Universities. They are still actively involved in teaching and conducting excellent research in Bolivia and Peru. J.A. Meredith, a nematologist from the United States, spent almost 20 years in Venezuela where she taught nematology at the 214

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Central University of Venezuela and studied, together with F. Dao, the response and tolerance of Venezuelan populations of the golden nematode to elevated soil temperatures. Since her retirement, her work has been continued by R. Crozzoli. In Chile, I. Moreno, in collaboration with European nematologists, is assessing potato responses to Chilean populations of G. rostochiensis and G. pallida. In Panama and Ecuador surveys of potato cyst nematodes were conducted by R. Tarté and J. Revelo, respectively. Studies on N. aberrans s.l. populations infecting potatoes have been conducted in Argentina, Bolivia, Mexico and Peru. In Argentina, M. Costilla investigated extensively the parasitism of N. aberrans s.l. on potato. He made available useful techniques for the extraction of this nematode from soil and potato tubers. Costilla dedicated his life to nematological studies and teaching. He died prematurely shortly after providing a valuable contribution to a cooperative study on N. aberrans s.l. that was published by Manzanilla-López et al. (2002). The applied work of Costilla on N. aberrans s.l. was expanded by M. Doucet and P. Lax (Fig. 5) (University of Cordoba, Argentina) who conducted studies on the molecular and morphological characterization of Argentinean populations of N. aberrans s.l. Additional work was done by G. Cap and E. Chaves (Fig. 5). In Bolivia, J. Franco is involved in applied and basic research on Nacobbus. His research studies were facilitated by the experience he acquired working on potato cyst nematodes in Peru. Franco moved to Bolivia in 1989, and is conducting N. aberrans s.l. management studies in open field and field plots at very high elevations in the Bolivian Andes. In Mexico, the work conducted on Nacobbus by R.H. ManzanillaLópez (Fig. 5) in cooperation with other nematologists, such as I. Cid del Prado-Vera, J. Cristóbal and E. Franco (Postgraduates” College), J. Rowe (see Fig. 17) and K. Evans (Rothamsted) (see Fig. 16), involved several vegetable crops, including potatoes. However, infestations of N. aberrans s.l. on potato in Mexico are less frequent and not economically important in comparison with those in the Andean Region. Her molecular work with A.P. Reid and D. J. Hunt (CABI) has emphasized the importance of understanding the relationship and taxonomic status of Nacobbus s.l. populations in Latin America. She is a native of Mexico but currently lives in England and works at Rothamsted Research. CONTRIBUTIONS BY LATIN AMERICAN NEMATOLOGISTS

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In Peru, P. Jatala (see Fig. 10) studied the biology and biotypes of N. aberrans s.l. He was the first to consider this nematode a species complex. Jatala was born in Iran. He obtained a Ph.D. in nematology at Oregon State University under the supervision of H. Jensen. He moved to Peru in the 1970s where he worked and trained students at the International Potato Center.

Concluding remarks The spectrum of this review has been limited to some of the more important nematological problems that are characteristic of Latin America and Caribbean Islands. There are many other nematological problems important to agriculture in Latin America and the Caribbean that are not mentioned in this chapter. This region is afflicted by serious crop losses caused by root-knot nematode species due to its warm climate. However, Ditylenchus dipsaci is also a major agricultural pest, but in the cooler regions and at higher elevations. Indigenous nematode species such as Punctodera chalcoensis and Thecavermiculatus andinus damage indigenous crops including maize and oca (Oxalis tuberosa). In recent years, fruit crops, grapes, vegetables, and ornamentals have become major Latin American export crops. They are damaged by other plant nematode species and many of the nematologists listed above are also involved in studying these nematological problems. Many phytopathologists, such as G. Múnera Uribe, M. Pizano and F. Varón in Colombia conduct integrated management programs that include nematodes. The contributions provided by Latin American scientists have had a great impact in developing and increasing the production of export crops which, in turn, has greatly benefitted the economy of this region. In spite of recent progress made in some sectors of Latin American agriculture, basic yield production is still below that obtained in European and North American countries and nematode damage to staple food crops and vegetables remains very high in many areas. The banning of certain nematicides due to regulatory and environmental concerns makes the chemical management of nematodes more difficult and opens new challenges to scientists. Basic research is still needed to provide alternative management strategies to the chemical control of nematodes and to ensure safe and high quality products. However, this need cannot be fully met 216

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by the nematologists who are working today in Latin America because their number and research funding have decreased, as has the awareness of growers and politicians of the impact of these pests on intensive, monoculture systems. The decrease in research funding and the lack of replacement of retiring nematologists will have a negative effect on the production of tropical crops such as bananas, pineapple and coffee which will not continue to be marketed at today’s relatively low prices. Training a new generation of nematologists capable of using new technologies for the identification and management of nematode pests in Latin America is not an easy task. However, we are confident that Latin american universities and research agencies in collaboration with nematologists from more technologically advanced countries can achieve this goal.

Acknowledgements Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.

Selected references ANTHOINE, G. & MUGNIÉRY, D. 2005. Nematology 7: 503–516. BRODIE, B.B., EVANS, K. & FRANCO, J. 1993. In: Plant Parasitic Nematodes in Temperate Agriculture. K. Evans, D. Trudgill and J. M. Webster (eds). CAB International, Wallingford, UK, pp. 87–132. BRUCHER, H. 1960. Naturwissenshaften 47: 21. CAMPOS VELA, A. 1967. Taxonomy, life and host range of Heterodera mexicana. Ph.D. Thesis. University of Wisconsin, USA, 65 pp. CAMPOS, V.P. AND VILLAIN, L. 2005. In: Plant Parasitic Nematodes in Subtropical and Tropical Agriculture. M. Luc, R.A. Sikora, J. Bridge (eds.). 2ndedition. CAB International, Wallingford, UK, pp. 529–579. CHAMPION, J. 1968. El Plátano. R. Cote, (ed.) Colección Agricultura Tropical. Editorial Blume, Barcelona, Spain, 247 pp. CIP.1978. Developments in the control of nematode pests of potato. Report of the 2nd nematode planning conference 1978. Lima, Peru, 193 pp. FRANCO, J., RAMOS, J., OROS, R., MAIN, G. & ORTUÑO, N. 1998/1999. Revista Latinoamericana de la Papa 11: 40–66.

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GOWEN, S., QUÉNÉHERVÉ, P. A. & FOGAIN, R. 2005. In: Plant Parasitic Nematodes in Subtropical and Tropical Agriculture, M. Luc, R.A. Sikora, J. Bridge, (eds). 2nd edition. CAB International, Wallingford, UK, pp. 611–643. GRIFFITH, R., GIBLIN-DAVIS, R., KOSHY, P.K. & SOSAMMA, V.K. 2005. In : Plant Parasitic Nematodes in Subtropical and Tropical Agriculture. M. Luc, R.A. Sikora, J. Bridge, (eds). 2nd edition. CAB International, Wallingford, UK, pp. 493–627. IVERSON, L.G. 1972. American Potato Journal 49: 281. MANZANILLA-LÓPEZ, R.H., COSTILLA, M.A., DOUCET, M., FRANCO, J., INSERRA, R.N., LEHMAN, P.S., CID DEL PRADO-VERA, I. & EVANS, K. 2002. Nematropica 32: 149–227. MONTES-BELMONT, R. 2000. Nematología vegetal en México (investigación documental). Sociedad Mexicana de Fitopatología. Ciudad Obregón, Sonora, México, 98 pp. ORTUÑO, N., FRANCO, J. RAMOS, J., OROS, R. MAIN, G. & MONTECINOS, R. 2005. Desarrollo del manejo integrado del nematodo rosario de la papa. Fundación PROINPA-Proyecto PAPA ANDINA. Documento de trabajo No. 26. Cochabamba, Bolivia, 124 pp. REID, A.P., MANZANILLA-LÓPEZ, R.H. & HUNT, D.J. 2003. Nematology 5: 441–451. ROMÁN, J. 1978. Fitonematología Tropical. Universidad de Puerto Rico, Recinto Universitario de Mayagüez, Colegio de Ciencias Agrícolas, Estación Experimental Agrícola, Río Piedras, Puerto Rico, 256 pp. SIAP 2002. Panorama mundial de la papa. 11 pp. [http://www.siap.sagarpa.gob.mx/InfOMer/analisis/Anpapa.htlm]. SOSA-MOSS, C. 1987. Nematologia Mediterranea 15: 1-12.

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14. QUARANTINE NEMATODES DAVID MCNAMARA Formerly: East Malling Research Station, UK and European and Mediterranean Plant Protection Organization, Paris, France.

Introduction The major stages in the history of quarantine nematodes are the same as those of other types of quarantine pests, that is, a sequence of 1) ignorance of the dangers of transferring plant pests from one region of the world to an other, followed by 2) recognition of the dangers (usually resulting from some major problem caused by an introduced pest), then 3) early, and largely ineffective, attempts by national authorities to prevent further introductions, and finally 4) international cooperation to exchange information on quarantine pests and methods to prevent spread. The story of potato cyst nematodes is probably the earliest and most dramatic example of what can happen if nematodes are transported from their area of original distribution to another part of the world. When potatoes were carried from South America to Europe in the 17th century, to provide a cheap and nourishing staple food, they carried with them the seeds (in a figurative sense) of their own potential destruction: they were infested with one or more species of Globodera. At that time, of course, the people who transported them knew almost nothing of nematodes or other minute pests, and they certainly did not recognise that the potatoes that they brought with such hope to Europe were infested with Globodera spp. which would become established in certain countries and would soon spread to virtually every European country. This resulted, eventually, in it becoming increasingly difficult to grow adequate amounts of potatoes. The financial cost to Europe, for the period between the 17th century and the present day, of the introduction of Globodera spp. is incalculable. Certainly it has been an enormous amount, taking into account the loss of potential yield, both quantitative and qualitative, QUARANTINE NEMATODES

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the cost of research into the cause of yield loss and into possible control measures, the cost of applying chemical nematicides, the cost of internationally co-ordinated management strategies, and the loss of potential foreign markets for potatoes and other agricultural produce that could possibly carry the nematodes to other, as-yet, un-infested areas. The lesson that was learnt from the history of Globodera spp. and from other similar pests was: – that plants and/or soil, when transported by human activity from one region of the world to another, can very easily, and unnoticed, carry pests such as nematodes and cause their longterm establishment in the new region; – that it is virtually impossible to eradicate an introduced pest after it has become established and spread; – that these exotic pests can have a devastating impact in the new region, even more so than in their area of origin; – that, without a knowledge of pests, their geographic distribution, their biology and host range, it is impossible to prevent their spread to other regions, short of stopping all international trade in plants or other commodities that could possibly carry the pests.

Early quarantine legislation Stimulated by several other extremely damaging pest introductions (e.g. Phytophthora infestans, the causal agent of late blight of potato and the cause of the Irish Famine of the mid 19th century, and Viteus vitifoliae, the phylloxera insect which destroyed European grapevine production during the same period), several countries began, in the late 19th and early 20th century, to put in place specific legal measures to try to prevent the introduction of exotic pests into their territories. Measures for this purpose were described as “quarantine“ measures. The name “quarantine” derives ultimately from the Italian word for forty – quaranta, and means “approximately forty“. The use of this word originated in the fourteenth century during the terrible human epidemic, the Plague or Black Death, which swept through Europe, killing an estimated 25 million people. Certain coastal cities in the Mediterranean remained free of the disease and, in order to prevent infection from outside, their rulers required that 220

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ships and their passengers and crews should be kept in isolation outside the cities (often on islands) until it was certain that they were free from the disease. It is not clear why forty days was chosen as the period of isolation; it was perhaps related to the mystical significance that forty days appears to have in religious contexts. Or possibly, experience of the disease had indicated that people who were infected, but in whom the disease was latent, would develop symptoms within approximately forty days of isolation. The meaning of “quarantine” later expanded to include any measures intended to prevent the introduction of diseases or pests, not only of humans but also of animals and plants; the word also retains its original sense of keeping any suspect disease carrier in isolation and in secure conditions for a defined period of time within which symptoms should appear. In fact, the word “quarantine” in its broad sense has been going out of fashion in recent time; it has been partly replaced by “phytosanitary” (so that quarantine measures are known as “phytosanitary measures”) and, more recently, by “plant health”, but it is still retained when referring to organisms against which measures are taken (as in “quarantine pest“ or “quarantine nematode”); it is also used as a noun (e.g. “a quarantine”), especially in North America, to refer to a particular regulation. The phytosanitary measures established by national governments relied on the expertise of their own national experts in plant pathology and entomology to recognise which exotic pests might present a potential risk and to devise appropriate measures to prevent possible introduction. The consequence of such a system was that there were great variations between the measures of different countries, even between neighbouring countries where the climatic and agricultural conditions were largely similar. The reasons for this were that different experts inevitably have different opinions about the importance of a particular pest, and each expert tends to exaggerate the importance of the organism(s) included in his/her expertise! Different experts are likely also to minimise the danger of a pest present in their own country, and to exaggerate the risk of a pest in another country. (Incidentally, before I became involved in plant quarantine and after many years of nematological research, I was under the naïve impression that science was always the pursuit of an absolute truth; my first discussions about Bursaphelenchus xylophilus with nematologists from other parts of the world demonstrated to me that the interpretation of scientific data is relative and QUARANTINE NEMATODES

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depends on the nationality of the scientist!). The result of the differences of expert opinion in different countries was that many of the measures targeted unimportant pests, they were often not effective or they were unnecessarily restrictive (and, as such, they constituted barriers to trade).

Internationalization of quarantine To try to overcome these problems, it was recognised that some form of international agreement was needed in order to ensure harmonization between the regulations of different countries. The first attempt at such an agreement (The International Phytopathological Convention of Rome) was drawn up at a most unfortunate time – in 1914 just at the beginning of the First World War. It was never ratified. A second attempt was made in 1929, with the International Convention for the Protection of Plants, but, although signed by many countries, it did not receive much subsequent support and was soon forgotten. It was not until after the Second War, when Europe, and some other parts of the world, faced severe food shortages due to land destruction and lack of a labour force, that a more permanent international collaboration developed. At that time, it was recognised that it was absolutely essential to avoid any possible threat from plant pathogens to agricultural food supplies. The International Plant Protection Convention (IPPC), was drawn up by concerned countries in 1951, under the aegis of the Food and Agricultural Organization (FAO) of the United Nations. It required each country to: – establish a competent national plant protection service which could survey its own territory to determine what pests were present, and that could inspect plants and plant products to ensure absence of pests and issue phytosanitary certificates to attest to this fact; – publish its phytosanitary regulations, including a list of the quarantine pests against which measures were taken; – to collaborate with neighbouring countries to establish regional plant protection organizations which could coordinate all phytosanitary activities. A major element of the IPPC was the concept that a particular country does not have to act alone in defending its borders from invading pests, but also can depend on exporting countries to take 222

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effective action to ensure that their exports are free from pests. Thus, a certain level of competence was required on the part of national plant protection organizations so that importing countries could have confidence in the thoroughness of the phytosanitary inspection of the exporting country. The IPPC also urged governments to create regional plant protection organizations (RPPOs) to coordinate the quarantine activities within specific geographic regions, where the environmental and climatic conditions and the pest spectrum would be similar for the countries in the region. RPPOs have been established for all parts of the world, and their establishment has contributed massively to the exchange of information about potentially dangerous exotic pests, about their distribution and about methodology that could be applied to prevent their spread. The RPPOs also allowed countries to act together to protect the region as a whole from alien pests; it is clearly a more effective strategy to prevent the initial introduction into a region rather than trying to prevent spread from country to country after the pest has become established somewhere in the region. The concept of quarantine pest was established by the IPPC and it can be defined as any pest (that is, any animal, plant or pathogenic agent injurious to plants or plant products; note that the European Union uses “harmful organism” with the same meaning) against which official measures are taken by a country to prevent its introduction to that country; it should be of potential economic importance to the country, and it should be absent from the country or, if present, should be of limited distribution and subject to official national measures to prevent spread. Thus a quarantine pest only has relevance in relation to a particular country. The concept of quarantine nematode is, of course, included with this definition, but the name is often used also for nematodes where the measures to prevent introduction have already failed and now efforts are being made to eradicate, to limit spread or to reduce the damage caused by the nematode. It should be noted that, from a global perspective, the principles of the IPPC were not applied equally diligently throughout the world. There were many countries, who, although signatories of the IPPC, did not fully trust either their trading partners or their neighbouring countries in the region to provide protection from exotic pests and they, therefore, maintained their traditional, phytosanitary policy of relying only on their own, thorough, border inspections. Such a policy tended to slow trade unnecessarily. QUARANTINE NEMATODES

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After approximately 40 years of operation of the IPPC, during which time the convention had been once revised and most countries of the world had signed the convention, the World Trade Organization (WTO) came to the conclusion that the IPPC (as well as other international agreements on human and animal health) was ineffective. The WTO was of the opinion that countries were using the pretext of protecting themselves from exotic pests of humans, animals and plants in order to block undesirable imports into their territories, for example, imports that might be in direct competition with their own national industries. In other words, they were using sanitary and phytosanitary regulations as non-tariff barriers to trade. In 1994, the WTO drew up the Agreement on Sanitary and Phytosanitary Measures (the SPS Agreement) which aimed to establish more rigorous criteria for devising and implementing such measures. The SPS Agreement recognised that countries have the sovereign right to take phytosanitary measures to protect plant health in their territory and that the country can decide on the level of protection that they consider appropriate. However, the measures should be based on international standards, guidelines or recommendations, or, if not, they should be based on scientific principles. As well, perhaps most importantly for the WTO, they should have a minimal effect on trade. The SPS Agreement allowed countries to challenge each other’s phytosanitary regulations in a WTO court, with possible sanctions if severe measures are maintained unjustifiably. The arrival of the SPS Agreement was a “wake-up call” for those of us involved in plant quarantine. It indicated to us that we had been concentrating on maximising the efficacy of quarantine measures and ignoring the fact that phytosanitary regulations were being used by some countries to block unwanted trade. Plant quarantine can be seen as the attempt to balance the two aims of preventing, as much as practical, the spread of plant pests while, at the same time, allowing international trade to proceed as fully as possible. We did not have the balance right. The other lesson learnt from the advent of the SPS Agreement was that, in any confrontation between plant protection and world trade, it will be trade that will always win. Particularly as the World Trade Organization can impose punitive sanctions on any transgression, whereas, the IPPC can only suggest an arbitration procedure between disputing countries. The plant quarantine community moved quite rapidly in reac224

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tion to the SPS Agreement and set about revising the IPPC to bring it into line. The revision was completed in 1997. One of the requirements in the SPS Agreement was that phytosanitary measures should be based on international (that is, global) standards, but, in fact, no such global standards existed. Regional standards had previously been produced by some of the regional plant protection organizations. In particular, EPPO (the regional organization for the European and Mediterranean region) had produced quite a number of such standards but these could not be considered as global standards without having been approved and accepted by all countries of the world. The revision of the IPPC envisaged a system to develop global standards; this involved a Secretariat of the IPPC working within FAO with a coordinator responsible for a programme on the development of standards. The Commission on Phytosanitary Measures (CPM) supervises the creation and international approval of these standards. All member countries of FAO are given the opportunity to study the standards during their preparation, to comment on them at this stage and to approve them on completion. One of the most important international standards required by the SPS Agreement was on pest risk analysis (PRA). This is needed in order to decide which pests should be quarantine pests for a particular country, or part of a country, and which measures should be taken to prevent their introduction. A standard based on the principles of PRA was, therefore, one of the first to be produced by the Secretariat of the IPPC. PRA, in a standardized format, has become the basis for modern phytosanitary measures. This is not to say that risk analysis had not previously been used in deciding on quarantine pests and in developing phytosanitary measures. In fact, the process was generally performed in a non-structured way, either by the intuitive opinion of a particular expert or as a result of discussion “in smoke-filled rooms” between several experts on different aspects of plant quarantine. Either way, the steps by which the final decisions were taken did not usually follow a systematic sequence, were seldom accurately recorded and were unknown to other people who were not involved. Within the formalized PRA process developed by the Secretariat of the IPPC, there are three stages: 1. INITIATION, which identifies the pest that should be subjected to the process because it might present a problem if introduced into the area being considered; 2. RISK ASSESSMENT, in which an estimate of risk is obtained by QUARANTINE NEMATODES

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combining the probability of introduction and the potential economic impact; it has not been possible to agree on any form of quantitative estimate of risk and it is usually expressed in relative terms. The first two stages require a considerable amount of biological data on the pest and its host plants and can be performed only by experts with the appropriate knowledge. For example, when analysing the risk from a particular nematode species, a nematologist would be required to have available, and to take account of, information on the nematode’s geographic distribution, host range, host/parasite relations (including economic impact on specific hosts; possibly under different climatic/edaphic conditions), survival ability during transport of the host plants and of other commodities, the possibility of it becoming established in the area under consideration, and the efficacy of measures that could be used to ensure that commodities are nematode-free. In addition, information is needed on patterns of trade and on ecological conditions in the country of origin and country at risk. 3. RISK MANAGEMENT, in which a decision is taken as to whether the risk is sufficiently great to require measures and, if so, which measures could, or should, be taken. This final stage usually includes some political decision-making concerning the risk that a country could consider to be acceptable. All the steps in this process must be recorded and may be re-examined later if new information becomes available. It is clear that this version of PRA is much more objective and transparent than the previous procedure described above. Other standards so far developed by the IPPC Secretariat (which are named International Standards for Phytosanitary Measures or ISPMs) include general standards on the principles of plant quarantine, export certification, a phytosanitary import regulatory system and a glossary of phytosanitary terms, as well as standards on more specific subjects, such as, how to conduct surveillance for pests, how to establish and maintain pest-free areas and areas of low pest prevalence, the use of irradiation as a phytosanitary measure, and the regulation of packaging wood in international trade. Twentyfour ISPMs have been published (Table 1) and others are in the pipeline. As the development of ISPMs continues, it is obvious that the subject matter will become more specific. For example, at present, there is a programme in progress for producing diagnostic protocols each of which will focus on individual pests, or on groups of related pests, and will provide guidance on how to 226

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detect the pest in question in traded commodities, and then how to identify the pest. All of these standards are developed with the active participation of relevant experts worldwide. Diagnostic protocols are planned for the nematode species Ditylenchus destructor, D. dipsaci, Bursaphelenchus xylophilus and Xiphinema americanum. It is obvious that, to ensure that dangerous pests are prevented from being introduced to new areas and to avoid cargoes being unnecessarily blocked, it is essential that suitable methodology should be used to detect quarantine pests during inspection at export or import and that any organism found should be correctly identified. The more scientifically developed countries may not see the need for such diagnostic protocols when expert taxonomists for all the major pest groups are available to their phytosanitary inspectors, but it should be recognised that most countries of the world do not have such a well developed support service. This programme should continue with the aim of providing, in the short term, diagnostic protocols for those quarantine pests where there are recognised taxonomic difficulties (and, among the quarantine nematodes, there is a surprising number of such difficult cases), and, in the long term, protocols for all quarantine pests.

Table 1. International Standards for Phytosanitary Measures (ISPMs) produced by the Secretariat of the IPPC. See www.ippc.int for further information. ISPM 1

Principles of Plant Quarantine as related to international trade ISPM 2 Guidelines for Pest Risk Analysis. ISPM 3 Code of conduct for the import and release of exotic biological control agents ISPM 4 Requirements for the establishment of pest-free areas. ISPM 5 Glossary of phytosanitary terms. ISPM 6 Guidelines for surveillance. ISPM 7 Export certification system. ISPM 8 Determination of pest status in an area. ISPM 9 Guidelines for pest eradication programmes. ISPM 10 Requirements for the establishment of pest free places of production and pest free production sites. ISPM 11 Pest risk analysis for quarantine pests. QUARANTINE NEMATODES

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ISPM 12 Guidelines for phytosanitary certificates. ISPM 13 Guidelines for the notification of non-compliance and emergency action. ISPM 14 The use of integrated measures in a systems approach for pest risk management. ISPM 15 Guidelines for regulating wood packaging in international trade. ISPM 16 Regulated non-quarantine pests: concept and application. ISPM 17 Pest reporting. ISPM 18 Guidelines for the use of irradiation as a phytosanitary measure. ISPM 19 Guidelines on lists of regulated pests. ISPM 20 Guidelines for a phytosanitary import regulatory system. ISPM 21 Pest Risk Analysis for regulated non-quarantine pests. ISPM 22 Requirements for the establishment of areas of low pest prevalence. ISPM 23 Guidelines for inspection. ISPM 24 Guidelines for the determination and recognition of equivalence of phytosanitary measures.

Quarantine nematodes If one examines the quarantine lists of those countries that publish such lists (and there are still several major countries of the world that do not yet publish their quarantine lists), one can usually find a number of quarantine nematodes. The species most commonly found probably reflect the views of nematological advisors to the quarantine authorities as to which are the most important species or the most likely to be transported with international trade. The following species are the ten most commonly represented in lists: Globodera rostochiensis, G. pallida, B. xylophilus, Aphelenchoides besseyi, Radopholus similis/citrophilus, D. dipsaci, D. destructor, Heterodera glycines, X. americanum and Nacobbus aberrans. These species are probably justified to be quarantine nematodes, but the justification for other nematode species found on the quarantine lists of some countries is difficult to imagine. For example, some European countries have the following list entries: 1) “Meloidogyne spp.”, 2) “Hirschmanniella spp. other than H. gracilis” and 3) “Longidorus diadecturus. In my personal opinion, 228

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these nematodes could only have been declared quarantine nematodes as a result of a failure to apply PRA or a lack of detailed information, and phytosanitary measures taken against them might constitute unjustified barriers to free trade. Taking them in order: 1) There is probably not a country in Europe that does not have some Meloidogyne spp. present; 2) This list entry is probably intended to prevent the introduction of species that attack rice, but, in fact, there are numerous Hirschmanniella spp. living in aquatic environments throughout the world, that have little or no effect on the aquatic plants on which they feed. There are several species in Europe, in addition to H. gracilis; 3) L. diadecturus is listed as a quarantine nematode because of its supposed ability to transmit peach rosette mosaic virus (PRMV). But PRMV belongs to the group of nepoviruses that are transmitted by Xiphinema spp. and is, therefore, unlikely to be transmitted by a species of Longidorus. Furthermore, the published methodology used to demonstrate transmission is open to question.

Conclusions This brief history of the development of plant quarantine demonstrates that the early attempts by individual governments acting alone to prevent the spread of dangerous pests from one region of the world to another was largely unsuccessful. This was mainly because the expertise necessary to recognise potentially dangerous pests and to design suitably effective measures to prevent their movement without hindering trade were not widely available. The first exploitation of international collaboration, through the International Plant Protection Convention improved the efficacy of quarantine measures but was not, apparently, satisfactory to those involved in world trade. Stimulated by the SPS Agreement of the World Trade Organization, international bodies engaged in plant quarantine have set in motion a process of development of international standards that will surely lead to a more objective, transparent and harmonized system of plant quarantine. However, now in 2007, we have not reached that goal, as there are many countries which have not yet fully adopted the philosophy of international collaboration, and there are still examples of quarantine legislation, for nematodes at least, that need to be reviewed in the light of current more enlightened thinking. QUARANTINE NEMATODES

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There is a need for more input from nematologists into the internationalization process so as to provide information regarding potential quarantine nematodes on all aspects that could influence plant quarantine decisions. Furthermore, although most of this history relates to nematodes (or other types of organisms) as plant pests, it should be noted that nematode species that are not parasites of plants but are fungal feeding or saprophagous members of the soil ecosystem are becoming increasingly examined as potential alien invasive species, that is, as organisms that could be introduced into new areas and have the potential themselves or through the organisms that they carry to disrupt the existing ecological balance. Such alien invasive species are often covered by the same types of legislation and the same agencies as quarantine pests. Much more information is needed about these nematodes, but they have been very little studied as compared with the parasitic species; this needs to be remedied.

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15. THE PINEWOOD NEMATODE: A PERSONAL VIEW HELEN BRAASCH Biologische Bundesanstalt, Stahnsdorfer Damm 81, D-14532 Kleinmachnow, Germany. &

MANUEL M. MOTA Departmento Biologia, Universidade de Évora, 7000 Évora, Portugal

The first report of the disease (“pine wilt disease”) associated with the pinewood nematode, goes back to 1905, when Yano, Japan, reported an unusual decline of pines from Nagasaki. For a long time thereafter, the cause of the disease was sought, but without success. Because of the large number of insects that were usually seen around and on infected trees, it had always been assumed that the causal agent would prove to be among these insect species. However, in 1971, Kiyohara and Tokushike found a nematode, of the genus Bursaphelenchus, in infected trees. This nematode multiplied on fungal cultures, was inoculated into healthy trees and then re-isolated from the resulting wilted trees. The subsequent published reports were impressive: this Bursaphelenchus species could kill fully-grown trees within a few months in the warmer areas of Japan, and could destroy complete forests of susceptible pine species within a few years. Pinus densiflora, P. thunbergii and P. luchuensis were particularly affected. In 1972, Mamiya and Kiyohara described the new species of nematode extracted from the wood of diseased pines; it was named Bursaphelenchus lignicolus. Since 1975, the species has spread to the north of Japan, with the exception of the most northerly prefectures. In 1977, the loss of wood in the west of the country reached 80%. Probably as a result of unusually high summer temperatures and reduced rainfall in the years 1978 and 1979, the losses were more than 2 million m3 per year. From the beginning, B. lignicolus was always considered by Japanese scientists to be an exotic pest. But where did it come from? That this nematode could also cause damage in the USA THE PINEWOOD NEMATODE: A PERSONAL VIEW

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became clear in 1979 when B. lignicolus was isolated in great numbers from wood of a 39 year-old pine tree (Pinus nigra) in Missouri which had died after the colour of its needles changed to a reddish brown colour (Dropkin and Foudin, 1979). In 1981, B. lignicolus was synonymised by Nickle and colleagues with B. xylophilus, which had been found for the first time in the USA as far back as 1929, and reported by Steiner and Buhrer in 1934. It had originally been named Aphelenchoides xylophilus, the wood-inhabiting Aphelenchoides, but was recognised by Nickle, in 1970, to belong in the genus Bursaphelenchus. Its common name in the USA was the “pinewood nematode” (PWN). After its detection in Missouri, it was found that B. xylophilus was widespread throughout the USA and Canada. It occurred there also on native species of conifers where, as a rule, it did not show the symptoms of pine wilt disease unless the trees were stressed e.g., by high temperature or lack of water. This fact that North America could be the homeland of PWN was an illuminating piece of evidence. Dwinell (1993) later reported the presence of B. xylophilus in Mexico. The main vector of the PWN in Japan was shown to be the longhorned beetle, Monochamus alternatus, belonging to the family Cerambycidae. This beetle lays its eggs in dead or dying trees where the developing larvae then feed in the cambium layer. It was already known in Japan in the 19th century, and by the 1930s, it was said to be present in most areas of Japan, but was generally uncommon. However, with the spread of the pine wilt disease, and the resulting increase of weakened trees that could act as breeding sites for beetles, the populations of Monochamus spp. increased significantly. In North America, other Monochamus species transmit PWN, and the main vector is M. carolinensis. There are also other, less efficient vectors in the genus Monochamus. Possibly, all Monochamus species that breed in conifers can transmit the PWN, but the occasional transmissions by less efficient species of Monochamus or by some other wood or barkbreeding beetles are of little significance. In Europe, M. galloprovincialis and M. sutor are known to transmit the closely related nematode species B. mucronatus. Some speculate that these two insect species are “standing by” and waiting for the arrival of B. xylophilus. The fear became true, in 1999, in Portugal, where M. galloprovincialis transmits B. xylophilus with “great success”. In 1982, the nematode was detected in China. It was first found in dead pines near the Zhongshan Monument in Nanjing 232

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(Cheng et al., 1983); 265 trees were then killed by pine wilt disease. Despite great efforts at eradication in China, the nematode spread further and pine wilt disease has been reported from parts of the provinces of Jiangsu, Anhui, Guangdong, Shandong, Zhejiang and Hubei (Yang, 2003). In 1986, the spread of the PWN to Taiwan was discovered and, in 1989, the nematode was reported to be present in the Republic of Korea where it was detected first in P. thunbergii and P. densiflora. It was thought to have been introduced with packaging material from Japan. PWN was advancing! In 1984, B. xylophilus was found in wood chips imported into Finland from the USA and Canada, and this was the impetus to establish phytosanitary measures to prevent any possible spread into Europe. Finland prohibited the importation of coniferous wood chips from these sources, and the other Nordic countries soon followed suit. EPPO (the European and Mediterranean Plant Protection Organization) made a recommendation to its member countries, in 1986, to refuse wood imports from infested countries. With its Directive of 1989 (77/93 EEC), the European Community (later called the European Union or EU) recognized the potential danger of B. xylophilus for European forests and imposed restrictions on imports into Europe. PWN was placed on the quarantine list of the EU and also of other European countries. Later, in 1991, a dispensation was allowed by the Commission of the EU (92/13 EEC) for coniferous wood from North America provided that certain specified requirements were fulfilled that would prevent introduction of B. xylophilus. Helen Braasch: “The pinewood Nematode has been particularly attractive to me ever since I learned of the enormous damage it had caused in Japan in the 1970s. Damage by the potato cyst nematode, by Meloidogyne species, by the stem and bulb nematode and by other nematode pests of agricultural or horticultural importance were well known to us in Europe, but that a nematode could cause the death of great pine trees was almost unbelievable. The only other equivalent case was that of Rhadinaphelenchus (Bursaphelenchus) cocophilus, the causal agent of red ring disease of palms in the Caribbean Islands. At this time, I was in charge of the Quarantine Laboratory in the Central Plant Protection Office of the former East Germany (GDR) in Potsdam, close to Berlin. Being always on the lookout for new threats in plant quarantine, it was quickly clear to me that PWN in Japan represented a major new THE PINEWOOD NEMATODE: A PERSONAL VIEW

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quarantine problem. In 1983, I published, in Nachrichtenblatt für den Pflanzenschutz in der DDR, the first European report on PWN, entitled: “The pine wood nematode, Bursaphelenchus xylophilus (Steiner und Buhrer, 1934) Nickle, 1970, from the point of view of plant quarantine” (in German). I was concerned with PWN, in one way or another, for more than 10 years without ever having seen it. Even obtaining the relevant literature, for those of us “behind the Wall” often required it to be obtained by circuitous routes! When David McNamara, as Assistant Director of EPPO visited our institute at this time (the GDR had become a member of EPPO) I took the opportunity to ask him (quietly!) to support my participation in an EPPO Panel of Experts on PWN. What a request! For me, any participation in scientific meetings with the “capitalist abroad“ was forbidden. My boss at the time, the head of the Central Plant Protection Office, considered my interest in Bursaphelenchus to be an unnecessary interference with my work as a quarantine nematologist. How often did I hear the expression: “This is not the EPPO Panel on Bursaphelenchus!” The belief somehow persisted that the “Iron Curtain“ would keep the PWN out! But they did not reckon with my stubbornness. As I could not obtain a sample of the PWN, I concerned myself with its relatives. Thus, I discovered that B. mucronatus, the nearest relative of the PWN, was not only present in Germany but also frequently detected in pine timber imported from Russia; I published this in 1979. The crucial and dangerous fact about several Bursaphelenchus spp., including the PWN and B. mucronatus, is that they can survive for a very long time within wood, together with the larval stages of their insect vectors. The sampling of the Russian sawn timber at our wood storage sites was spectacular. We found boards which looked like sieves as a consequence of the Monochamus infestation, and the workers at the sites spoke of “swarms of flying beetles”. The beetles would be able to obtain their maturation feeding in the surrounding Brandenburg pine forests and later would be able to lay their eggs in weakened trees. The dauer larvae of B. mucronatus, which are mainly carried in the tracheae of the insects, would be transported to the trees where they could possibly find a new vector and perhaps mingle with native populations. This could happen in a similar way, we believed, with the PWN which would then find the required environmental conditions to take up residence in the dry pine forests of Brandenburg where summer temperatures are relatively high for Germany. 234

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My relationship with PWN improved with the collapse of the Berlin Wall and the unification of Germany. The first personal contact that I had thereafter with the “western world” was in the Institute for Nematology and Vertebrate Research of the BBA (Federal Biological Research Centre for Agriculture and Forestry) in Münster, where Dieter Sturhan and Marlies Schauer-Blume had also been concerning themselves with the Bursaphelenchus problem, and had demonstrated the presence of B. fraudulentus in deciduous trees. The title of their publication: “The occurrence of pinewood nematodes (Bursaphelenchus spp.) in the Federal Republic of Germany ?” (in German) (1989) led later to the misunderstanding in certain parts of the world that it was the pinewood nematode (B. xylophilus) itself that was present in Germany. The readers had failed to recognize the question mark in the title. I was later required to provide clarification to the resulting enquiries. I obtained Bursaphelenchus cultures from Münster and I could, at last, study the PWN “in person“. New doors were opened to me and I obtained a new position in the Kleinmachnow Branch of the Federal Biological Research Centre for Agriculture and Forestry. During my vacation, I made the first personal contacts with Canadian ‘Bursaphelenchists’, being received very amicably in Vancouver by John Webster and Jack Sutherland. Now, I could also establish my membership of the EPPO Panel of Experts on the Pinewood Nematode, which was chaired by David McNamara. The Department for Economic and Legal Affairs in Plant Protection of the BBA in Braunschweig, with an external branch in Kleinmachnow, in which I was employed as a nematologist from 1990, had the task, among others, of taking responsibility for fulfilling the requirements of Annexes I–V of EU Directive 77/93/EEC (Plant Quarantine Directive) and contributing to related international working groups. The requirements concerning coniferous wood from North America needed particularly intensive action. Even if the occasional journeys to Brussels to participate in the EU Standing Committee on Plant Health or in expert groups were inevitably stressful due to the need to leave home at 5 o’clock in the morning and to return at about 11 o’clock in the evening, I was always interested in the statements from the representatives of other countries on the time-consuming and expensive implementation of the requirements concerning PWN. The Sword of Damocles was hanging over Europe: coniferous THE PINEWOOD NEMATODE: A PERSONAL VIEW

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wood from infested countries was still being imported, although under certain conditions. The phytosanitary measures considered necessary by the EU (especially heat treatment of wood to prevent introduction of B. xylophilus) were accepted only reluctantly by the exporting countries, because of the extra expenditure needed to be applied to their wood exports. Surveillance of the cargoes imported into Europe was limited to an insufficient number of samples, while the examination of documentation continued at a high rate. The danger of introduction with packaging wood was not adequately recognized. Meanwhile, some experts in North America began to question whether the PWN could survive in Europe and, even if it did, would it cause damage anywhere there apart from the Mediterranean region where the temperature was sufficiently high? There were even some suggestions that the PWN might be already present in Europe and that the import restrictions were, therefore, unnecessary! One good thing came out of this situation: although the main function of my department was to provide high quality scientific advice, I was also placed in the position to be able to conduct research on the PWN problem in order to provide scientific evaluation concerning this quarantine organism, and to present the results at international conferences. This privilege was not always free from envious glances from some of my colleagues, and also some obstacles needed to be overcome, or simply ignored. I surveyed the German States for Bursaphelenchus species, studied their taxonomy and biology, compared the morphology and damage caused by B. xylophilus and B. mucronatus, conducted inter-species crossing experiments, researched the variability and climatic needs, the means of transmission and spread, and collaborated with foreign nematologists. With John Philis from Nicosia, I studied the Bursaphelenchus fauna of Cyprus; B. xylophilus was, luckily, not found there, despite patches of dead pines. From a damaged pine in South Africa, an isolate of Bursaphelenchus was sent to me by A. Swart which, again fortunately, proved not to be B. xylophilus, but B. leoni. Several international conferences between 1994 and 2004, with sections on pine wilt disease, all contributed not only to the international exchange of experience with the Bursaphelenchus problem, but also led to the foundation of a pan-European research collaboration on Bursaphelenchus. This collaboration reached its high-point at the end of the 20th century with the completion of the EU Project RISKBURS (1996-2000). During a visit to our partner in the research project in Vienna, I met 236

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Heinrich Schmutzenhofer, Secretary of the International Union of Forestry Research Organisations (IUFRO). In 1981, Dr. Schmutzenhofer had reported finding a Bursaphelenchus species associated with decline of fir trees in Austria; he made available to me the nematode material that he had collected at the time. During the ‘Symposium on sustainability of pine forests in relation to pine wilt and decline’ in Tokyo, Japan (1998) I was able, for the first time, to hold discussions with Yasuharu Mamiya – an impressive, modest and yet radiating personality. In the 1990s, it was recognized that a scientific response should be given to the question of how dangerous would the PWN really be to Europe and to try to convince the doubters, especially in North America, of the need for the EU’s quarantine measures. The EU, therefore, established an Expert Group from the member states under the leadership of Hugh Evans of the U.K. The results of the Group’s deliberations were published in 1996 in the EPPO Bulletin as a formal pest risk analysis (Evans, H. F.; McNamara, D. G.; Braasch, H.; Chadoeuf, J.; Magnusson, C.: Pest Risk Analysis (PRA) for the territories of the European Union (as PRA area) on B. xylophilus and its vectors in the genus Monochamus). The most important conclusions of this analysis were that the whole of the PRA area is suitable for colonisation by B. xylophilus, but that the dry Mediterranean and continental regions would be in particular danger for the occurrence of pine wilt disease, that the occurrence in Europe would have important economic consequences, and that the greatest danger from wood imports would be when the nematode and its vector were both present at the same time. The final conclusion was that shipments of coniferous wood from infested areas required phytosanitary measures. The EU-financed project RISKBURS, of which I was the project leader, included biologists, forest scientists and molecular biologists from Germany, Greece, Ireland, Italy and Austria. The project allowed, for the first time in Europe, large-scale detection surveys to be conducted to determine the Bursaphelenchus species present in Europe. Fortunately, B. xylophilus was not detected in the surveyed areas in Germany, Greece, Italy and Austria. Numerous pathogenicity tests conducted on young conifer plants with 15 Bursaphelenchus species suggested that B. mucronatus and B. sexdentati could also have a potential for pathogenicity, although, so far, no reliable confirmation of these laboratory data have been observed on forest THE PINEWOOD NEMATODE: A PERSONAL VIEW

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trees. May collaboration with Wolfgang Burgermeister (BBA Braunschweig) and his colleagues, which had begun before the start of the project, on the molecular characterization of Bursaphelenchus spp., proved to be fruitful. The characterization of species that had previously been identified morphologically, first used RAPD-PCR, later ITS-RFLP and finally sequencing. The methodology provided an essential contribution to the results of the project and then, later, to the confirmation of the first introduction of B. xylophilus into Portugal; the methods are still used today. Techniques for molecular identification of B. xylophilus and B. mucronatus were also elaborated in Ireland, France and North America. The reference pictures built up in Germany, with the aid of the extensive BBA collection of Bursaphelenchus cultures (now continued by Thomas Schröder), permitted the differentiation of B. xylophilus from about 30 other Bursaphelenchus spp. by means of the ITS-RFLP technique.” Manuel Mota: “In 1999, B. xylophilus was detected for the first time within the territory of the EU, more precisely in an area in the Setúbal Peninsula in Portugal. I am proud to have led a team of researchers from Portugal, which included Maria Antónia Bravo and Edmundo Sousa, from INIA. The discovery was made in May 1999 while we were surveying cerambycid beetles and associated aphelenchid nematodes, in the area of Pegões, a town located in the Setúbal Peninsula, 30 km southeast of Lisbon. In collecting samples of wood and insects, our intention was to establish which species of nematode were present and with which species of tree and insect they were associated. We had no thought that B. xylophilus might be present. To our great surprise (and alarm), one of the samples yielded a tremendous number of specimens of a species of Bursaphelenchus. It was my M.Sc. student, Ana Catarina Penas who made this observation during research work for her thesis, and she called me to confirm. We also asked Maria Antónia Bravo if she had seen the same nematode in her lot of samples and she said ‘yes’. The nematode appeared to us to be B. xylophilus, but we needed confirmation as soon as possible. So, we contacted Helen Braasch and Wolfgang Burgermeister in Germany who, more than anyone else, had the expertise to confirm quickly this initial diagnosis, using molecular techniques (ITS-RFLP).”

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Fig. 1. In a forest in Portugal, Manuel Mota tells the story of finding the first infestation of pine wood nematode in Europe. Helen Braasch and Wolfgang Burgermeister are to the right of the picture.

Helen Braasch: “The greatest surprise of my nematological life was when I looked through my microscope, in spring 1999, at a sample from a killed pine tree (Pinus pinaster) which had been sent to me by Manuel Mota from the University of Évora, Portugal, and saw thousands of wriggling specimens of B. xylophilus. The pinewood nematode in Europe! It was hard to believe. Morphologically, there was little doubt, but a molecular study by Wolfgang Burgermeister’s team would confirm the identification. Within a week, we were able to confirm Manuel’s suspicions.” Manuel Mota: “The results arrived back from Germany with the message that the molecular analyses confirmed our worst fears: we had detected pinewood nematode for the first time in the EU! Following a team meeting, we proceeded to inform our institutional authorities (University president, Research directors) about this issue and immediately contacted the national plant protection authority (DGPC), who would have to inform the EU Commission in Brussels about this. The initial intention of the EU, in September 1999, was to impose a general embargo on pinewood exports from Portugal to other countries of the EU. Fortunately, and following some intense political lobbying, the quarantined area was restricted to the Setúbal THE PINEWOOD NEMATODE: A PERSONAL VIEW

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Peninsula. On a short anecdotal note, I remember my friend José Francisco Fernandes, owner of a large property inside the affected area, who in the past had joked and pulled my leg for studying these strange little animals under the microscope, with no apparent practical interest. Well, once his pine stand was subjected to strict and expensive quarantine measures, he suddenly became very interested in nematology, and wanted to learn everything about this new “enemy” which caused major economic damage to his financial situation!” Helen Braasch: “I felt that I must go to Portugal as soon as possible, and, shortly thereafter, I travelled there with Wolfgang Burgermeister and Kai Metge. We were met at the airport by Manuel Mota and a delegation of important people and very soon, during a working lunch, we exchanged our information. I insisted on seeing the infected tree, and after the meal we drove on the Setúbal Peninsula towards Évora to the affected forest plot. Of course, it was not really an amusing situation but we laughed when Manuel could not, at first, find the infected tree! After walking back and forth along the forest path, the tree was at last found; it had been sawn down and the stump was still standing; the tracks of beetle larvae could be seen on it. We were shown a photograph of the tree as it appeared while still standing, but it did not look as though it had died only in 1999. As we stood there in sad discussion around the remains of the fallen tree, I heard the repeated expression from the representative of the Portuguese Ministry: “It’s a nightmare”! Samples taken from the site, from the stump and from nearby trees confirmed the infestation. As well, in a second area lying close to a timber storage site, we made another find. A forest worker had noticed that the pines appeared to die unusually quickly. The results of the sampling did not permit any doubt, and they soon appeared in print (Mota, M. M.; Braasch, H.; Bravo, M. A.; Penas, A. C.; Burgermeister, W.; Metge, K.; Sousa, E., 1999, First record of Bursaphelenchus xylophilus in Portugal and in Europe. Nematology 1: 727-734). Shortly afterwards, the vector was recognised to be M. galloprovincialis which had previously been found only rarely in Portugal. The discovery of PWN in Portugal dramatically altered the European view of the problem. The EU quarantine machinery rolled into action and the Portuguese did their best to destroy any infected trees, delimit the infested area, and prevent the spread of the pest to other areas of Portugal and Europe. Although the spread of pine wilt disease has 240

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been prevented, it has proved impossible to eradicate the PWN infestation, despite the felling and destruction of all suspected trees in the demarcated zone and the buffer zones. The EU carried out inspection visits to evaluate the situation in Portugal. During the first inspection visit, we were pursued by the paparazzi, but our delegation was not prepared to speak to the press. The press photographers succeeded in taking pictures and, on 16th September, 1999 an article appeared in the Portuguese press entitled: ‘Inspectores Europeus em Silencio’ (‘The European Inspectors remain silent’). It included a picture of our delegation, under which my name was given with the additional information that it was I who had confirmed the presence of the pest in Portugal. I had hardly returned to Kleinmachnow before the telephone began to ring incessantly: the European press wanted to know something about the PWN. The German Press Agency spread the news about the occurrence of B. xylophilus in Europe. The press reports ranged from more or less scientifically correct to rather distorted, and had headlines such as: “New Pest spreads Shock and Awe“, “The Advancing Worm”, “The Invasion of the Worms”, “Pine Stands in Portugal Dying of Thirst”, “Worm becomes a Global Threat”, “Tiny Worm Destroys Complete Pine Forests”, “Scientist from Kleinmachnow Protects the World’s Coniferous Forests”– and these are just a few of the many headlines. According to EU Directive 2000/29/EC, which replaced the earlier Directive 77/93 EEC, the most important quarantine requirements for imported conifer wood from infested countries were (depending on the commodity type): heat treatment (56°C in the centre of the wood for at least 30 minutes), debarking, drying and freedom from bore holes (galleries of cerambycid larvae and exit holes of beetles). Only the last three requirements were applied to coniferous packaging wood, which is frequently used to contain and support other commodities and which is often of inferior quality; this proved to be a serious mistake. There is considerable circumstantial evidence that PWN was probably introduced to Portugal with packaging wood from East Asia. The ports of Setúbal and Lisbon are both near to the infested zone and there is a strong suspicion that infested wood passed through these ports. The impression is certainly strengthened that importation of packaging wood played a key role in the spread of PWN. For example, in China, saw mills, building sites and storage areas for packing wood are considered to THE PINEWOOD NEMATODE: A PERSONAL VIEW

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be sources for new infestations. In recent years, in many European and Asiatic countries, samples were taken from packaging wood coming from infested areas, and PWN and living larvae of the vector beetles were found. This is the greatest danger; both organisms together in the wood! In Brussels, the experts from the member states discussed how the deplorable situation in Portugal should be combated. These meetings are among the most interesting memories of my professional career. It required intense concentration to ensure that the right thing was done, nothing should be overdone, nothing should be neglected, and that the correct procedures should be completed. The decision of the EU of January 11th, 2000 (2000/58/EG) and its modified version of March 12th, 2001 (2001/218/EG), as well as later adjustments, established the regulations whereby the transfer of the pest with coniferous wood and plants from the infested zone to pest-free areas in Portugal and in other countries should be prevented. At the same time, member states of the EU were obliged to conduct surveys in their countries to officially determine the distribution of PWN. The diagnostic protocol for B. xylophilus, which had been developed by EPPO, would facilitate identification. Many of the non-EU countries in Europe also decided to examine their pine stands. The results of our research from previous years indicated that B. xylophilus had not, until the discovery in Portugal, been found in any European country.” In order to prevent further introductions of the dangerous pest into Europe with packing wood, the EU Commission decided, on March 12th,, 2001, to apply immediate but temporary measures (2001/219/EG) requiring packaging wood from Canada, USA, China and Japan to be specifically treated by heat, fumigation or pressure impregnation and to be marked by the exporting country to show the origin and treatment applied. Consignments from China had to be accompanied by a phytosanitary certificate indicating the origin of the packing wood and the identity of the executer of the measures carried out. Furthermore, the member state receiving the consignment must confirm by sampling that the required conditions have been met. But these requirements would not be sufficient. It has long been clear that packaging wood is often re-used and, therefore, moves around the world, and that Bursaphelenchus spp. survive such travel, as do many other pests. Because the origin of wood packaging mate242

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rial is often difficult to determine, globally applied measures are necessary in order to significantly reduce the risk of pest spread. FAO Guidelines for Regulating Wood Packaging Material in International Trade (ISPM 15: FAO, 2003) are recognized as the basis for phytosanitary measures applied by members of the World Trade Organization (WTO); non-contracting parties are also encouraged to observe these standards. These standards describe phytosanitary measures to reduce the risk of introduction and/or spread of quarantine pests associated with packaging material (including dunnage, crating, pallets, packing blocks, drums, cases etc.) made of coniferous and non-coniferous raw wood, in use in international trade.” Helen Braasch: “After my retirement, I conducted a course in China on the identification of Bursaphelenchus spp., together with Wolfgang Burgermeister and Thomas Schröder of the BBA Braunschweig; this took place in October, 2002 in Shanghai and Nanjing, and was organised by Professor Maosong Lin of Nanjing. The course led to the development of useful collaboration with several Chinese scientists, especially with Jianfeng Gu of the Technical Centre, Ningbo Entry-Exit Inspection and Quarantine Bureau, Ningbo, Zhejiang, China. Almost all wooden packages imported through Ningbo harbour since 1997 have been sampled and inspected. Bursaphelenchus xylophilus has been detected many times in large numbers in wood samples from different countries, and a considerable number of other Bursaphelenchus species, among them several undescribed species, were found. The results are alarming: The percentage of batches of packing wood containing any nematodes averaged 21.3% (between 2000 and 2005), despite the claims on the accompanying phytosanitary certificates that the wood had been heat treated. The fact of recording B. xylophilus in 40 (1.2%) out of 3416 samples from eleven different countries or regions, including six countries where the pinewood nematode is not known to occur, underlines the necessity of rigorous application of international agreements on the phytosanitary treatment of packing wood in international trade. Furthermore, the effectiveness of the quarantine measures agreed upon should be investigated and their application should be controlled more intensively. In recent years, several new Bursaphelenchus species from East Asia have been added to the already large number within this genus. Is it possible that we may find more species of Bursaphelenchus that THE PINEWOOD NEMATODE: A PERSONAL VIEW

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are pathogenic to trees? An example of the difficulty of disease detection and diagnosis occurred in Vietnam (Lam Dong province) where symptoms similar to pine wilt disease, as well as dead Pinus kesiya trees were found in several locations, and were apparently associated with a Bursaphelenchus species (EPPO Reporting Service 2003). The disease was found in four locations in Lam Dong province in 36-48% of the trees. The species found in Vietnam is morphologically distinct from B. xylophilus but shares the same vector, M. alternatus.” Manuel Mota: “In 2001, my colleagues and I organized a scientific meeting in Évora, Portugal, for the international community working on the pinewood nematode in order to exchange views on recent research and to discuss control measures. About 50 researchers from 14 countries attended the symposium. It was noted that pine species native to North America and growing in the warmer regions are resistant to pine wilt disease, whereas two of the most common pine species native to Europe, Pinus sylvestris and P. pinaster, are highly susceptible to the disease and P. pinaster, in particular, occurs in the hotter, southern region. Pine wilt disease is a threat to pine forests in southern and possibly eastern Europe, and the predicted climatic changes, such as increasingly warm and unusual weather conditions, may significantly influence the incidence of pine wilt disease. The conclusions arrived at in Évora indicate that the final resolution for controlling pine wilt should rely on eradication of the nematode or resistance-breeding strategies. Control measures should be aimed at breaking the pine tree/pinewood nematode/pine sawyer disease triangle. Spraying of insecticides, trunk injection, cutting and destroying of trees presumed to be infected, restrictions in transporting wood, heat treatment and fumigation of timber are the control measures most applied in countries with pine wilt disease. In spite of these various efforts, however, the total amount of pine timber lost to the disease is not decreasing conspicuously. This is also true for the newly invaded region in Portugal. Therefore, strong quarantine measures and a particularly rigorous response at the early stage of any new occurrence of the pinewood nematode are essential to hinder the spread of this economically important disease in Europe. A better understanding of the inter-relationships between the nematode, its vectors and the host trees is clearly a precondition for limitation of damage by B. xylophilus. Additionally, an improvement 244

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in pest risk analysis systems would help to prevent further spread and, hopefully, the EU project ‘Development of improved Pest Risk Analysis techniques for quarantine pests, using pinewood nematode, Bursaphelenchus xylophilus, in Portugal as a model system’ (PHRAME) will refine PRA techniques. Future symposia of the leading scientists working in this field will give us ideas for better managing the problem in all its aspects.” Helen Braasch: “An excellent monograph on PWN resulted from the conference in Évora in 2001, containing all the presentations. I am confident that researchers working in this field will find the booklet invaluable. But I hope also that they do not look too closely at the picture of Bursaphelenchus on the cover; it is not a picture of B. xylophilus! But maybe only someone with my continuing fascination with the taxonomy of the pinewood nematode and its relatives would notice.”

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16. HISTORY OF THE DEVELOPMENT OF NEMATODES AS BIOCONTROL AGENTS PARWINDER S. GREWAL Department of Entomology, Ohio State University, Wooster, Ohio, USA.

& HARRY K. KAYA Department of Nematology, University of California at Davis, Davis, California, USA.

Nematodes attack a wide range of organisms, and in doing so, can be detrimental or beneficial to humans. They parasitize plants and animals, including many invertebrates, and as many invertebrates are pests, those nematodes that attack them are beneficial to humans. Our focus is on those nematodes that benefit us, especially those that can be used in biological control against pest organisms. The earliest record of association between a nematode (worm – probably a mermithid parasite) and an invertebrate (grasshopper) was reported in the 16th century by Aldrovandus (see Stock, 2005, Journal of Invertebrate Pathology 89: 57–66). Subsequent insect-nematode associations were reported in the 17th to 19th centuries, but the use of nematodes as biocontrol agents did not occur until the 20th century when Steinernema glaseri was applied in an augmentative biological control program against larvae of the Japanese beetle that had been introduced into the USA (Glaser & Farrell, 1935, Journal of the New York Entomological Society 43: 345–371). A spectacular success was achieved when an insect-parasitic (entomogenous) tylenchid nematode species was used as a classical biocontrol agent against an exotic Sirex woodwasp species in Australia in the 1970s. In the mid-1980s, a steinernematid species was introduced as a classical biological control agent from Uruguay into Florida, USA for the control of an invasive mole cricket. However, the number of projects using entomopathogenic or entomogenous nematodes as classical biological control is limited. Commercial interest in using nematodes as biological insecti246

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cides surged in the 1980s. Private companies refined technologies for mass production of nematodes based on the initial research by university and government researchers. But mass production is only one facet of using nematodes as biocontrol agents. Both fundamental and applied research programs have provided a wealth of knowledge that has led to successful commercialization of these nematodes. For example, CSIRO scientists in Australia discovered the two different life cycles and morphologies of the tylenchid nematode used in classical biological control against the Sirex woodwasp which eventually led to the first commercial nematode product. In the USA, researchers demonstrated that a mermithid nematode had considerable promise against mosquitoes and was briefly commercialized in the 1970s. Although this effort failed, because of production/storage/transport problems and the discovery of a potent bacterium that was more effective against mosquitoes than the nematode, much information about the mermithid’s biology, host range and survival was obtained. In the late 1970s and early 1980s, research in mass production technology with entomopathogenic nematodes and efficacy tests against various soil insect pests paved the way to successfully commercialize the nematodes. Today, a number of companies are producing and marketing entomopathogenic nematodes in Asia, Europe, and North America, and there is interest in other parts of the world to produce these nematodes commercially. In the 1990s, a nematode species that parasitizes slugs was found in the United Kingdom and mass production and efficacy tests resulted in its commercial production. These accomplishments demonstrate that nematodes can be important biocontrol agents of insects and slugs. In fact, with the spread of the Sirex woodwasp into other parts of the world, the tylenchid nematode continues to be used as a classical biological control agent. Scientists continue to do novel research that furthers the use of nematodes as biological control agents. For example, using predatory nematodes as biocontrol agents against plant parasitic nematodes and plant pathogenic fungi has generated interest among researchers. In this chapter, we identify the nematodes and the researchers by providing a brief overview of events that led to the development of nematodes as biocontrol agents. In presenting this historical perspective, we could have discussed the significant findings chronologically as events occurred, but this would have resulted in a discontinuous presentation of several nematode species and pest organisms. A more HISTORY OF THE DEVELOPMENT OF NEMATODES AS BIOCONTROL AGENTS

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readable approach, which we will use, is to do a treatise on the development of biological control of insects and some other organisms by a range of nematode species. Accordingly, we divide our presentation into four categories – entomopathogenic nematodes, entomogenous nematodes, malacogenous (malacopathogenic) nematodes, and predatory nematodes. For more complete information of the various nematodes used as biocontrol agents, the reader is referred to “Nematodes as Biocontrol Agents” by Parwinder Grewal, Ralf-Udo Ehlers and David I. Shapiro-Ilan (eds) (2005).

Entomopathogenic nematodes Entomopathogenic nematodes belong to the families Steinernematidae and Heterorhabditidae which are mutualistically associated with bacteria in the genera Xenorhabdus and Photorhabdus, respectively. Once the infective juvenile nematodes enter a susceptible host, the mutualistic bacteria are released resulting in host death within 2 days, hence the term, entomopathogenic. The nematodes develop and reproduce within the insect cadaver, feeding on the mutualistic bacteria and degraded host tissues. There are now over 36 species of Steinernema and 10 of Heterorhabditis described from around the world. George O. Poinar Jr. wrote the first book on this topic, “Nematodes in Biological Control” in 1975. This was followed by “Entomopathogenic Nematodes in Biological Control” edited by Randy Gaugler and Harry K. Kaya (1990), “Entomopathogenic Nematology” by Randy Gaugler (ed.) (2002) and “Nematodes as Biocontrol Agents” by Parwinder Grewal, Ralf-Udo Ehlers and David I. Shapiro-Ilan (eds) (2005). Seminal discoveries that had significant impact on the commercial development of entomopathogenic nematodes are listed in Table 1, and we describe below some of the historic details behind some of these discoveries. Rudolf Glaser was the first to establish a culture of the entomopathogenic nematode, S. glaseri, and to conduct field trials for the control of the introduced Japanese beetle in New Jersey. The remarkable discovery of the symbiotic relationship between Steinernema and the bacterium Achromobacter nematophilus was made by George O. Poinar Jr. and G. M. Thomas (1966, Parasitology 56: 385). This bacterium was later renamed, Xenorhabdus nematophilus. Another nematode genus, Heterorhabditis, with biology similar to 248

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that of Steinernema, was described by George O. Poinar Jr. in 1976 (Nematologica 21: 463–470). The bacterial symbiont of this nematode was first described as X. luminescens, but was later transferred to a new genus, Photorhabdus. It is now understood that all Steinernema species have mutualistic symbioses with species of Xenorhabdus and all Heterorhabditis species with Photorhabdus species (see Noel Boemare’s chapter in Entomopathogenic Nematology, 2002). Indeed, the discovery of symbiosis between entomopathogenic nematodes and bacteria was a major turning point in the development of the nematodes as commercial biological control agents. Exploiting the discovery of the symbiotic relationship between the nematodes and bacteria, Robin Bedding (1981, Nematologica 27: 109–114) was the first to establish a successful mass-production system which has come to be known as a solid culture due to his innovative use of polyether polyurethane sponge as a three-dimensional support structure allowing nematodes to move through the matrix and provide air exchange. He demonstrated that the nematodes can be mass-produced on symbiotic bacteria by impregnating the sponge with an artificial diet. This led to the formation of the first commercial company, Biotech Australia, selling nematodes for control of the black vine weevil in Australia and Europe. The first commercial production of entomopathogenic nematodes in liquid culture was established by a team of researchers led by Milton Friedman at Biosys Inc., in Palo Alto, California. This was soon followed by MicroBio, a company based in Littlehampton, UK, which established liquid production of Steinernema feltiae. Ralf-Udo Ehlers (1998, Biocontrol 43: 77–86) led the development of the first commercial scale production of heterorhabditids in liquid culture. Formulation development for the application of entomopathogenic nematodes has been slow. John Webster and Joan Bronskill (1968, Journal of Economic Entomology 61: 1370–1373) were the first to use a water-holding polymer and a UV protectant mix with the nematodes for application. Robin Bedding (1988, World Patent No. WO 88/08668) developed a “clay sandwich” formulation in which nematodes were placed in layers of clay to remove surface water. This formulation formed the basis for the commercial introduction of the first entomopathogenic nematode product against the black vine weevil in Australia. Scientists at Biosys developed an alginate formulation in which sheets of calcium alginate spread over plastic HISTORY OF THE DEVELOPMENT OF NEMATODES AS BIOCONTROL AGENTS

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screens were used to trap nematodes. This formulation was introduced in the USA in the late 1980s. Continuous efforts to improve nematode formulations led Biosys to develop the first water dispersible granular formulation in which the nematodes were encased in 10–20 mm diameter granules consisting of a mixture of various types of silica, clay, cellulose, lignin, and starches (Silver et al., 1995, World Patent No. WO 95/0577). This was the first formulation in which up to 7 months of room temperature shelf-life was achieved for the commercially produced S. carpocapsae. Quality control is an important activity in the commercial success of any product in the market. Rick Miller (1989, Journal of Nematology 21: 574) reported the development of the first quality control method to assess the virulence of commercially produced S. carpocapsae. This method, called the one-on-one Galleria mellonella bioassay, spurred the development of several other methods to enhance nematode quality control. As nematodes can be easily applied using conventional pesticide application equipment, the advances in nematode application technology have been limited. P.S.P. Rao (1975, Indian Journal of Agricultural Science 54: 275) was the first to show that S. carpocapsae is compatible with certain insecticides and can be tank mixed. Research has occurred more recently on the effects of pumps, pressure differentials, contraction flow fields, agitation, and hydraulic nozzles on entomopathogenic nematodes. Although, nematodes are still not used extensively against foliar pests, studies have been conducted on the use of spinning disks and the application of additives such as desiccation and UV protectants. Harry Kaya and C. Nelson (1985, Environmental Entomology 14: 572–574) suggested that the nematodes could be applied to the soil in alginate gels for increased persistence, and this concept was commercialized in the application of nematodes to tree trunks. Demonstrations that the nematodes can be applied through trickle, center-pivot and furrow irrigation systems have enhanced the commercial utility of nematodes. It has been demonstrated that the infected insect cadavers can serve as slow release systems for nematodes (Jansson & Lecrone, 1994, Florida Entomology 77: 281–284) and formulating nematode infected cadavers for application has been attempted (Shapiro-Ilan et al., 2001, Journal of Invertebrate Pathology 78: 17–23). Studies demonstrating the safety of entomopathogenic nematodes to mammals and soil invertebrates were instrumental in 250

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obtaining exemption of registration requirements for their commercial use in the USA, while studies demonstrating the susceptibility of scores of insect pests to nematodes led to the introduction of nematode products in various markets round the world. Predictability of control was first addressed in a seminal paper summarizing data from 82 field trials conducted on the use of nematodes against white grubs by Georgis, R. and Gaugler R. (1991, Journal of Economic Entomology 84: 713–720). A major advance in the field of entomopathogenic nematology has been the recognition of dichotomy in the host-finding behavior of entomopathogenic nematode species. Studies, particularly in Professor Gaugler’s laboratory, have shown that nematode species that use the ambushing type of foraging behavior are better adapted to finding and parasitizing hosts that are highly mobile and remain on the soil surface while nematode species that use the cruising type of foraging behavior are more adapted to finding and parasitizing sedentary hosts that feed deep in the soil. These studies have been instrumental in effective matching of the appropriate nematode species with the target insect pests for most effective insect control.

Entomogenous nematodes Entomogenous nematodes are true parasites that do not kill their hosts quickly. The infective nematode enters a host and obtains nutrients, affecting host fitness by reducing fecundity or causing sterility as well as reducing longevity, flight or causing other aberrant behaviors. Nematodes in this group are diverse and include the families Neotylenchidae [e.g., Beddingia (= Deladenus)] and Mermithidae (e.g., Romanomermis). Unlike the neotylenchids, the mermithids kill their hosts when they exit as 4th stage post-parasites. Although there are a large number of entomogenous nematodes including those in the family Allantonematidae (e.g., Thripinema), they have not been used extensively in biological control programs. The best example of successful use of entomogenous nematodes in biocontrol is Beddingia siricidicola against the woodwasp, Sirex noctilio (see Bedding & Iede, 2005, in Nematodes as Biocontrol Agents). The nematode, B. siricidicola, was initially discovered parasitizing S. noctilio in New Zealand in 1962, and was later described as a new species by Bedding (1968, Nematologica 14: 515–525). HISTORY OF THE DEVELOPMENT OF NEMATODES AS BIOCONTROL AGENTS

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Bedding (1967, Nature 214: 174–175) made the startling find that this nematode had two, complex and separate life cycles. It has a freeliving and a parasitic life cycle with two types of morphologically different adult female. The use of B. siricidicola as a biological control agent against S. noctilio was greatly facilitated by the discovery of the free-living life cycle. This nematode was mass produced monoxenically on autoclaved, hydrated wheat in flasks that had been inoculated with the mutualistic fungus. Beddingia siricidicola was introduced experimentally into Tasmania in 1970. It established, spread, and achieved high levels of parasitism. By 1974, over 70% of the Sirex-infested trees contained nematodes and 90% of the emerging S. noctilio adults where parasitized by them. The following year, the number of killed trees dropped dramatically. In 1970, 1000 inoculated logs were sent from Tasmania to Victoria. Subsequently, millions of nematodes were sent to Victoria and released into the pine plantations. The nematode became established and was the major factor in suppressing S. noctilio populations. In Brazil, B. siricidicola from Australia were released in 1989. Bedding and Iede (2005, in “Nematodes as Biocontrol Agents”) reported that in one area where Sirex infestations were high, the nematode was released from 1990 to 1993 and resulted in levels of parasitism of 45% in 1991, 75% in 1992, and more than 90% in 1994. In 1995, it was difficult to find Sirex-infested trees in this area. There can be variation in nematode parasitism depending on the prevalence of Sirex infestation and the locality. Nematode parasitism varied from 17% to 65% in four different areas of Sirex infestation, but parasitism as high as 92% has been recorded from three other areas. Since 1995, only the Kamona strain has been released in Brazil. Although B. siricidicola has been a highly successful biocontrol agent against S. noctilio, human intervention is required to monitor and assist the spread of the nematode. In Australia, there is a National Sirex Coordination Committee, which developed a National Sirex Strategy. Detailed standard operating procedures for rearing, storing, formulation, and quality control have been established. In new areas of infestation, worksheets covering various aspects of Sirex control, including inoculation and distribution of nematodes, are needed to ensure continued success. Commercial developments for the entomogenous nematodes have been reviewed by Platzer et al. (2005, in “Nematodes as 252

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Biocontrol Agents”). In particular, nematodes belonging to the family Mermithidae have potential for the control of mosquito larvae. One species, Romanomermis culicivorax (syn. Reesimermis nielseni), which was initially isolated from mosquito larvae in Lake Charles, Louisiana and Gainesville, Florida (Petersen, 1985, in Plant and Insect Nematodes. W.R. Nickle (ed.), Marcel Dekker Inc., New York. pp. 797–820), was commercially available in the USA for a short period of time. At least two companies pursued the development and commercialization of this mermithid nematode. However, shipping and packaging problems (keeping the nematode eggs viable) plagued the first company and this along with financial constraints, caused the cessation of mermithid production. The second company was able to develop an effective shipping container after a couple of years of research, but changed its program before test marketing the container and dropped all biological control products. The discovery and eventual registration of another biological control agent for mosquitoes, Bacillus thuringiensis subsp. israelensis, in 1981, probably added to the decision not to produce R. culicivorax as a biological control agent for mosquitoes. Overall, the use of either R. culicivorax or R. iyengari as inundative biocontrol agents may not be cost effective in developed countries, but may have greater potential for use as inoculative agents for long-term control.

Malacogenous (or Malacopathogenic) nematodes Only one nematode species has been commercially developed for the control of pest molluscs. This species, Phasmarhabditis hermaphrodita, was first described as being associated with the slug Arion ater. Although an artificial culture of P. hermaphrodita in ‘‘rotting flesh” was developed in the 1900s, the biocontrol potential of this species was not discovered until the 1990s when Wilson et al. (1993, Biocontrol Science and Technology 3: 503–511) patented the use of Phasmarhabditis nematodes as biological molluscicides, following a 5-year research program supported by MicroBio Ltd (now Becker Underwood). This company developed the first commercial product ‘NemaSlug’, in 1994, for sale in Europe for the control of slug and snail pests. Much of the early research on the biology, culture, host range, and biocontrol potential of P. hermaphrodita was conducted by Wilson and his associates. They noted that the nemaHISTORY OF THE DEVELOPMENT OF NEMATODES AS BIOCONTROL AGENTS

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todes produce an infective juvenile stage that enters the shell cavity of the slug where they multiply and cause the characteristic symptoms in the form of a swollen mantle. Methods used for mass-production and formulation of P. hermaphrodita are similar to those developed for entomopathogenic nematodes (see Wilson & Grewal, 2005, in Nematodes as Biocontrol Agents). Improvements in application methods and field efficacy of P. hermaphrodita have been demonstrated against several slug species in diverse cropping systems and horticultural settings (see Ester & Wilson, 2005, in Nematodes as Biocontrol Agents).

Predatory nematodes Nematophagy Cobb (1917, Soil Science 3: 431–486) was the first to note predatory activity among nematodes. He reported that some mononchid nematodes ferociously feed on other small animals in the soil, including nematodes. Predatory nematodes belong to several orders including Mononchida, Dorylaimida, Diplogasterida, Aphelenchida, Enoplida, and Rhabditida. Although mononchs have several limitations as inundative biocontrol agents, studies have demonstrated that they are natural enemies of plant parasitic nematodes. Bilgrami and Brey (2005, in Nematodes as Biocontrol Agents) make a good case for commercial development of predatory nematodes for the control of plant-parasitic nematodes. Mycetophagy Nematodes in the genera Aphelenchus, Aphelenchoides, Filenchus, Iotonchium, and Tylenchus are some of those known to feed on diverse species of fungi. Apart from their role in nutrient cycling, some species have the potential to reduce intensity and incidence of root diseases caused by fungi. Most studies have concentrated on Aphelenchus avenae which is ubiquitous in temperate zones and feeds on over 76 species of fungi. Mankau and Mankau (1962, Phytopathology 52: 741) reported on the ability of A. avenae to reproduce on phytopathogenic fungi, and Barker (1964, Plant Disease Reporter 48: 428–432) reported a reduction of Rhizoctonia solani induced disease on plants due to feeding by A. avenae. Ishibashi et al. (2000, Japanese Journal of Nematology 30: 8–17) were the first to report on the development of mass-production of 254

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A. avenae for commercial application. Ishibashi and his colleagues have conducted much of the recent research on field efficacy of A. avenae in controlling fungal pathogens of plants, and he makes a convincing case for further evaluation and the potential development of A. avenae as a prophylactic biocontrol agent of root diseases of plants caused by soil borne fungi (see Ishibashi, 2005, in Nematodes as Biocontrol Agents). Bacteriophagy Bacteriophagy is common in rhabditid and diplogastrid nematodes in the soil. However, there are reports of both a decrease and an increase in bacterial biomass in the presence of bacteria-feeding nematodes. Grewal (1991, Annals of Applied Biology 118: 47–55) has demonstrated the potential of the bacteria-feeding nematode, Caenorhabditis elegans, to spread the antagonistic bacterium, Pseudomonas flourescens, for suppressing the bacterial blotch disease of mushrooms, caused by Pseudomonas tolaassii. This area definitely requires additional research.

Table 1. Some seminal discoveries that led to commercial development of entomopathogenic nematodes as biocontrol agents. Discovery

Year

Reference

Discovery of Steinernema

1923

Steiner, G., Zentralblatt für Bakteriologie,Parasitenkunde, Infektionskrankheiten und Hygiene Abt. 1 orig., 59: 14.

Establishment of Steinernema in culture

1931

Glaser, R.W., Science 73: 614.

Susceptibility of the first pest species

1932

Glaser, R.W., New Jersey Department of Agriculture. Circ. 211.

First field application against a pest

1935

Glaser, R.W., Farrell, C.C., Journal of New York Entomological. Society 43: 345–371.

Discovery of a symbiotic bacterium

1966

Poinar, G.O., Jr., Thomas, G.M., Parasitology 56: 385.

First prototype formulation

1968

Webster, J.M., Bronskill, J.F., Journal of Economic Entomology 61: 1370–1373.

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256

Desiccation, survival of Steinernema

1973

Simmons, W.R., Poinar, G.O. Jr., Journal of Invertebrate Pathology 22: 228–230.

Safety to mammals

1975

Poinar, G.O., Jr., In: Entomogenous Nematodes. E. J. Brill, Leiden.

Description of the Galleria-bait technique

1975

Bedding, R.A., Akhurst, R.J., Nematologica 21: 109–110.

Pesticide compatibility of Steinernema

1975

Rao, P.S.P., Indian Journal of Agricultural Sciences. 54: 275.

Establishment of the first commercial mass-production system (solid culture)

1981

Bedding, R.A., Nematologica 27: 109–114.

First demonstration of effective control of black vine weevil with nematodes

1981

Bedding, R.A., Miller, L.A., Annals of Appied. Biology 99: 211–216.

Encapsulation of nematodes in calcium alginate

1985

Kaya, H. K., Nelsen, C.E., Environmental Entomology 14: 572–574.

Development of liquid culture of nematodes

1986

Pace, G.W., Grote, W., Pitt, D.E., Pitt, J.M., World Patent No. WO 86/01074.

Development of clay-based formulations

1988

Bedding, R. A., World Patent No. WO 88/08668.

Development of the one-on-one quality control bioassay

1989

Miller, R., Journal of Nematology 21: 574.

Establishment of the first commercial liquid mass-production for Steinernema

1990

Friedman, M.J., In: Gaugler R., Kaya, H.K., (eds.), Entomopathogenic Nematodes in Biological Control. CRC Press, Boca Raton, Florida, pp. 153–172.

Safety to invertebrates

1991

Georgis, R., Kaya, H.K., Gaugler, R., Environmental Entomology 20: 815–822.

HISTORY OF THE DEVELOPMENT OF NEMATODES AS BIOCONTROL AGENTS

Development of water dispersible granules

1995

Silver, S.C., Dunlop, D.B., Grove, I.D., World Patent No. WO 95/0577.

Establishment of the first commercial liquid mass-production for Heterorhabditis

1998

Ehlers, R.-U., Lunau, S. Krasomil-Osterfeld, K., Osterfeld, K.H., Biocontrol 43: 77–86.

Synergism with nicotinoid insecticides

1998

Koppenhöfer, A.M., Kaya, H.K., Journal of Economic Entomology 91: 618–623.

Development of the sand-well bioassay

1999

Grewal, P.S., Converse, V., Georgis, R., Journal of Invertebrate Pathology 73: 40–44.

First assessment of the quality of commercially produced nematodes

2000

Gaugler, R., Grewal, P., Kaya, H.K., Smith-Fiola, D., Biological Control 17: 100–109.

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17. DYNAMICS OF NEMATOLOGICAL INFRASTRUCTURE ROLAND N. PERRY Plant Pathogen Interactions Division, Rothamsted Research, Harpenden, Hertfordshire, UK

& JAMES L. STARR Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas, USA

Introduction In common with all scientific groups, exchange of research and associated information in nematology relies to a considerable extent on journals, newsletters and conferences as essential components of an interlinking infrastructure. In the last 50 years, there have been several important changes and developments to facilitate information exchange, the most important of which is the advent of the internet and the associated repository of freely available information, amongst which molecular data are arguably the most significant. In this short overview, we aim to trace the development of the different aspects of the infrastructure of the European Society of Nematologists (ESN) and the Society of Nematologists (SON), and make some educated guesses about future changes.

Societies The first international conference for nematology (called the ‘International Nematology Symposium and Training Course’) was held at Rothamsted Experimental Station (now Rothamsted Research) in Harpenden, UK in 1951. Twelve subject areas were covered, each being examined in relation to Heterodera (=Globodera) 258

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rostochiensis, Ditylenchus dipsaci, Aphelenchoides spp., and Pratylenchus spp. The 46 participants came from different European countries and also from countries such as Egypt, Haiti, Indonesia, Uganda; USA nematology was represented by Gerald Thorne. The meeting was notable for including a one-day excursion to Cambridge. This inclusion of a tour day, in which the spouses and family of nematologists also participate has become an enjoyable, standard feature of almost all conferences organised by the ESN. However, it was not until 1953, at the International Congress of Zoology in Copenhagen, that the idea of a European nematology society was envisaged. Two years later at the ‘International Symposium on Plant Nematodes and the Disease they Cause’ in Wageningen, Prosper Bovien from Denmark was a prime mover in establishing the Society of European Nematologists, as it was initially called. The minutes of the discussion record that terms such as ‘white cysts’ and ‘nematocide’ should be avoided. The first meeting of the Society of European Nematologists took place in Hamburg 1957 and was numbered subsequently as the 4th International Symposium for Nematology. During the 9th European symposium, in Warsaw in 1967, the general meeting decided to change the name of the society from the Society of European Nematologists to the European Society of Nematologists. In 1958, the US nematologists in the American Phytopathological Society discussed the formation of a separate society. The first officers of the SON were elected in 1961, with Merlin Allen as the President. The first meeting was held the following year at Oregon State University. Unlike the ESN, which holds meetings biennially (Table 1), the SON meetings are held annually (Table 2). The initial meetings of the SON, in the 1960s and 1970s, were held predominately on college campuses, whereas for the last 15 years the majority of the meetings have been held in luxury hotels. There is a greater tendency amongst North American nematologists, compared with nematologists from elsewhere, to incorporate the conference into a family vacation. Many of the ESN meetings have included accommodation in University halls of residence, which in recent years have been built more specifically to attract conferences and provide en suite facilities, a great rarity in the early years. The frequent encounters in those years with distinguished scientists plodding down the hall to a common bath/shower facility was a great equaliser, although not always appreciated by some senior nematologists! DYNAMICS OF NEMATOLOGICAL INFRASTRUCTURE

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Table 1. List of the International Symposia of the European Society of Nematologists. 1st Symposium 2nd Symposium

3rd Symposium

4th Symposium

5th Symposium 6th Symposium 7th Symposium 8th Symposium 9th Symposium

10th Symposium 11th Symposium 12th Symposium 13th Symposium 14th Symposium

15th Symposium 16th Symposium 17th Symposium

18th Symposium 19th Symposium 20th Symposium

260

Harpenden, England, UK Copenhagen, Denmark (part of the International Congress of Zoology) Wageningen, The Netherlands (part of the International Symposium on Plant Nematodes and the Diseases they Cause) Hamburg, Germany (the first meeting after the official formation of the Society of European Nematologists) Uppsala, Sweden Gent, Belgium Auchincruive, Scotland, UK Antibes, France Warsaw, Poland (the Society’s name was changed to the European Society of Nematologists) Pescara, Italy Reading, England, UK Granada, Spain Dublin, Ireland Munich, Germany (as the Nematology section of the 3rd International Congress of Plant Pathology) Bari, Italy St Andrews, Scotland, UK Guelph, Canada (as part of the 1st International Congress of Nematology) Antibes-Juan-les-Pins, France Uppsala, Sweden Veldhoven, The Netherlands (as part of the 2nd International Congress of Nematology)

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1951 1953

1955

1957

1959 1961 1963 1965 1967

1970 1972 1974 1976 1978

1980 1982 1984

1986 1988 1990

21st Symposium 22nd Symposium 23rd Symposium

24th Symposium 25th Symposium 26th Symposium

27th Symposium 28th Symposium

Albufeira, Portugal 1992 Gent, Belgium 1994 Guadeloupe, Antilles 1996 (as part of the 3rd International Congress of Nematology) Dundee, Scotland, UK 1998 Herzliya, Israel 2000 Tenerife, Canary Islands 2002 (as part of the 4th International Congress of Nematology) Rome, Italy 2004 Blagoevgrad, Bulgaria 2006

Table 2. List of the Meetings of the Society of Nematologists. 1st Meeting 2nd Meeting 3rd Meeting 4th Meeting 5th Meeting 6th Meeting 7th Meeting 8th Meeting 9th Meeting 10th Meeting 11th Meeting 12th Meeting 13th Meeting 14th Meeting 15th Meeting 16th Meeting 17th Meeting 18th Meeting 19th Meeting 20th Meeting 21st Meeting 22nd Meeting

Corvallis, Oregon Amherst, Massachusetts Boulder, Colorado Urbana, Illinois Daytona Beach, Florida Washington, D.C. Columbus, Ohio San Francisco, California Washington, D.C. Ottawa, Canada Raleigh, North Carolina Minneapolis, Minnesota Riverside, California Houston, Texas Daytona Beach, Florida East Lansing, Michigan Hot Springs, Arkansas Salt Lake City, Utah New Orleans, Louisiana Seattle, Washington Knoxville, Tennessee Ames, Iowa

1962 1963 1964 1965 1966 1967 1968 1969 1970 1971 1972 1973 1974 1975 1976 1977 1978 1979 1980 1981 1982 1983

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23rd Meeting

24th Meeting 25th Meeting 26th Meeting 27th Meeting 28th Meeting 29th Meeting

30th Meeting 31st Meeting 32nd Meeting 33rd Meeting 34th Meeting 35th Meeting

36th Meeting 37th Meeting 38th Meeting 39th Meeting 40th Meeting 41st Meeting

42nd Meeting 43rd Meeting 44th Meeting 45th Meeting

262

Guelph, Canada (as part of the 1st International Congress of Nematology) Atlantic City, New Jersey Orlando, Florida Honolulu, Hawaii Raleigh, North Carolina Davis, California Veldhoven, The Netherlands (as part of the 2nd International Congress of Nematology) Baltimore, Massachusetts Vancouver, Canada Nashville, Tennessee San Antonio, Texas Little Rock, Arkansas Guadeloupe, Antilles (as part of the 3rd International Congress of Nematology) Tucson, Arizona St Louis, Missouri Monteray, California Quebec City, Canada Salt Lake City, Utah Tenerife, Canary Islands (as part of the 4th International Congress of Nematology) Ithaca, New York Estes Park, Colorado Fort Lauderdale, Florida Kauai, Hawaii

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1984

1985 1986 1987 1988 1989 1990

1991 1992 1993 1994 1995 1996

1997 1998 1999 2000 2001 2002

2003 2004 2005 2006

Fig. 1. Roland Perry (left), current co- Editor-in-Chief of Nematology and James Starr (right), current Editor-in-Chief of Journal of Nematology and past President of the Society of Nematologists.

The SON council is more structured than the equivalent in ESN. As well as an executive board of the SON, with elected and nominated members, there are numerous committees that make recommendations on various aspects from awards to specialist topics and policy. The President of the Society is elected by the Society’s members. Traditionally, Presidents and executive board members have been from North America, although Roland Perry (Fig. 1) became the first person from Europe to serve on the executive board (2000–2003) and it is possible that a future President may be from the wider membership outside North America. The ESN President is the person in whose country the next ESN meeting will take place. Thus, participants at the annual general meeting vote on the venue for the next meeting and the President is from the country of the successful bid. The board of ESN comprises people nominated by the membership and recently has included a nematologist from the USA; currently this is James Starr (Fig. 1). An essential component of all scientific meetings is the availability of an appropriate place where liquid refreshment facilitates exchange of information and discussion of research ideas. This has always been a challenge on US college campuses but changes, especially the provision DYNAMICS OF NEMATOLOGICAL INFRASTRUCTURE

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of microbreweries, have been beneficial. Such changes are not just confined to campuses: in 1979, the SON meeting was held in Salt Lake City and it was an extreme challenge to find any suitable establishment but, by contrast, when the Society met there again, in 2001, there were two microbreweries in the city and several ideal bars. In European conferences, there were few problems finding suitable bars. The 14th symposium in Munich in 1978, which formed part of the 3rd International Congress of Plant Pathology, was memorable for the welcome reception in the famous Hofbräu Haus! This meeting included, for the first time, posters and a paper on biological control. Looking through the programmes over the years, it is evident that the emphasis in the early years on papers about chemical control gradually changed, and by the 15th symposium in Bari, in 1980, there was a session on alternative control methods. This trend has continued in meetings of both ESN and SON and in recent conferences there are few papers on chemical control per se. As the societies developed there were increasing interactions among individuals and groups from Europe and North America and the advent of relatively cheap flights, especially within Europe, generated closer links among nematologists world wide. Several individuals perceived a need for an official forum to link the nematology societies more closely. This was partially realised when the three largest societies, the ESN, the SON and the Organisation for Nematologists of Tropical America (ONTA; organized in Puerto Rico in 1967 and first meeting in 1968), joined in the organization of the 1st International Congress of Nematology in Guelph, Canada in 1984. The growing awareness of the adverse effects of nematicides was reflected in a colloquium, at the Congress, on ‘Pesticides in groundwater’. At the Congress dinner, one enthusiastic after-dinner speaker, whose native language was not English, caused some amusement when he exhorted the audience to publicise the science of nematology by telling them to “go out and expose yourselves”! During this Congress the demand for an integrated forum resulted in a proposal for an International Nematological Society. Subsequently, the second International Congress of Nematology, at Veldhoven, The Netherlands in 1990, established a pattern of holding these meetings every six years. The 21st ESN Symposium in Albufeira, Portugal in 1992, after the “Iron Curtain’ had been lifted, was notable for the plenary address from Eino Krall on ‘A personal perspective on nematology in 264

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Eastern Europe”. The gradual incorporation of molecular and biochemical studies into mainstream nematology is evident from the programme and was further developed in the 22nd ESN Symposium in Gent, Belgium, 1994. Belgium is justly famous for its extensive range of excellent beers and the beer and cheese evening, with 11 different types of beer, certainly stimulated some animated discussions, some of which were scientific. A similar effect was achieved at the 24th Symposium, in Dundee in 1998, with whisky as the essential catalyst. In between these two meetings, the third International Congress of Nematology was held in luxurious surroundings in Guadeloupe, French West Indies, in 1996. A hurricane and an earthquake caused some problems during the first couple of days, with the welcome reception being cancelled and several delegates unable to travel. The hardy individuals who endured the initial travails were rewarded with a scientifically stimulating congress in a most revealing tropical setting. The lifting of the Iron Curtain had a positive effect on some other nematology societies. In 1995, The Russian Society of Nematologists was able to hold its 1st English Language International Symposium in St. Petersburg with 30 invited delegates from the West. This was very successful and subsequent conferences have been held biennially with delegates also from outside Russia. These meetings have been instrumental in underpinning joint research projects between scientists from Europe and the USA and Russia. The increased interaction between nematology societies and the success of the first three International Congresses of Nematology, were facilitated by the establishment of the International Federation of Nematology Societies (IFNS) as a global communications forum with the stated aim ‘to foster communication among nematologists world-wide’ (www.ifns.org). IFNS comprises 14 individual societies with a total of about 2,500 members (Table 3). There was a long gestation period (the seed of the idea was sown at the 1st International Congress of Nematology in Guelph, Canada in 1984) before the birth of the IFNS because of differences in the decision making processes of the various societies. The establishment, in 1996, and success of the Federation were due primarily to the hard work, tact and persistence of Kenneth Barker (Fig. 2), of North Carolina State University, who became the first President of the IFNS. An enjoyable and scientifically excellent 4th International Congress of Nematology was held in Tenerife, Canary Islands in 2002 and the 5th is to be held in DYNAMICS OF NEMATOLOGICAL INFRASTRUCTURE

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Brisbane, Australia in 2008. This will be the first opportunity for the nematology societies to meet in Australia, and it should provide an ideal forum for enhancing links between nematologists from Australasia and other parts of the world. Table 3. The 14 nematology societies which are affiliated to the International Federation of Nematology Societies. Afro-Asian Society of Nematologists Australasian Association of Nematologists Brazilian Nematological Society Chinese Society of Plant Nematologists Egyptian Society of Agricultural Nematology European Society of Nematologists Italian Society of Nematologists Japanese Nematological Society Nematological Society of India Nematological Society of Southern Africa Organization of Nematologists of Tropical America Pakistan Society of Nematologists Russian Society of Nematologists Society of Nematologists The International Congresses of Nematology have been very successful, despite some initial growing pains, caused mainly by individual societies insisting on adhering to their own traditional formats. This was especially evident in the banquets of the first two congresses where the after-dinner presentations were inordinately long and tedious. Sense has prevailed and the banquets are now an enjoyable part of the meetings. Undoubtedly, the International Congresses will continue to be important adjuncts to the conferences of individual societies.

Newsletters Society newsletters have traditionally been used to convey to the membership minutes of executive meetings, announcements of future conferences and general items of interest to members. The ESN newsletter (initially called Nematology News) was first sent to 266

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Fig. 2. Kenneth Barker, first President of the International Federation of Nematology Societies.

Fig. 3. Michel Luc, founding editor of Revue de Nématologie and a current member of the editorial board of Nematology.

forty colleagues as a few typed A4 sheets stapled together. Subsequently, in the first issue of Nematologica, Nematological News was incorporated as an appendix of the journal. Later it was separated again, and in the 1990s it developed into a much more extensive and professionally produced document, including summaries of Ph.D., theses, articles commenting on specific aspects of nematology and news from various centres of nematology. The latter items were often a catalogue of overseas trips made by travelmad nematologists and degenerated, in some places, to one-upmanship contests! The ESN newsletter is now much slimmer and more focused than in previous years, with interesting contributions such as laboratory profiles. The SON newsletter is somewhat similar in content to the ESN newsletter but is used more frequently as a forum for discussion of specific topics; for example, correct terminology of races, pathotypes and strains etc., journal publication policy and scientific ethics. Both newsletters are now available as electronic versions and sent by e-mail. Although, newsletters still have a role in alerting the membership to meetings of interest, their future existence as hard copy offerings is in doubt. The advent of the internet and the Societies’ websites now fill much of the previous role of newsletters. DYNAMICS OF NEMATOLOGICAL INFRASTRUCTURE

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Journals The primary method of information exchange of any scientific discipline is through the printed word via scientific journals. This is still true, although, as we discuss below, the use of electronic transfer of information may supersede the traditional journal format. For plant nematology, the importance of information transfer with developing countries still justifies the traditional methods. It is always slightly frustrating that nematology as a discipline is divided into different subdisciplines, such as plant nematology, animal nematology (usually as a component of parasitology), Caenorhabditis elegans groups, free-living nematology and, increasingly, entomopathogenic nematology. Each sub-discipline has its preferred conferences and the fragmentation is also reflected in the journals, as each group has its own preferred journals for publishing. In universities, nematology is rarely taught as a separate discipline, but the sub-disciplines are often included as components of other courses. In the USA, plant nematology is usually taught as part of a plant pathology degree course, whereas in the UK and some other parts of Europe, plant nematology was included as a topic in zoology and biology degree courses, often as a “pure science” discipline. In the past, this was reflected in a more field-based approach to nematology research in the USA, whereas the European nematologists had a far more laboratory-based, pure science research approach. In the past, the fragmentation of nematology had three major effects. First, research developments in one area, such as animal nematology, were often not utilised in others, such as plant nematology. In the past, a paper given by a plant nematologist at a parasitology meeting would result in the speaker being deafened by the noise of people exiting the lecture theatre. Now, the use of common molecular techniques to examine a wide range of host-parasite interactions, for example, means that the research on plant parasitic nematodes has more universal appeal. Second, the content of some journals reflected, to a great extent, only one of the sub-disciplines of nematology. Thus, reliance on abstracting services, e.g. CABI abstracts, was necessary to keep abreast of the literature. As the electronic era progressed, more effective access to a wide range of other journals was ensured. Now, although work on molecular biology of plant parasitic nematodes may be published in molecular journals, it is a simple task to access the information quickly on-line. The third effect is that the journals publishing most of the plant nematology papers have a low impact factor. 268

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In the present era, when impact factors are considered (by administrators and grant review panels!) to be of paramount importance as indicators of research output quality, such journals may not be able to attract the top quality papers, especially those with molecular biology content. However, they are still an important part of the nematology infrastructure and it is interesting to examine the evolution of the journals and to consider their possible future. The two main international journals for papers on plant nematology are currently Nematology and the Journal of Nematology, with Nematropica, the Russian Journal of Nematology (first appeared in 1993) and Nematologia Mediterranea (1973) providing a forum that includes papers on more regional aspects. Nematology is owned and published by Brill, The Netherlands, and there are no page charges for publication but a high subscription fee. By contrast, Journal of Nematology (JON) is owned by the Society of Nematologists and imposes page charges but is sent to all members of the society as a part of the modest membership fee. In the 1970s and 1980s, many European universities and research establishments were reluctant to finance page charges if a paper could be published at no cost elsewhere. Consequently, there were few papers from European authors in JON. The situation has changed slightly as research scientists have to obtain grants to justify their continued existence and grant money can, in some cases, provide European nematologists the opportunity to fund publication costs. In the future, journals, whether wholly on-line or hard copy and on-line, may move towards the JON format, with the authors bearing the publication costs and the journals provided free of charge. JON first appeared in 1969 with Seymour D. Van Gundy as its first editor. The first issue of Nematology was published in 1999, but the history of this journal goes back to the 1950s when the journal Nematologica (also published by Brill) first appeared, in 1956, with J.H. Schuurmans Stekhoven as Editor-in-Chief. Nematologica was established as a journal to cover the field of nematology in general, except for papers on medical and veterinary subjects. It attracted papers on plant nematology that previously would have been submitted to the Journal of Helminthology, a journal that still exists but which is now more mainstream parasitology. In 1978, Michel Luc (Fig. 3) started a second journal, published in France by the Office de la Recherche Scientifique et Technique d’Outre-Mer (ORSTOM), called Revue de Nématologie, with an A4 format (compared to the DYNAMICS OF NEMATOLOGICAL INFRASTRUCTURE

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smaller Nematologica). Revue de Nématologie quickly became a very successful journal but its title counted against it. At one promotion interview in the UK, the candidate was asked why he published in a French national journal; it took some persuasion to convince the questioner that Revue de Nématologie was a well respected international title. Partly because of this type of perception and also to ensure that the journal was included in Current Contents, the title was changed, in 1992, to Fundamental and Applied Nematology, published by Gauthier-Villars – ORSTOM and, subsequently, Elsevier. When Brill acquired the title, Fundamental and Applied Nematology was amalgamated with Nematologica to form the present journal, Nematology. From 1987 until 2001, the SON published also the Annals of Applied Nematology, which was a single issue per year produced as a supplement to the Journal of Nematology. Annals of Applied Nematology served as an outlet for strictly applied aspects of plant nematology. Publication ceased because of insufficient submissions to support both SON publications. Just prior to this action, the JON changed to the larger A4 page format and discontinued its traditional orange coloured cover. These changes caused great concern to some members of the society! In terms of impact factors, neither Nematology nor JON rates very highly, rarely breaking the 1.0 barrier, but this is not a reflection on the quality of the science. In part, it is due to the small number of plant nematologists but it also is a consequence of the need for scientists to publish in high impact journals, and many of the papers on molecular nematology, for example, are published elsewhere. However, on the plus side, the citation life of papers in the plant nematology journals is long. On-line publishing is likely to be the scenario of the future, although there will continue to be a demand for hard copies of the journals for several more years. However, with libraries increasingly cutting back on journal subscriptions and the escalating cost of publishing, production of hard copies of nematology journals may, in the long term, not be economically viable.

Websites In the 1990s, both ESN (www.esn-online.org) and SON (www.nematologists.org) set up websites. These are gradually taking over the role of newsletters and they also provide registration facilities for confer270

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ences and access to relevant sources of information. Manuscript submission for JON is now through the SON website. Such sites will become more important in the future. With the demise of the Annals of Applied Nematology and the concomitant need for a place to publish large data sets, such as results of germplasm screening and nematode distribution, websites are likely to become the medium of choice. This mirrors the access to genetic information, such as DNA and protein sequence data, that is freely available on the web. Electronic communications, whether to replace journals or for access to data sets, may take some years to realise fully their potential. In part, this is because the Societies have to consider the needs and resources of all interested parties and, in part, because of the undoubted reluctance of some nematologists to embrace the advances of the 21st century.

The future Both the ESN and the SON, independently and as part of the INFS, have a bright future in our opinion. Clearly, as we have alluded to in the above sections, electronic communication will be central to the future infrastructure of the Societies but conferences will continue to be essential. One aspect we have not mentioned: the fact that all facets of communication essential to science depend on the time and effort of volunteers. Moving to a predominantly electronic forum will not ease this burden. The present generation of nematologists should be grateful to all those who have ensured the healthy development of the ESN and SON and the journals over the past 50 years. The future will still depend on the goodwill and effort of the next generation of nematologists to keep the Societies and their associated activities alive and active. At the end of the day, we all get fun out of nematology!

Acknowledgements We are grateful to Maurice Moens, Merelbeke, Belgium for making available the text of his address to the ESN meeting in Israel on the history of the ESN.

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18.

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(i) A NEMATOLOGY DREAM: MISCALCULATIONS AND FALSE PROPHECIES? ERNEST C. BERNARD Department of Entomology and Plant pathology, The University of Tennessee, Knoxville, Tennessee, USA

Predicting the course of a science over the next 50 years is a hopelessly risky business, and is more likely to produce laughter at the 2057 meetings of the nematology societies than acknowledgement of a (by then very old) nematologist’s gift of prophecy. I do have some meager credentials, however, as I made a few predictions for the following ten years at the 1993 APS/SON meeting in Nashville. Those predictions have come at least partially true, although I must concede that they were not difficult to foresee. Ten years and fifty years are vastly different lengths of time. When I was born, the Korean War had just erupted. There was no interstate highway system in the U.S. at that time, and Studebakers, DeSotos, and Packards were still being manufactured, B.G. Chitwood had just published a paper that placed root-knot nematode taxonomy onto a sound footing, soybean cyst nematode was not yet known in the U.S., and trichodorids were not recognized as significant plant pathogens. Advances did not come at such a rapid rate as they do now. The pace of all aspects of life was much slower and more relaxed 50 years ago. Do we willingly speed up our pace as we grow older or do we do it because we must? I tend to believe that our sense of life’s pace is set early in our lives, and so I suspect that the younger contributors to this compilation may do a better job of hitting the technical high spots as they gaze into the crystal ball. I could beat the original Mario Brothers, but I don’t even try the stuff my sons play. The advantage to nematologists who span eras is that they may see the whole picture a little more 272

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clearly, and can engage in sweeping generalities without being held to specifics! These generalities should provide lots of amusement for mid-century colleagues. The science of nematology is viable primarily because of the importance of crop-parasitic nematodes in agriculture. Without this economic connection we would be individuals in a zoology or ecology department with an absorbing interest in obscure creatures. Nematologists are products of educational systems and of societies that place great value on the application of knowledge to crop production and protection. Therefore, the future of nematology is tied closely to the future of agriculture, and as we cannot do away with agriculture, nematologists will always exist. The difficulty is knowing the course of agriculture. The ability to grow surplus (more than subsistence) crops is dependent on a suitable climate with sufficient rainfall, but at no time since the medieval Little Ice Age has humanity faced a challenge like the next 50 years will pose. If current scientific consensus is correct, runaway production of carbon dioxide will profoundly alter world agriculture by making some areas hotter and drier, and others hotter but wetter. Agricultural lands just a little above sea level may be inundated as the Antarctic and Greenland ice caps melt. I suspect the nematology lessons that are being learned now in dryland agriculture in Africa, Asia, and the American Southwest will be put to good use over a much larger area of the world. On the other hand, some water-loving crops will shift northward. The wetter and warmer Canadian Plains will become immensely important producers of soybeans that will still be in Maturity Groups 0-II, but will be infested with Heterodera glycines. However, arable land and aquifer capacity are likely to be diminished worldwide within 50 years, reducing food surpluses and requiring the utmost scientific effort and ingenuity to improve and protect crops. Nematode management in 50 years will be much more sophisticated and environmentally friendly than it is today, largely because the trend toward truly committed environmentalism is building in developed societies. My guess is that we will be decidedly “green” in 50 years. Students will shudder when really old nematologists reflect wistfully on the days when they injected chlorinated and brominated hydrocarbons into soil. Crop protection will center mostly on the manipulation of plant and pest genomes. However, if there is anything we should have learned already, it is that nematodes often can adapt NEMATOLOGY: DREAMS AND VISIONS OF THE FUTURE (i)

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and select themselves faster than we can change cultivars. Economic nematology will be tied even more closely to cultivar development and testing in 50 years than it is today. I suspect that the sources for new germplasm and cultivars will have swung back to university and experiment station laboratories. Growers will have become extremely sophisticated in their approaches to growing plants, partly because crop prices will be higher due to the contraction of arable land. Producers will turn to the very best, most reliable, and least biased science they can obtain to optimize their yields, and this will be forthcoming from the universities. There will be minor though significant work still being done on the implementation of biological control and the transfer of soil suppressiveness, but soil stasis will still be a difficult nut to crack. However, the maturing interest in and love of environmental harmony will lead to nematode synecology being studied to a far greater degree than it is today. Fifty years from now, universities will remain the centers for training nematologists, but the distribution of strong programs will be much different from what it is today. We can expect to see world-class institutions well-distributed around the world. Instruction in nematology will, of course, still be available, but in a form completely different from what we now have. Our ability to store and process huge quantities of data will continue to grow together with concomitant leaps in the sophistication of computer generated graphics. This means that students in 2057 will learn almost totally by immersion in virtual reality scenarios, where they can be a nematode living its life – whether it be penetrating root epidermis, sucking on a fungal hypha, scooping up bacteria or being attacked and destroyed by another nematode. Students will be able to try out different stylets for feeding or wiggle through soils of different textures. For that matter, they will also be able to be a plant, insect or vertebrate reacting to invasion of a parasitic nematode. If this vision of instruction by virtual reality proves to be correct it will result in instruction and research being fused – acting out different scenarios will result in the generation of hordes of testable hypotheses that may themselves be tried by simulation. Another fundamental change in instruction is that by 2057, nematology students will no longer be dependent for most of their instruction on their home institutions; rather, there will be a world curriculum, taught primarily through simulation, to get the best knowledge from every institution. For instance, taxonomy instruc274

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tion may come from Europe, organic amendment knowledge from India, potato nematode instruction from Peru, cropping systems from the U.S., ecology principles from Poland, dryland farming from Africa and New Mexico. Going a step further, virtual reality-based instruction will enable experts from all continents to cooperate in the development of courses on an intimate scale not previously achieved. Today, we can link together for video conferencing, but the technology is clumsy and prone to breakdowns – or at least will be considered clumsy and fragile 50 years from now! When I was a cute little newborn more than 50 years ago, my parents had just managed to obtain a private telephone line to replace the party line that they had been sharing with three other households. The most glorious event I can remember from my preteen years was the 1962 launch of the Telstar 1 satellite, riding a mighty Delta-Thor rocket into space. It seems almost paltry now to talk of advances in communications. Rather, we are barrelling headlong (not just advancing) into a future where the pace of change will be breathtaking; the changes wrought in that future will be much greater than what has occurred since 1950. I plan to be at the 2057 SON meeting (maybe all meetings will be virtual by then!) to review the predictions that my colleagues and I have made, and to drink a toast to the next 50 years after that.

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(ii) VISION OF NEMATOLOGY IN CANADA IN THE NEXT 50 YEARS GUY BÉLAIR Agriculture and Agri-Food Canada, 430 Gouin blvd. Saint-Jean-sur-Richelieu, Quebec, Canada

What do I see in my crystal ball in the domain of nematology for the next 50 years? Well, as the weather man would say, it’s going to be a mixture of sun and clouds with some possibility of rain or snow. Which basically means that “Everything is possible.” It is almost impossible to predict the political choices that will be made at national and international levels. However, one can only hope that a high priority is given to research and that significantly greater financial resources will be injected into research by industrialized countries (10% or more of GDP per country) to pursue both fundamental and applied research in nematology. Over thousands of years of human activity on our planet mankind has been able to adapt to numerous changes along his/her evolution. Now, what we are experiencing is something quite unique; a phenomenon most probably occurring for the first time in our planet’s long history. The planet earth is trying to adapt to environmental changes caus ed by centuries of human activity. From what we have seen in the last decade, it’s having a hard time coping. Yes, global warming is a huge challenge, with enormous consequences across the planet. We can hear it almost every day on the news report. Somewhere, someplace, the earth is shivering, sneezing and desperately trying to adapt to global warming. What does this phenomenon have to do with nematology? Well, I believe that in the next few decades, or even sooner, this global change will have a tremendous impact on pests, and diseases in agriculture and forestry. Temperate agricultural production areas will see their short list of pests and diseases expanding significantly due to milder tempera276

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tures during the winter and more heat during the summer. For example, in Canada, the soybean cyst nematode, Heterodera glycines, and the root-knot nematode, Meloidogyne incognita, will most probably become common residents of agricultural soils together with many new root and foliar fungal diseases. After a continuous and steady decline over the next 10-20 years, the worldwide number of nematologists will expand again to reach new peaks as the necessity for both basic and applied research in agriculture, forestry, and medicine will be identified as top priorities by universities, industry and governmental institutions. Field extension nematology will advance to meet the challenges by using more accurate tools for monitoring damaging nematode populations. From a soil sample, both scientists and extension workers will be able to make an assessment of the number of pathogenic nematodes using quantitative molecular kits. Traditional morphological identification will still be required to keep track of new species in a given territory. Using internet and interactive web sites, agronomists will have access to information on damage thresholds and also predictive models for managing major pathogenic species. New regulations and rules between industrialized countries on global market issues will be implemented in order to maintain and assure the survival of agriculture in all producing countries. The demand for healthier, quality foods will be booming and chemical pesticide applications reduced as Governments legislate restrictions in their use. The outcome will require increased research on alternative control methods, including biological controls, and this will generate new tools. The list of entomopathogenic nematodes available for insect control will grow, and the in-vitro multiplication of several species of insect parasitic mermithids will be achieved and made available commercially. Steinernematid and heterorhabditid nematodes, better adapted to drought and active at cool temperatures, will be developed and extensively used. The symbiotic bacteria, Xenorhabdus and Photorhabdus, will have revealed their active ingredients which will be applied as environmental friendly compounds with increased specificity against harmful pests. Other basic research on these bacteria will be pursued to successfully introduce the bacterial genes into plants to provide high resistance to both foliar and root insect feeders. The availability of fresh water is already a major issue in numerous countries. Irrigation of large production areas will be reduced NEMATOLOGY: DREAMS AND VISIONS OF THE FUTURE (ii)

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and in some areas prohibited in order to maintain sufficient amounts of water for human consumption. Research on drought resistant crops will be included in breeding programs, together with multiple resistance to plant parasitic nematodes and diseases, and this will be the major tool for maintaining high yield production in industrial and, more importantly, in third-world and developing countries. I believe GMO’s will be extensively used, providing all major crops such as wheat, rice, sorghum, millet, etc., with multiple resistance to plant pathogens. Basic research on model organisms, such as Caenorhabditis elegans, will be expanded as numerous revelations in intra- and extra-cellular communications and gene expression will have contributed to the development of new ways of detecting and curing medical disorders, such as cancer and AIDS. Studies on cryptobiosis and anhydrobiosis of nematodes such as, Anguina tritici, will be pursued by both medical and NASA research teams. This ametabolic state of life could essentially permit life to persist indefinitely until environmental conditions are hospitable. The increasing energy costs for producing food for humanity will significantly reduce the livestock industry and stimulate the creation of alternative protein sources. New sources, such as nematode-based tofu, will be investigated by the food industry. Basic new mathematic models based on the study of the development and growth of C. elegans will explain the secret to the formation of life on our planet and, ultimately, will provide all the information needed to recreate life under laboratory conditions from basic molecules. Many of my colleagues may consider this wish list to be somewhat out of proportion … and they are probably right. But when you think about it, life is actually all a matter of choices. One of our priorities as human beings is to take full responsibility for our actions as creators of change. Every day, we create our own lives. Imagination and dreaming are also part of our divine capabilities for creating a “today”, a “tomorrow” and a “future”. On this same level of thinking, we must imagine (John Lennon, 1971) and integrate into our own lives, now more than ever, unconditional love, tolerance and happiness for ourselves and for others in order to help make it happen in all mankind. Yes, I see a great future for nematology.

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(iii) THE FUTURE OF NEMATODE SYSTEMATICS AND PHYLOGENY OVER THE NEXT 50 YEARS VIRGINIA R. FERRIS Department of Entomology, Purdue University, Smith Hall, 901 W. State Street West Lafayette, Indiana, USA

As one who began her nematology career prior to academic use of computers or the discovery of the double helix, I assume that today’s young nematologists will experience comparable paradigm changing discoveries, inventions and ways of doing research during their careers. Rather than speculate about general changes in nematological research over the next 50 years, I prefer to reflect on the future impact on nematode systematics research of changes in thinking and technology that are already underway. Probably the most crucial of the coming innovations that will affect nematode systematics 50 years out cannot even be envisaged at the present time. For example, although wide-spread use of computers was probably predictable, as well as the unravelling of the nature of DNA, who could have predicted the discoveries of restriction endonucleases and DNA cloning, automated DNA sequencing, large array and high throughput molecular analysis, or genetic transformation for pest resistance in plants. We did not, perhaps could not, envision the extent and rapidity of technical improvements to computer hardware and the vast array of software developed to utilize such enormous quantities of new molecular data. Polymerase chain reaction (PCR) and the companion invention of the thermocycler alone were career-changing for me because these tools enabled me to obtain DNA from a single nematode specimen for use in molecular systematics and phylogeny. Molecular sequencing technologies will continue to improve. Just as we evolved from the period of large sequencing gels, laboriNEMATOLOGY: DREAMS AND VISIONS OF THE FUTURE (iii)

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ously poured, loaded and analyzed with light boxes in our laboratories to automated sequencing and computer generated output, nanosequencing is coming at breakneck speed. Because they can be produced so rapidly the generation of nano-sequences of increasing length will change the face of systematics and phylogenetic analysis. Large array molecular analyses of all kinds, generated to explain how organisms behave and function, are proliferating; and even large companies find crucial needs for people skilled in in silico research. Increasingly, graduate students are generating over a few weeks more data than can be analyzed in years. This will lead to increasingly more complex software to make sense of polybites of new data and whole new fields of research in bioinformatics. Many nematology graduate students will find that their entire research project revolves around data mining, not in libraries of books, but of computer data stored in electronic data repositories. They may never enter a lab or see a whole nematode under a microscope even though they are trying to understand living organisms. I can foresee continued heated discussion for the next decade or more about DNA barcoding and its place in systematics. The current discussions about DNA barcoding are an opening salvo in what I believe will be an ongoing and prolonged discussion about the kinds of data that will be utilized in future taxonomic and systematic research, i.e., the outcome of these important discussions will determine what nematode systematists of the future will be doing. A DNA barcode at the present time is defined as a specific subset of the DNA of an organism believed by barcode proponents to delimit species boundaries, facilitate rapid identification (by comparison of barcode data among all known species of interest), and provide sufficient data for phylogenetic analysis. A principal and early proponent of DNA barcodes has been the entomologist Paul Hebert, University of Guelph, Guelph, Canada. He is joined by many colleagues, especially people who have major interests in biodiversity, ecosystem inventories and conservation, e.g., Daniel Janzen, University of Pennsylvania, Philadelphia, USA, and Allen Herre, Smithsonian Institution, Panama City, Panama (both evolutionary biologists). Dr. Hebert has designated about 600 base pairs (bp) of the DNA of mitochondrial cytochrome oxidase I (COI) as the barcode of choice, and many concur. Often researchers predict that pocket gadgets will be available that will enable any researcher to input a tiny sample of the specimen into the gadget and, based 280

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on barcode data, quickly learn the identity of the specimen and its relationship to other taxa. Although I laughed with a recent class of mine at this notion, a few weeks later a student brought me a paper that suggested ongoing development of such a gadget by an entrepreneurial company. An important nematology spokesman for DNA barcodes has been Mark Blaxter, although his examples have emphasized barcode sequences of ribosomal DNA (rDNA) for species previously described. Others have suggested that barcodes from several genes will be needed for identification and analysis. Although barcode proponents often mention the value of “traditional taxonomy”, they likewise point out that experts in taxonomy are disappearing from university and other laboratories, and that rapid advances in cost-effective technology will likely force society to abandon traditional taxonomy for more economic approaches. Opponents of DNA barcoding provide vigorous arguments about the perils of an emphasis on DNA barcoding at the cost of a truly integrative taxonomy that includes traditional elements as well as new molecular sequence data. Among these spokesmen are entomologists Quentin Wheeler, Natural History Museum, London, UK, and Kipling Will, University of California, Berkeley. They argue forcefully that the notion that DNA barcodes can replace traditional taxonomy and systematics is a very bad idea. They are skeptical that a 600 bp piece of the mitochondrial COI gene can suffice to sort out all of the life forms that may be encountered (and this skepticism is shared even by some writers who basically like the idea of DNA barcodes to circumvent traditional systematic procedures). A number of writers express concerns that the “hype” over the DNA barcode approach will appeal to research grant panels and boards who distribute funds but who have little knowledge of systematics; and that such shifts in funding will result in even fewer practitioners of integrative taxonomy who use many different kinds of characters. This debate will continue and I am unable to predict the outcome. Currently, we are not able to sequence entire organisms quickly and efficiently in our own laboratories, and therefore systematists must use morphological, behavioral and other data, in addition to whatever molecular data we are able to acquire. When nano-sequencing techniques make it possible to easily and economically obtain the entire sequence for every specimen, and new hardware and software enable appropriate comparisons and phylogenetic analyses of these sequence data, the barcode controversies about NEMATOLOGY: DREAMS AND VISIONS OF THE FUTURE (iii)

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which piece of DNA to use may abate. However, the controversies regarding the need for an “integrative taxonomy” will likely persist. Will nematologists find a need to collect other kinds of data about their specimens and have the funds to do so? Will DNA sequences tell the whole story regarding species similarities, differences and phylogenetic relationships? Will a remnant of nematologists remain who take their students on collecting trips and marvel over the structural beauty of specimens they find in soil or stream? Will nematologists still have microscopes in their laboratories? Today’s youngest nematologists will know the answers in 50 years.

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(iv) A NEMATOLOGIST´S DREAM FLORIAN GRUNDLER Institute of Plant Protection, BOKU-University of Natural Resources and Applied Life Sciences, A-1190, Wien, Austria

I was scared and disturbed by a penetrating knocking on the door of my office. “Mr Grundler, are you ready? Your lecture will start soon and as it is for your farewell ceremony you should be on time!” My secretary – what good luck to have her around! I wonder what would have happened to me without her during the past 20 years. I glanced at my watch – two o´clock, 4th of October 2025 – and continued to go through my manuscript. It comprised an overview of the last 40 years of Plant Nematology and an outlook on the future. Since the late 1980s, plant nematology has received a strong boost triggered by the rapid developments in ecology, molecular biology and computer sciences. Nowadays, complete maps and sequenced genomes are available for many nematodes and for all important crop hosts, and routine nematode identification is almost completely based on sequence analysis. We have just established our most recent purchase in the lab: a fully automated “life analyzer” that identifies organisms and provides information on the most important biological parameters through the integrative analysis of nucleic acid sequences, transcription activities and metabolic products. A single nematode, a small sample of microbes or a piece of plant, and the name of the organism or organisms are presented together with all available information. Many problems in plant nematology that have kept generations of scientists busy, seem to be solved today: we know a lot about the interactions between plant and nematodes. For example the function of plant resistance genes are now well understood. It was very exciting to learn about the function of nematode compounds triggering feeding cell development in root-knot and cyst nematodes. Now they are identified and the cascades of events in plants leading to the development of feeding cells are well explained. NEMATOLOGY: DREAMS AND VISIONS OF THE FUTURE (iv)

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We also know a lot about the nematodes. For most of the described nematode species their relation with their host plants can be clarified and their economic and ecological impact identified. The knowledge gained also has been transferred into control measures. Transgenic approaches have been developed to engineer resistance in crop plants by the transfer of resistance gene components in a wide range of host plants. In other cases the signal cascade leading to the formation of feeding cells can be locally interrupted thus preventing the development of nematodes. In other cases, defence pathways are triggered whenever nematodes start to invade or feed on the particular plant. Nematicides are still available but their development has headed in a completely new direction. Some of them are harmless synthetic compounds that can be sprayed on plants. At sites of nematode infection, however, they are processed to effective nematicides by the action of plant or nematode compounds. An important role is played by microbial nematicides. After decades of research a number of highly active microbial nematicides are available on the market. But to be honest, there are still a lot of problems caused by nematodes and other pathogens. Although the ideological altercation between conventional and organic farming is now history and the real integration of culture methods and control methods has brought considerable improvement to the situation, our little worms are still almost omnipresent. The fact is that in a number of cases artificial or natural resistance has been overcome by new pathotypes, while on the other hand the establishment of new plant species as crops for food and industrial uses has put nematode species on the map that were “no names” previously. A problem that has become more and more important is the almost unpredictable occurrence of extreme weather events especially in moderate climates. While heat and drought has brought about a continuous and significant expansion of deserts and the Sahel, in our former moderate climatic zones the weather nowadays oscillates between extreme frosts or warmth in the winter and rapid changes from heavy rainfall to long drought periods in the summer. Unfortunately, our crops seem to be much less well adapted to these challenges than nematodes or pathogens which often find good conditions in the weakened plants and thus aggravate the already serious damage. It will be a challenge for all disciplines to improve plant production under these conditions. 284

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I settled back in my chair and deliberated again on the future as it would pass the half century. The number of described nematode species has increased especially over recent years when taxonomic procedures have become more and more automated. Nematodes were confirmed to live in nearly all environments and to be the “absolute exploration experts”. Some of them are excellent biological models in genetics and cell biology and these have been studied in great depth. However, the great wealth of variation in physiological and behavioural adaptations is still not fully realized and by far not fully used. I suggest that nematodes will be more and more used as a source of knowledge and a resource for industrial purposes. For example, high value proteins and other biologically active compounds will be produced by fermentation of specific nematodes for nutritional, pharmaceutical or technical application. On the other hand they will be employed e.g., to improve degraded soils or by releasing specific species combined with microbes. Many more applications will be found. Just recently… The telephone rings. Tapping around in the dark I try to find the receiver. “Mr Grundler, this is your wake up call, it is seven o´clock!” A look at the illuminated alarm clock confirms what the friendly voice said. I hesitate when reading the date on the display: 12th of October 2006. I try to remember. I have been somewhere else. What happened? Maybe a dream, but, unfortunately, I cannot remember what it was about.

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(v) MY NEMATOLOGY DREAM PAULO VIEIRA Lab. Nematologia/ICAM, Universidade de Évora, Évora, Portugal

As a graduate student, when I was asked to dream about the future of nematology, my first thought went to Jules Verne´s famous book, Twenty Thousand Leagues Under the Sea, to find inspiration. Although Verne was able to imagine a future for the world, he was unable to accurately portray the basic changes through which the twentieth century would go. It is also unclear to me what substantial advances will take place in nematology during the next fifty years. Nevertheless, in view of the knowledge that this new discipline has already reached we can expect a promising future. As a discipline and a society we should remember what has already been accomplished in the field of nematology in a relatively short time. Important resources are already available from the internet and from genome-based technologies, and are likely to increase. With globalization, nematology will face many challenges while minimizing the introduction of new nematode pests (“non-beneficial nematodes”). At the present time there are several diseases caused by nematodes that, although not completely under control, have been successfully managed. For some diseases, such as pine wilt disease, caused by Bursaphelenchus xylophilus, the best disease management is prevention such as minimizing introductions, forest management, tree species selection, etc. Nematologists will continue to participate in inter-disciplinary approaches to overcome the complexity of such diseases. Very recently, the secretary of FAO called for a new “green revolution” in the face of the growing human population and the need to obtain more food and fiber. It has been calculated that an additional 1 billion tons of cereals will be needed to feed the world. This increase in crop production carries a concomitant need to protect 286

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plants, be they field crops, horticultural crops, ornamentals or forest species. Nematodes, as major pests and pathogens, will certainly continue to play an important role in limiting the production of these essential products for human survival and economic growth. The progressive reduction and phasing out of many pesticides, essential in the 1970s and 80s to control soil pests, will make certain groups of nematodes, such as entomopathogenic nematodes (EPNs), more relevant. However, the public, as a whole, still needs to learn the importance and potential of nematodes as beneficial organisms (e.g., Caenorhabditis elegans and EPNs). The major developments in science and technology often draw from curiosity-driven research, and over time these developments have had a great impact on national interests. As a consequence of recognizing economic benefits, nematologists will face new paradigms, such as to perform research mainly for the economic interest and benefit of a region or country. I wonder if this will lead to inequality between those who have access to research and those who do not. Nematology could split into several sub-disciplines and thus lead to tough competition between them, which could ultimately lead to the disregard of some areas (e.g., classical taxonomy). In such a scenario, the future of this discipline should stimulate a better and more determined discussion among nematologists, and perhaps a better definition of itself. From the beginning of my career in science I have always had the following quote in mind – Science came into its own when it managed to refuse the subjective and embrace the objective. Looking ahead into the next 50 years my dream is that nematology will always keep this in mind, and embrace the objective (and “basis”), the nematode!

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(vi) THE DEVIL’S ADVOCATE DEREK J.F. BROWN College of Tourism, Bansko, Bulgaria

Nematologists are a small part of the general plant pathology community. Since the inception of the European Society of Nematologists in 1956, and up to the 1980s, plant pathologists, nematologists included, have largely achieved their purpose of increasing and protecting strategic world food supplies and commercial crops, including forestry. Much of the achievement was driven by staff from Europe and North America, where governments had invested heavily to i) increase strategic, national production and commercial returns from agriculture and forestry, and ii) circumvent a perceived world food shortage as a result of global population increase. In these two continents in more recent times there has been a progressive and increasingly rapid decline in numbers of these staff. To speculate on the future of nematology over the next 50 years the world can be divided into three socio-economic regions.

Regions “A” – Australasia, Europe, Japan, North America and Russia Globalisation, cheap international freight costs, and the emergence of international supermarket chains have each contributed to major changes in agricultural and forestry production to service populations in these regions. With high disposable incomes, basic food costs are increasingly becoming a smaller proportion of the individual’s budget. In these regions an increasing quantity of basic food products are imported due to lower production costs available in other regions and accompanying low international transport costs, whilst demand for alternative foods also increases. This results from higher 288

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production costs for home grown foods, and inexpensive international travel that exposes individuals to alternative foods. Substantial current emphasis in nematology in these regions is directed towards technology transfer and education, ecology and molecular biology, including genetic manipulation of crops. Nematodes are increasingly being removed from “funding” agendas, as they are not perceived by the public or governments to be of importance except in plant quarantine and to provide an opportunity for technology transfer and education, i.e., training of overseas postgraduate students that financially contribute to the national income. Technology transfer and education is short term, as trained foreign students returning to their home regions will supply this function locally. As a result of the non-recognition of nematology, funding for nematode ecology will rapidly decline as will granting for much molecular biology that is already considered “taxonomic tinkering”. Genetically manipulated crops have spectacularly failed to gain public acceptance, and this also has contributed to funding decline. International supermarket conglomerates that control global food production can, almost overnight, shift crop production from one country to another, and frequently between continents. In the future, global supermarkets will be the source of plant pathology/nematology funding, but such funding will be small, short term, highly focused on an end product result, and much of it provided to “local” staff. Plant quarantine services provide governments with a rationale to control global trade through prevention of the introduction of non-indigenous harmful pests and pathogens. Thus, plant pathologists with some nematology training, will be employed as nematode taxonomists in plant quarantine services. Consequently, there will be many fewer nematologists than currently employed working in these regions in 50 years time.

Regions “B” – Africa, Central and Latin America, China, India and Pakistan There is a requirement to increase food and forestry production to serve the populations in these regions. Also, there is an increasing demand by populations in Regions “A” for food and forestry products produced in Regions “B”. Nematologists are already working in these regions and will continue to be required to provide strategic input NEMATOLOGY: DREAMS AND VISIONS OF THE FUTURE (vi)

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to increase agricultural and forestry production. As well, global supermarkets will employ local nematologists for specific research when required. Plant quarantine personnel, including nematologists, are already working in these regions as part of the global endeavour to prevent the introduction of non-indigenous pests and pathogens. It appears unlikely that there will be any significant change in the number of nematologists employed in these regions during the next 50 years. In some countries, e.g., Brazil, there is likely to be a reduction in numbers whereas in others, such as China, there may be a modest increase.

Regions “C” – Others Few nematologists are employed in these regions, mainly as a result of local national economic situations. This situation may change if global supermarkets shift food production to these regions. Any increase in the number of nematologists that might be employed in these regions during the next 50 years will be relatively small.

The alternative scenarios Globalization, particularly as it affects agricultural and forestry production, is dependant on low income nations being used to provide food/services to high income nations and inexpensive transport is a key factor. With existing oil supplies steadily declining, combined with increasing demand from developing nations such as China and India, it can be expected that international transport costs will substantially increase during the next 50 years. When the importation costs become equal to, or greater, than local production costs it can be anticipated that home grown production will replace imports. In such a scenario there will be renewed interest in “regenerating” national plant pathology, including nematology, to help in increasing strategic national production and commercial returns from agriculture and forestry in Regions “A” Global climate change may provide incentives and opportunities for “regenerating” national plant pathology, including nematology. For example, it may become feasible to produce in Regions “A” some of the products that currently can be produced in only 290

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Regions “B” and “C” This change of production centers would require the assistance from plant pathologists, including nematologists, who would be employed in Regions “A”. With one, or other, or both of these scenarios the number of nematologists that might be employed in Regions “A” will be relatively small, and quite unlike the number of nematologists employed previously.

Conclusion It would be gratifying, having spent 32 years as a nematologist, to be positive about the future prospects for nematology. Sadly, with extensive global travel I have reached the painful conclusion that globally the science of nematology, as with plant pathology in general, is in increasingly rapid decline. There appears to be little chance of recovery, and by 2056, the 100th anniversary of the European Society of Nematologists, there will be many fewer nematologists than at present. It is not inconceivable that even within the next 25 years rather than waiting for 50 years, the European Society of Nematologists will itself have ceased to function.

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(vii) MY DREAM, NOT A NIGHTMARE A. FOREST ROBINSON USDA-ARS, 2765 F & B Road, College Station, Texas, USA

Here is my dream (hopefully not nightmare) of nematology 50 years from now. I hope my vision is not unduly skewed by my own research career having been focused on a single crop (cotton) and only two species of nematode. Fortunately, I’ll not be around to accept the blame for factual errors. Please ascribe occasional irreverence to my father, from whom it was inherited. I dreamt nematology had advanced … as a body of knowledge, a funded research endeavor and a community of scientists. Sequencing studies had long ago answered many central questions in nematode systematics, and functional genomics had provided a good framework for investigating and understanding at least in part, the basis of plant parasitism by nematodes, host plant resistance, and host specificity. Molecular methods had long ago identified numerous candidate suppressive agents within soil and provided regulatory agencies with powerful tools for identifying taxa of regulatory importance. The world (finally) had embraced transgenes in crop plants. Nematode resistance transgenes from fungi, not plants, had been successfully inserted into plant genomes with promoters from viruses, and these constructs (like their insect resistance transgene predecessors) made some plants but not others resistant to some nematodes, but not to others. Consequently, transgenes had found their place, the novelty was over, and nematologists were again intensely exploring fundamental (in some cases new and in some cases long-neglected) components of nematode biology – pheromones, hormones, the species concept, plant host finding (!), and the underlying biochemistry, morphology, toxicology, and physiology of parasitism of nematodes by antagonists, and ditto for parasitism of plants and insects by nematodes. 292

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Intensive studies had extended nematode ecology beyond comparative nematode community structure, to the exploration and modeling of dynamic interactions among all soil biota, as influenced, of course, by soil physics, nutrient cycling, carbon to nitrogen ratio, plant growth, moisture fluctuations, respiratory gas exchange and by diurnal thermal waves through soil. A burgeoning new field expanded the Bernardian rule to predict trophic and species diversity in disturbed and undisturbed soil-plant systems. (Apologies to Ernie Bernard, as it must have been his intriguing symposium at the Society of Nematologists meeting in Kauai that triggered this reflection) In applied nematology, a major breakthrough had created many new jobs, increasing teaching and research positions in nematology by several-fold (reminiscent of the demonstration of the huge yield responses to soil fumigation more than a century earlier), but when I started to read in detail about the breakthrough in a recent journal article, I awoke. In a (relatively) crazy dream, I saw new waves of nematologists sequentially clearing the way for new dogma by freeing themselves of the previous wave’s, starting with our own. One such wave viewed the plant damage models of today as antiquated oversimplifications suitable for management predictions only in Mediterranean environments, but provided no immediate substitute. Another wave discarded the concept of host race, returning it to plant pathology, placing plant host preference and virulence within a large set of genetic plasticity dimensions. I dreamed of a Journal of Nematology with expanded freedom of expression and no page charges (now, that was REALLY crazy, but probably inevitable, following the conversion to 100% on-line publication). As a new requirement, all field plot manuscripts had to include a small diagram illustrating the experimental design (This seems like an excellent idea to me!). In dreams of research funding (dreams about the long-term future, that is, not the short term!), I saw partial globalization of resources for agricultural research, and the consequent refocusing of costly, technologically elite methodology at centers of nematological excellence in tropical, subtropical, and Mediterranean latitudes (where nematological problems historically have been arguably the most important and certainly the most complex, and yet the least well funded). I saw stationery letterhead (in three colors of ink, which suggests to me, lots of money) for the European Research NEMATOLOGY: DREAMS AND VISIONS OF THE FUTURE (vii)

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Institute for Mediterranean Nematology (at Bari), and similarly colorful letterhead for the Global Center for Fundamental and Applied Nematological Research (at Delhi), and the International Tropical Nematology Research Center (at Turrialba). I also saw evidence of recognition, by research granting institutions, that plant nematodes are in fact animals (i.e., animals first, and plant pathogens second), whose ecology, development, and behavior had come to merit, incredibly, the highest of funding priorities. I also noted that major research grants were being (without precedent) routinely awarded for the study of non-“entomopathogenic” insect parasites, and the biology of marine, freshwater, and free-living soil nematodes. In a dream about the worldwide nematological community, I saw nematologists continuing to focus on specific groups of nematodes, for example, on entomopathogenic nematodes, on plant nematodes important to a specific agricultural commodity, on parasites of domestic animals, on nematodes as bio-indicators, etc., because nematologists continued to be funded for the most part to study specific groups. However, as might be projected from today’s situation, I saw geographically isolated nematologists outpacing other scientists by utilizing the internet as part of a global scientific community. This was characterized by refreshingly vigorous interaction among scientists studying the same group in widely separated localities. I also saw frequent collaborations among geographically separated scientists for the purpose of comparing ecologically diverse nematode taxa. These developments and universal conversion to on-line journals had led, essentially, to the establishment of a World Society of Nematology with its own on-line journals, replacing the International Federation of Nematology Societies, which had outlived its usefulness. Was this really all a dream? A nightmare? Or perhaps, just happy ruminations!

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(viii) MY DREAM OF THE FUTURE OF NEMATOLOGY AND CHEMICAL COMMUNICATION RESEARCH – 50 YEARS FROM NOW EKATERINA RIGA Washington State University, Prosser, Washington, USA

For several years nematologists have been working on nematode behavior, orientation and the mechanism of nematode orientation to a range of chemical and non-chemical cues. Cues that evoke nematode responses are potential candidates for integration into nematode control strategies for commercial use in crop protection. Disruption of nematode orientation in response to species-specific pheromones, sex specific pheromones or host chemical cues could have environmental, economic and biological importance because it could lead to the discovery of new tools to manage plant parasitic nematodes. Although behavioral assays to study pheromones and host chemical cues for several plant parasitic nematode species have been devised, the specific chemical structure of nematode pheromones and host chemical cues have not yet been identified. In the future, semiochemical cues will be identified that will disrupt or saturate nematode reception especially when the nematode is searching for a host plant or for a mate. Sex and species-specific pheromones will be used in the field as nematode control agents. For amphimictic nematodes, sex pheromones will be applied in the field to saturate the soil environment, thus the male nematode sensory organs will become saturated and unable to respond to the signals from their females. Therefore, mating will either be inhibited or delayed to the extent that males will die due to starvation. Since hermaphroditic nematodes do not use sex pheromones to communicate with each other, we could saturate their environment with compounds that are antagonistic to NEMATOLOGY: DREAMS AND VISIONS OF THE FUTURE (viii)

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their species-specific pheromone, thus preventing their members from communicating with each other. Plant host cues will be identified for both amphimictic and hermaphroditic nematodes and used to design antagonistic compounds that mask the host cues and lead to nematode disorientation. For example, plant seeds or seedlings could be coated with nematode antagonistic compounds for protection against plant parasitic nematode invasion during the plant’s establishing period. In addition, plant host antagonistic compounds will be applied in the field, the same way as sex pheromones, to saturate the soil environment and to disorientate the nematodes. Compounds mimicking plant host root exudates will be used to trigger egg hatching prior to planting, thus the juveniles would hatch and in the absence of plant hosts die from starvation. Alternatively, antagonistic compounds to plant host root exudates will be designed to block egg hatching during planting, thus protecting the young roots from juvenile invasion and feeding. We will be able to genetically modify non-host plants to release host exudates in yet another nematode control strategy. Host plants will be modified to release compounds that attract predators of plant parasitic nematodes to the host root zone. Thus, plant parasitic nematodes will be eliminated due to the presence of predatory nematodes (e.g., mononchids), collembolans, predatory mites and other soil invertebrates. Trap crops will be genetically modified to release exudates with nematode pheromone properties, thus attracting nematodes to non-host roots. For example, genetically modified non-host plants or trap plants could be planted prior to host plants or as intercrops to mislead nematodes. I predict that 50 years from now we will be using these semiochemicals e.g., sex pheromones, species-specific pheromones, and host root exudates, to control plant parasitic nematodes by altering their behavior. The semiochemicals will have a narrow target range as they will be species-specific and nematode developmental stage-specific. As well, in the future, I believe that effective nematode control based on semiochemical isolation and identification will be achieved with minimal environmental impact.

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(ix) C. elegans AS A MODEL SYSTEM FOR SPACE TRAVEL ROBERT JOHNSEN & DAVID BAILLIE Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada

A multi-national manned mission to Mars is being planned. Unfortunately, it’s not known if humans can survive the 35-60 days’ trip across 100-million kilometres of space to the intriguing red planet. We don’t even know how much space radiation they will be exposed to or what its effects may be. The Brookhaven National Laboratory used its particle accelerator to attempt to simulate the radiation exposure of a round trip to Mars. The results showed an expected exposure of approximately 130,000 millirem – more than 370 times the annual average dose for Americans (350 millirem). It gets worse. According to the University of California Davis’ environmental health and safety website, the human LD50 (dose lethal enough to kill 50 per cent of the people exposed) is about 500,000 millirem. So a round trip to Mars could expose the voyagers to more than one-quarter LD50, which means that one person in eight could die from radiation poisoning while the rest would probably be very sick. This does not even take into account unpredictable solar flares which could increase the dose many times. The Brookhaven results may or may not be true. Little is known about the effects of long exposure to space radiation either aboard the International Space Station (ISS) – where there has been continual habitation for a few years – or for long-duration spaceflights such as the planned Mars mission. While manned spacecraft have been equipped with excellent radiation detection devices, they don’t measure the biological damage caused by space radiation. Caenorhabditis elegans is a good model system for collecting NEMATOLOGY: DREAMS AND VISIONS OF THE FUTURE (ix)

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data on the biological effects of radiation in space. These data could lead to the development of radiation countermeasures enabling long-duration manned spaceflight and continual habitation aboard the ISS. C. elegans has many advantages. A prime one is its small size (adults about 1mm long) and this enables C. elegans experiments to be very compact. This is important because it is expensive to launch weight into orbit, and spacecraft and the ISS, are very cramped. In addition, C. elegans has a short generation time. Its two-week lifespan enables a single hermaphrodite to produce about 300 offspring, each with less than 1,000 somatic cells, which are easy to maintain. These advantages allow short, flexible and cost effective experimental procedures. The nematodes can be cultured in small bags so that all an astronaut has to do is inject liquid food into the bags every couple of weeks. Another advantage is that C. elegans is the simplest multi-cellular organism with a completely known genomic DNA sequence. Like humans, C. elegans has about 20,000 genes, about 4,500 of which are orthologous to human genes. These orthologs include a large set of DNA damage repair genes and thus C. elegans is an excellent model for predicting potential biological damage to humans in the space environment. Our laboratory has Canadian Space Agency funding to evaluate the use of C. elegans DNA-array chips, containing bits of every gene, for their efficacy in the rapid analysis of space radiation induced deficiencies. Caenorhabditis elegans has already travelled in space on several missions. These include 1993 and 1996 JPL flights (Nelson et al., 1994. Advances in Space Research 14: 87–91& 209–214; Hartman et al., 2001. Mutation Research 474:47–55); the 2003 Columbia flight (Szewczyk et al., 2005. Astrobiology. Dec: 5(6): 690–705), which crashed in Texas; and the 2004 First International C. elegans experiment (ICE-first) (Zhao et al. 2005. Gravitation Space Biology Bulletin 18: 11–16) on the Delta mission. Amazingly, living nematodes were recovered from the Columbia disaster. They survived an impact 2,295 times the force of Earth’s gravity. “This is a very exciting result,” said Catharine Conley of NASA, “It’s the first demonstration that animals can survive a re-entry event similar to what would be experienced inside a meteorite. It shows directly that even complex small creatures originating on one planet could survive landing on another without the protection of a spacecraft.” The two JPL experiments and ICE-first used the eT1 mutagen testing system (Rosenbluth et al., 1983. Mutation Research 110: 298

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39–48; Johnsen & Baillie. 1991. Genetics 129: 735–752). eT1 is a balancer that can capture mutations in the genome of C. elegans for laboratory analysis after a completed spaceflight. While the first nematodes-in-space experiments were for short-time (about one generation) exposure to space radiation, ICEfirst lasted a little longer – 11 days in space. These short experiments yielded only a few mutations, not much above the number of spontaneous mutations we would expect on Earth. This is probably because satellites in low Earth orbit (LEO), including the ISS, are protected by Earth’s magnetosphere. Trips to the Moon or Mars will last a lot longer and will not be protected by the magnetosphere. So far, all the C. elegans space flights have been to LEO. In the future we would like to send nematodes on a very long unmanned mission outside the Earth’s protective magnetosphere. We would use an automated feeding system and take advantage of fluorescent proteins such as green fluorescent protein (GFP) and a fluorescence detector. The system would be designed so that new mutations would alter the nematode’s fluorescence in a detectable manner. This would give much better data then the LEO missions on the biological effects of long term exposure to fluctuating dosages of different types of radiation in deep space. The data could lead to spacecraft designs that will protect humans as we move out through space exploring and colonizing our solar system. Nematodes led us in the human genome sequence project now they may lead us into space.

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